Pharmacodynamics Of Methotrexate Research Articles

The Influence of Methotrexate-Related Transporter and Metabolizing Enzyme Gene Polymorphisms on Peri-Engraftment Syndrome and Graft-Versus-Host Disease after Haplo-Hematopoietic Stem Cell Transplantation in Pediatric Patients with Malignant Hematological Diseases

Background: Methotrexate (MTX), utilized as a graft-versus-host disease (GvHD) prophylactic agent in allogeneic hematopoietic stem cell transplantation (allo-HSCT), has been proven to effectively decrease the occurrence of the peri-engraftment syndrome (Peri-ES) and acute GvHD (aGvHD). Changes in the pharmacodynamics of MTX are closely associated with gene polymorphisms in genes encoding drug-metabolizing enzymes and transporters. Nevertheless, the current studies mainly concentrate on leukemia or autoimmune diseases, and limited studies on the allo-HSCT were reported. Methods: Here, we retrospectively assessed the relationship between MTX-related transporter and metabolizing enzyme gene polymorphisms, clinical characteristics and outcomes in 57 pediatric patients, who received haploid HSCT (haplo-HSCT), with malignant tumors at a single center. Log-rank test and Cox proportional regression model were used for univariate and multivariate analysis, respectively. A significance level of 0.05 was used for all analyses. Results: At baseline, the mean age at HSCT was 5.1 years old, and more males (80.8%) were enrolled in our cohort. Acute leukemias, including mixed phenotype acute leukemia (MAPL), was the most common etiology, which constituted 94.7% (54/57) of the cohort. All patients achieved hematopoietic recovery from haploidentical donors. The median time to neutrophil and platelet engraftment was 12 days (range, 10-23), 12 days (range, 5-39), respectively. A total of nine kinds of gene polymorphisms were detected and the most common variant type was ABCB1 (1236C>T), followed by MTHFR (665C>T) and ABCB1 (3435C>T). All gene polymorphisms were in Hardy-Weinberg equilibrium in our cohort. We observed the median platelet recovery time of 16 days (range, 10-24 days) for ABCB1 (1236C>T) CC genotype was significantly longer than the median time of 11.5 days (range, 5-39 days) for TC/TT genotype (P = 0.042). In this study, 61.4% of patients (35/57) developed Peri-ES, and 36.8% patients (21/57) developed grade II-IV aGvHD, including 8 (14.0%) with grade II, 8 (19.3%) with grade III, and 5 (8.8%) with grade IV. Compared with SLCO1B1 (1865+4846T>C) TT, patients with SLCO1B1 (1865+4846T>C) TC/CC had the increased incidence of Peri-ES (75.9% vs 46.4%, P = 0.030). Based on the multivariate Cox analysis, we discovered that SLCO1B1 (1865+4846T>C) TT genotype was an independent protective factor for Peri-ES morbidity (hazard ratio (HR)= 0.464; 95% confidence interval (CI)=0.231-0.931; P=0.031). Additionally, when compared with mononuclear cells (MNC) low dose according to the median dose value (cutoff: 6.99×10 8/kg) in our cohort, patients with MNC high dose had the increased incidence of II-IV aGvHD (50.0% vs 24.1%, P = 0.030). Cox regression analysis identified that the dose of MNC reinfused was significantly related with II-IV aGvHD as an independent risk factor in multivariate analysis (HR = 2.604; 95% CI=1.049-6.463; P = 0.039). Unfortunately, no differences was discovered between aGvHD after haplo-HSCT and MTX-related gene polymorphisms. Conclusion: In summary, our findings prove that the host's genotypes might modify the risk of developing Peri-ES, contribute to a better understanding of the inter-individual difference in efficacy, and facilitate the development of individualized approaches to GvHD prophylaxis.

The influence of methotrexate-related transporter and metabolizing enzyme gene polymorphisms on peri-engraftment syndrome and graft-versus-host disease after haplo-hematopoietic stem cell transplantation in pediatric patients with malignant hematological diseases.

