MTX-treated Rheumatoid Arthritis Patients Research Articles

Assessment of liver fibrosis markers in people with rheumatoid arthritis on methotrexate.

Up to 3% of methotrexate (MTX)-treated rheumatoid arthritis (RA) patients might develop liver fibrosis or cirrhosis, requiring effective screening algorithms. To assess the utility of non-invasive liver fibrosis assessment in RA patients on MTX. Fifty-six patients were recruited from rheumatology outpatient clinics in a public tertiary centre from July 2017 to October 2018. Clinical data was collected. Screening for hepatic fibrosis was performed using transient elastography (TE), aminoaspartate transaminase to platelet ratio index (APRI), Hepascore and Fibrosis-4 index (FIB-4). Those with suspected significant liver fibrosis based on these screening tests were assessed by a hepatologist. Twenty-seven patients were suspected to have liver fibrosis on screening, including 10/56 (18%) by TE, 20/56 (36%) by Hepascore, 2/56 by APRI (4%) and 1/56 by FIB-4 (2%). Of these 27 patients, 11 were reviewed by a hepatologist and one diagnosed with significant liver fibrosis. TE, but not APRI, Hepascore or FIB-4, was found to have 100% sensitivity and 84% specificity (P = 0.029) for hepatologist-diagnosed liver fibrosis. Liver fibrosis develops in a minority of MTX-treated RA patients. The present study suggests that TE is a more sensitive screening test than APRI, FIB-4 or Hepascore in the identification of people with RA at risk of hepatic fibrosis.

Development and Validation of a Sensitive UHPLC-MS/MS-Based Method for the Analysis of Folylpolyglutamate Synthetase Enzymatic Activity in Peripheral Blood Mononuclear Cells: Application in Rheumatoid Arthritis and Leukemia Patients.

Folylpolyglutamate synthetase (FPGS) is a crucial enzyme in both cellular folate homeostasis and the intracellular retention of folate analogue drugs such as methotrexate (MTX), which is commonly used for the treatment of (pediatric) leukemia and the anchor drug in rheumatoid arthritis (RA) treatment. To date, assessment of FPGS catalytic activity relies on assays using radioactive substrates that are labor-intensive and require relatively large numbers of cells. Here, we describe a nonradioactive, ultra-high-performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS)-based method allowing for sensitive and accurate measurements of FPGS activity in low cell numbers (ie, 1-2 × 10) of biological specimens, including leukemic blast cells of acute lymphoblastic leukemia patients and peripheral blood mononuclear cells of patients with RA. The UHPLC-MS/MS assay was validated with 2 CCRF-CEM human leukemia cells, one proficient and one deficient in FPGS activity. Linearity of time and protein input were tested by measuring FPGS activity at 30-180 minutes of incubation time and 10-300 mcg protein extract. In addition, FPGS enzyme kinetic parameters were assessed. The FPGS enzymatic assay showed a linear relation between FPGS activity and protein input (R ≥ 0.989) as well as incubation time (R ≥ 0.996). Moreover, the UHPLC-MS/MS method also allowed for evaluation of FPGS enzyme kinetic parameters revealing Km values for the substrates MTX and L-glutamic acid of 64 µmol/L and 2.2 mmol/L, respectively. The mean FPGS activity of acute lymphoblastic leukemia blast cells (n = 4) was 3-fold higher than that of CCRF-CEM cells and 44-fold and 88-fold higher than that of peripheral blood mononuclear cells from MTX-naive (n = 9) and MTX-treated RA patients (n = 6), respectively. Collectively, given its sensitivity with low cell numbers and avoidance of radioactive substrates, UHPLC-MS/MS-based analysis of FPGS activity may be eligible for routine therapeutic drug monitoring of MTX in RA and leukemia for therapy (non)response evaluations.

Non-alcoholic steatohepatitis-like pattern in liver biopsy of rheumatoid arthritis patients with persistent transaminitis during low-dose methotrexate treatment.

ObjectiveThe mechanism of liver injury with low-dose methotrexate (MTX) is incompletely understood. This study was designed to evaluate the association between non-alcoholic fatty liver disease (NAFLD) and liver injury during MTX treatment for rheumatoid arthritis (RA).MethodsBetween October 2014 and May 2015, we enrolled all MTX users for RA and monitored participant serum hepatic transaminase levels for 1 year. All patients had normal transaminase levels before the first MTX prescription. Using diagnostic criteria for non-alcoholic steatohepatitis (NASH), we performed histological analyses for patients presenting persistent transaminitis, defined as elevations of hepatic transaminases in four of six determinations during the follow-up period. Possible risk factors for persistent transaminitis were also examined.ResultsWe followed 846 RA patients with a mean cumulative MTX dose of 2.48 g and identified 51 patients presenting persistent transaminitis. According to multivariate logistic regression analysis, obesity (odds ratio [OR] 3.23, p < 0.001), type 2 diabetes (OR 3.52, p = 0.001), hypercholesterolemia (OR 2.56, p = 0.004), and hyperuricemia (OR 3.52, p = 0.019), which are recognized as risk factors for NAFLD, were independently associated with a risk of persistent transaminitis. Among patients with persistent transaminitis, 42 showed fatty liver at ultrasonography. These patients had no evidence of alcoholic fatty liver, chronic viral hepatitis, autoimmune liver diseases, or hereditary liver diseases. Biopsy specimens were obtained from 32 patients, and we found that a NASH-like pattern was the most prevalent histological abnormality. There was no significant impact of MTX dose and duration on the histological severity.ConclusionRisk factors and histological findings are similar between NAFLD/NASH and liver injury during low-dose MTX treatment for RA, which suggests a strong association between both entities. NAFLD/NASH may be an underlying condition causing persistent transaminitis in MTX-treated RA patients. The results of this study illustrate the need for monitoring liver injury in RA patients with NAFLD risk factors during MTX treatment.