Methotrexate (MTX), utilized as a graft-versus-host disease (GvHD) prophylactic agent in allogeneic hematopoietic stem cell transplantation (allo-HSCT), has been proven to effectively decrease the occurrence of the peri-engraftment syndrome (Peri-ES) and acute GvHD (aGvHD). Changes in the pharmacodynamics of MTX are closely associated with gene polymorphisms in genes encoding drug-metabolizing enzymes and transporters. Nevertheless, the current studies mainly concentrate on leukemia or autoimmune diseases, and limited studies on allo-HSCT were reported. Here, we retrospectively assessed the relationship between MTX-related transporter and metabolizing enzyme gene polymorphisms, clinical characteristics, and outcomes in 57 pediatric patients who received haploid HSCT (haplo-HSCT) with malignant tumors at a single center. We discovered all gene polymorphisms were in the Hardy-Weinberg equilibrium in our cohort. We discovered a significant correlation between platelet recovery time and ABCB1 (1236C>T) (p = 0.042). Compared with patients with SLCO1B1 (1865+4846T>C) TT, patients with SLCO1B1 (1865+4846T>C) TC/CC had an increased incidence of Peri-ES (p = 0.030). Based on the multivariate Cox analysis, we discovered that SLCO1B1 (1865+4846T>C) TT genotype was an independent protective factor for Peri-ES morbidity (hazard ratio (HR) = 0.464, p = 0.031), and the dose of mononuclear cells reinfused was significantly correlated with II-IV aGvHD (HR = 2.604, p = 0.039). In summary, our findings prove that the host's genotypes might modify the risk of developing Peri-ES, contribute to a better understanding of the inter-individual difference in efficacy, and facilitate the development of individualized approaches to GvHD prophylaxis.

Suppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells of rheumatoid arthritis patients.

To investigate the immunosuppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients. Concanavalin A-stimulated PBMC culture procedure was used to evaluate the pharmacodynamics of vitamin K2 in vitro. Methotrexate was set up as the positive control. The proliferation of PBMCs was detected by MTT assay. Relationship between IC50 values of drugs on PBMC proliferation and patient-related factors including laboratory data was analyzed by nonparametric Spearman correlation test. Vitamin K2 inhibited the proliferation of mitogen-activated PBMCs of RA patients with an IC50 value of 3,288.47±4,910.02 ng/mL (mean±SD). There was a significant correlation between IC50 values of vitamin K2 and patient-related factors of RA patients (p<0.05), such as C-reactive protein (CRP), rheumatoid factor, anti-cyclic citrullinated peptide antibody (ACPA), matrix metalloproteinase-3, Pre-DAS-28 (CRP), and ∆DAS-28 (CRP). It would be possible to predict the pharmacodynamics of vitamin K2 in RA patients according to the above factors. Methotrexate inhibited the proliferation of mitogen-activated PBMCs of RA patients with a IC50 value of 22.83±12.47 ng/mL (mean±SD). IC50 values of methotrexate only showed significant correlation with ACPA (p=0.0158, r=0.6905), which suggests that ACPA might be a suitable predictor of the pharmacodynamics of methotrexate. The above information suggests that vitamin K2 could provide a benefit for the treatment of RA patients via its immunosuppressive function.

Methotrexate and Cardiovascular Protection: Current Evidence and Future Directions

Patients with autoimmune rheumatic conditions, particularly rheumatoid arthritis, have an increased cardiovascular risk when compared with the general population. Methotrexate is a relatively old, yet effective, immunomodulatory drug for the management of autoimmune and chronic inflammatory disorders, such as rheumatoid arthritis, particularly in terms of symptom control, quality of life, and disease progression. Recent meta-analyses have also shown that methotrexate treatment is associated with a lower risk of cardiovascular events when compared with other disease-modifying antirheumatic drugs. This suggests that methotrexate might exert specific protective effects against atherosclerosis and thrombosis. This mini-review discusses the mechanisms associated with the increased cardiovascular risk in rheumatoid arthritis, the pharmacokinetics and pharmacodynamics of methotrexate, the available evidence on the in vitro and in vivo effects of methotrexate on modifiable cardiovascular risk factors, and suggest...

Prediction of the therapeutic response to methotrexate at 24 weeks by methotrexate–polyglutamates concentration in erythrocytes at 8 weeks in patients with rheumatoid arthritis

Objectives: The objective of this study is to evaluate the pharmacokinetics and pharmacodynamics of methotrexate–polyglutamates (MTX-PGs) in erythrocytes in patients with rheumatoid arthritis and correlate them with the efficacy.Methods: MTX-PG concentrations in erythrocytes were measured in 42 MTX-naïve patients repeatedly for 24 weeks by high-performance liquid chromatography. In 56 patients receiving stable MTX doses for at least 12 weeks, the correlation between MTX doses and MTX-PG concentrations was examined. The efficacy was measured by the change of DAS28CRP (ΔDAS28CRP).Results: There were moderate correlations between MTX dose and MTX-PG 3, 4, and 5. At 24 weeks, MTX-PG2, 3, 4, and 1–5 were higher in patients with ΔDAS28CRP >1.2 than in those with ≤1.2. The cutoff value of MTX-PG1-5 to discriminate ΔDAS28CRP >1.2 from ≤1.2 at 24 weeks was 68.7 nM. Among 20 patients with MTX-PG1-5 > 50.6 nM at 8 weeks, seven already improved at 8 weeks and additional 11 improved at 24 weeks (p < 0.001). On the contrary, among the nine patients with MTX-PG1-5 ≤ 50.6 nM at 8 weeks, none improved at 8 weeks and only one improved at 24 weeks (p = 0.500).Conclusions: Erythrocyte MTX-PGs might be a potential indicator and predictor of MTX efficacy.

Chemotherapy Pharmacodynamics and Neuroimaging and Neurocognitive Outcomes in Long-Term Survivors of Childhood Acute Lymphoblastic Leukemia.

To examine associations among methotrexate pharmacodynamics, neuroimaging, and neurocognitive outcomes in long-term survivors of childhood acute lymphoblastic leukemia treated on a contemporary chemotherapy-only protocol. This longitudinal study linked pharmacokinetic assays collected during therapy to neurocognitive and brain imaging outcomes during long-term follow-up. A total of 218 (72.2%) of 302 eligible long-term survivors were recruited for outcome studies when they were more than 5 years post-diagnosis and older than 8 years of age. At long-term follow-up, survivors were an average of 13.8 years old and 7.7 years from diagnosis, and 51% were male. Neurocognitive testing, functional magnetic resonance imaging (MRI) during an executive function task, and structural MRI with diffusion tensor imaging were conducted. Generalized linear models were developed to identify predictors, and models were adjusted for age at diagnosis, sex, and parent education. Intelligence was within normal limits (mean, 98; standard deviation, 14) compared with population expectations (mean, 100; standard deviation, 15), though measures of executive function, processing speed, and memory were less than population means (all P < .02 after correction for false discovery rates). Higher plasma concentration of methotrexate was associated with a poorer executive function score (P < .02). Higher plasma methotrexate was also associated with higher functional MRI activity, with thicker cortices in dorsolateral prefrontal brain regions, and with white matter microstructure in the frontostriatal tact. Neurocognitive impairment was associated with these imaging findings as well. Associations did not change after adjustment for age or dose of leucovorin rescue. Survivors of childhood acute lymphoblastic leukemia treated on contemporary chemotherapy-only protocols demonstrate executive dysfunction. A higher plasma concentration of methotrexate was associated with executive dysfunction as well as with a thicker cortex and higher activity in frontal brain regions, regions often associated with executive function.

Genetic determinants for methotrexate response in juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIAs) is the most common chronic rheumatic disease of childhood and is an important cause of disability. The folic acid analog methotrexate is the first choice disease-modifying anti-rheumatic drug in this disease, however, 35–45% of patients fail to respond. Molecular elements, such as variants in genes of pharmacological relevance, influencing response to methotrexate in JIA, would be important to individualize treatment strategies. Several studies have evaluated the effects of candidate genetic variants in the complex pathway of genes involved in methotrexate pharmacodynamics and pharmacokinetics, however, results are still contrasting and no definitive genetic marker of methotrexate response useful for the clinician to tailor therapy of children with JIA has been identified. Recently, genome-wide approaches have been applied, identifying new potential biological processes involved in methotrexate response in JIA such as TGF-beta signaling and calcium channels. If these genomic results are properly validated and integrated with innovative analyses comprising deep sequencing, epigenetics, and pharmacokinetics, they will greatly contribute to personalize therapy with methotrexate in children with JIA.

Understanding the variation in response to methotrexate

ORIGINAL ARTICLE, p 165 Methotrexate (MTX) has been used in the management of psoriasis since the 1960s and despite the increasing use and success of newer biologic agents, it remains a first‐line treatment in patients with moderate to severe psoriasis, due to its efficacy and cost‐effectiveness.1 It is used as a standard comparator in randomized controlled trials of biologic agents in psoriasis. However, a significant number of patients on MTX do not experience benefit and up to 30% of patients are unable to tolerate the drug because of toxicity including nausea, myelosuppression or hepatotoxicity.2 Reasons for this inter‐individual variability in both toxicity and response are partially unknown but may include demographic factors (such as patient age and weight), external or environmental influences (for instance adherence to treatment, drug dosage), and genetic variation in pharmacokinetics (for instance, drug metabolizing enzymes) or pharmacodynamics (drug targets). Pharmacogenetics is the study of those inherited factors that influence the inter‐individual variability in response to drugs and is a rapidly developing field. The pharmacokinetics and pharmacodynamics of MTX, as have recently been unravelled, have proved to be complicated, and early attempts at pharmacogenetic studies in both rheumatology and dermatology using a candidate gene approach have not been definitive. MTX is a pro‐drug which is actively transported into the cell via a transmembrane reduced folate carrier protein (SLC19A1) and actively exported from the cell by ATP‐binding cassette transporters ABCC1 and ABCG2. Polymorphisms in ABCC1 and ABCG2 have been found to correlate with a better response to MTX (with odds ratios of about 2) and polymorphisms in SLC19A1 and ABCC1 have been associated with MTX toxicity. Within the cell, MTX is activated by the sequential addition of between two and seven glutamic acid residues under the action of the enzyme folylpolyglutamate synthetase, thus converting the parent drug to the active and more stable polyglutamated form. MTX polyglutamates (MTXPGs) persist intracellularly, exerting a sustained effect which allows for its once‐weekly administration.3 Some studies of patients with rheumatoid arthritis have shown a correlation between intracellular MTXPG levels and MTX treatment efficacy.4, 5

PharmGKB summary

Mikkelsen, Torben S.a,b; Thorn, Caroline F.c; Yang, Jun J.b; Ulrich, Cornelia M.h; French, Deborahb; Zaza, Gianluigib; Dunnenberger, Henry M.b; Marsh, Sharoni; McLeod, Howard L.f; Giacomini, Kathye; Becker, Mara L.g; Gaedigk, Rogerg; Leeder, James Steveng; Kager, Leoj; Relling, Mary V.b; Evans, Williamb; Klein, Teri E.c; Altman, Russ B.c,d Author Information

Differential in vitro cytotoxicity does not explain increased host toxicities from chemotherapy in Down syndrome acute lymphoblastic leukemia

Treatment-related toxicities such as mucositis and infections are both more frequent and more severe in children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) compared to non-DS ALL. Altered methotrexate pharmacodynamics play a role, but severe toxicities also occur in treatment courses that lack methotrexate. We hypothesized that this might be attributable to heightened cytotoxic effects of other ALL chemotherapeutic agents on DS versus non-DS host tissues. Panels of DS and non-DS lymphoblastoid cell lines (LCLs) and primary fibroblast cell lines were treated with asparaginase, dexamethasone, doxorubicin, mafosfamide and vincristine. LCL survival was assessed using the MTT assay, and fibroblast proliferation using the clonogenic survival assay. No significant differences were observed between DS and non-DS cell lines using either assay. Both DS and non-DS cell lines were resistant to dexamethasone at the maximal concentrations tested, and did not differ significantly in sensitivity to the other drugs studied. Thus, heightened in vitro cytotoxicity does not appear to account for the increased treatment-related toxicities observed in patients with DS ALL.

Serum levels and pharmacodynamics of methotrexate and its metabolite 7-hydroxy methotrexate in Japanese patients with rheumatoid arthritis treated with 2-mg capsule of methotrexate three times per week

Methotrexate (MTX) is the first-choice drug for rheumatoid arthritis (RA); however, the pharmacodynamics of MTX in Japanese patients with RA treated legitimately according to the government recommended dosage, 6 mg per week, are unknown. Methotrexate and its metabolite, 7-hydroxy MTX (7-OH MTX), were measured in sera of 16 outpatients with active RA in the first week of MTX treatment and 4-12 weeks after the introduction at 0, 1, 2, 4, and 8 h after administration of the first and the third 2-mg capsule, followed by sampling at 48, 96, and 168 h. The mean maximal serum drug concentration (mean C(max)) of MTX attained at 1-2 h after ingestion of the first capsule was 0.215 and 0.252 microM, respectively, in the first and the follow-up week. The mean C(max) after ingestion of the third capsule was 0.223 microM and 0.357 microM. The mean C(max) of 7-OH MTX was 0.0334 and 0.0289 microM for the first capsule, and 0.0495 and 0.0672 microM for the third capsule. The results indicate that MTX does not accumulate or deposit in the body of Japanese patients with RA when treated with 6 mg per week, and pharmacodynamics of MTX are comparable to those in overseas patients treated with 7.5 mg per week.

Serum levels and pharmacodynamics of methotrexate and its metabolite 7-hydroxy methotrexate in Japanese patients with rheumatoid arthritis treated with 2-mg capsule of methotrexate three times per week

Methotrexate (MTX) is the first-choice drug for rheumatoid arthritis (RA); however, the pharmacodynamics of MTX in Japanese patients with RA treated legitimately according to the government recommended dosage, 6 mg per week, are unknown. Methotrexate and its metabolite, 7-hydroxy MTX (7-OH MTX), were measured in sera of 16 outpatients with active RA in the first week of MTX treatment and 4–12 weeks after the introduction at 0, 1, 2, 4, and 8 h after administration of the first and the third 2-mg capsule, followed by sampling at 48, 96, and 168 h. The mean maximal serum drug concentration (mean Cmax) of MTX attained at 1–2 h after ingestion of the first capsule was 0.215 and 0.252 µM, respectively, in the first and the follow-up week. The mean Cmax after ingestion of the third capsule was 0.223 µM and 0.357 µM. The mean Cmax of 7-OH MTX was 0.0334 and 0.0289 µM for the first capsule, and 0.0495 and 0.0672 µM for the third capsule. The results indicate that MTX does not accumulate or deposit in the body of Japanese patients with RA when treated with 6 mg per week, and pharmacodynamics of MTX are comparable to those in overseas patients treated with 7.5 mg per week.

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics.

The ability of leukemia cells to accumulate methotrexate polyglutamate (MTXPG) is an important determinant of the antileukemic effects of methotrexate (MTX). We measured in vivo MTXPG accumulation in leukemia cells from 101 children with acute lymphoblastic leukemia (ALL) and established that B-lineage ALL with either TEL-AML1 or E2A-PBX1 gene fusion, or T-lineage ALL, accumulates significantly lower MTXPG compared with B-lineage ALL without these genetic abnormalities or compared with hyperdiploid (fewer than 50 chromosomes) ALL. To elucidate mechanisms underlying these differences in MTXPG accumulation, we used oligonucleotide microarrays to analyze expression of 32 folate pathway genes in diagnostic leukemia cells from 197 children. This revealed ALL subtype-specific patterns of folate pathway gene expression that were significantly related to MTXPG accumulation. We found significantly lower expression of the reduced folate carrier (SLC19A1, an MTX uptake transporter) in E2A-PBX1 ALL, significantly higher expression of breast cancer resistance protein (ABCG2, an MTX efflux transporter) in TEL-AML1 ALL, and lower expression of FPGS (which catalyzes formation of MTXPG) in T-lineage ALL, consistent with lower MTXPG accumulation in these ALL subtypes. These findings reveal distinct mechanisms of subtype-specific differences in MTXPG accumulation and point to new strategies to overcome these potential causes of treatment failure in childhood ALL.

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics

Folate Pathway Gene Expression Differs in Genetic Subtypes of Acute Lymphoblastic Leukemia and Influences Methotrexate Pharmacodynamics.

Methotrexate (MTX) is an essential treatment component for acute lymphoblastic leukemia (ALL). The ability of leukemia cells to accumulate MTX in its polyglutamylated form (MTXPG) is recognized as an important determinant of its antileukemic effect. We measured in vivo MTXPG accumulation in leukemia cells from 101 children with ALL, and established that blasts of B-lineage ALL with either the TEL-AML1 (n=24 patients, median 911, range 338 to 5906 pmol/109 blasts) or E2A-PBX1 gene fusion (n=5, median 553, range 364 to 800 pmol/109 blasts) or T-lineage ALL (n=14, median 572, range 284 to 1468 pmol/109 blasts) accumulate significantly lower MTXPG, compared to those of other B-lineage ALL (BNHD, n=39, median 2210, range 186 to 9722 pmol/109 blasts) or hyperdiploid ALL (BHD, n=19, median 4375, range 377 to 9206 pmol/109 blasts) (E2A-PBX1 versus BHD, p=0.008; E2A-PBX1 vs. BNHD, p=0.010; TEL-AML1 vs. BHD, p<0.001; TEL-AML1 vs. BNHD, p=0.004; T-ALL vs. BHD and BNHD, p<0.001; p-values are from pair-wise comparisons using Wilcoxon rank sum test, adjusted for multiple testing using Holm's method). To elucidate mechanisms underlying these differences in MTXPG accumulation, we used oligonucleotide microarrays (Affymetrix® HG-U133A) to analyze expression of 32 folate pathway genes (53 probe sets) in diagnostic bone marrow blasts from 197 children with ALL. This revealed ALL subtype-specific patterns of folate metabolism gene expression and identified differences in gene expression that discriminated the MTXPG accumulation phenotype in ALL cells. We found significantly lower expression of the reduced folate carrier (SLC19A1, MTX uptake transporter) in E2A-PBX1 ALL; significantly higher expression of breast cancer resistance protein (ABCG2, MTX efflux transporter) in TEL-AML1 ALL; and lower expression of FPGS (catalyzes formation of MTXPG) in T-ALL; consistent with lower MTXPG accumulation in these ALL subtypes. These findings reveal distinct mechanisms of subtype-specific differences in MTXPG accumulation and point to new strategies to overcome this potential cause of treatment failure in childhood ALL.

New aspects of clinical and cellular pharmacodynamics of methotrexate with special emphasis on its role in the treatment of acute lymphoblastic leukemia in children.

New aspects of clinical and cellular pharmacodynamics of methotrexate with special emphasis on its role in the treatment of acute lymphoblastic leukemia in children.

Clinical pharmacokinetics of methotrexate in children.

Among the few antineoplastic agents investigated pharmacologically in children and adults, methotrexate has been clearly demonstrated to be handled differently in the two age groups. Age has in fact proved to be a major determinant, exerting an effect on both the pharmacokinetics and pharmacodynamics of methotrexate. Its pharmacokinetics, in turn, determine the drug toxicity. The beta-phase of methotrexate clearance, represented by the plasma drug concentration 48 hours from the start of a 6-hour infusion in a high dose treatment regimen, appears to be constant with age. In children, an increasing plasma drug concentration is apparent with increasing age, but whether this trend reflects a potential increase in the area under the plasma concentration-time curve of methotrexate has yet to be defined. Recent investigations have suggested that the drug is more completely distributed in the tissues of children than adults at the same infused dosage. This may explain the increased tissue toxicity caused by methotrexate. However, other observations suggest a faster drug turnover rate in the tissues of children. This may prevent the drug from concentrating in vital organs. Whether the metabolism of methotrexate, particularly the biosynthesis of methotrexate polyglutamates, plays a role in the biological effect of the drug is worthy of further investigation. The high brain tissue concentration after systemic methotrexate infusion and the slower efflux of methotrexate from brain tissues and cerebrospinal fluid make these tissues vulnerable to methotrexate toxicity.