Are gene polymorphisms related to treatment outcomes of methotrexate in patients with rheumatoid arthritis? A systematic review and meta-analysis.

Identifying the predictors of responsiveness and adverse events in methotrexate (MTX) treated patients with rheumatoid arthritis (RA) has been the focus of most concern, but still without consistent consensus. PubMed and OVID EMBASE were searched to collect relevant studies that addressed correlations between gene polymorphisms and efficacy and/or toxicity in MTX-treated RA patients. Allelic, recessive, dominant and over-dominant model were applied. A total of 68 studies were included. For associations with efficacy, AMPD1 34C>T polymorphism was related to responsiveness in dominant model (odds ratio [OR]: 1.77; 95% CI: 1.19-2.63) and over-dominant model (OR: 1.59; 95% CI: 1.04-2.45). ATIC T675C polymorphism had association with responsiveness in recessive model (OR: 2.54; 95% CI: 1.23-5.26). For associations with toxicity, polymorphisms in TYMS 1494 del6 and FPGS rs10106 were correlated to absenting overall adverse events in recessive model (OR: 0.68; 95% CI: 0.49-0.95) and dominant model (OR: 0.54; 95% CI: 0.35-0.83) respectively while MTHFR C677T was associated with presenting overall adverse events in allelic model (OR: 1.29; 95% CI: 1.02-1.63), recessive model (OR: 1.38; 95% CI: 1.00-1.89) and dominant model (OR: 1.41; 95% CI: 1.02-1.94). Polymorphisms in AMPD1 34C>T and ATIC T675C predict responsiveness. The absence of TYMS 1494 del6 and FPGS rs10106 and presence of MTHFR C677T predict adverse events in RA patients treated with MTX. Moreover, variations of the associations were found between Caucasians and non-Caucasians.

Analysis of Mitogen-Activated Protein Kinases in Bone and Cartilage of Patients with Rheumatoid Arthritis Treated with Abatacept.

The aim of this study was to analyze the histological changes related to mitogen-activated protein (MAP) kinases in bone and cartilage treated with abatacept for rheumatoid arthritis (RA). A total of 20 patients of bone and cartilage were assessed: 10 abatacept with methotrexate (MTX)-treated RA patients were compared with 10 MTX-treated RA patients (control). The histology of bone and cartilage was observed by staining with hematoxylin and eosin and analyzed immunohistochemically for the expression of tumor necrosis factor-α, interleukin-6, CD4 (T cell), CD68 (macrophage), receptor activator of nuclear kappa-B ligand, osteoprotegerin, osteopontin, CD29 (β-1 integrin), phospho-p38 MAPK (Tyr180/Tyr182), phospho-p44/42 MAPK (extracellular signal-regulated kinase, ERK1/ERK2), and phosphor-c-Jun N-terminal kinase. The expressions of CD29 known as mechanoreceptor and ERK known as mechanotransduction signal protein in MAP kinases in the bone and cartilage of patients treated with abatacept were significantly different from those of control. These findings suggest that increases in CD29 and ERK in MAP kinases may change the metabolism of bone and cartilage in RA patients treated with abatacept.

Global Molecular Effects of Tocilizumab Therapy in Rheumatoid Arthritis Synovium

To investigate the global molecular effects of tocilizumab (TCZ) in comparison with methotrexate (MTX) treatment in synovial biopsy tissue obtained from patients with previously untreated rheumatoid arthritis (RA) before therapy (T0) and 12 weeks after the initiation of therapy (T12), and to compare the results with previous gene expression data obtained in synovial biopsy tissue from adalimumab (ADA)- and rituximab (RTX)-treated patients with RA. Paired synovial biopsy samples were obtained at T0 and T12 from the affected knee of TCZ-treated RA patients and MTX-treated RA patients. Gene expression studies were performed using GeneChip Human Genome U133 Plus 2.0 microarrays, and confirmatory quantitative real-time reverse transcription-polymerase chain reaction experiments were performed on selected transcripts. The effects of TCZ and MTX on synovial cell populations and histologic characteristics were assessed by immunohistochemistry. Gene expression studies showed that blockade of the interleukin-6 receptor (IL-6R) gene (IL6R) using TCZ induced a significant decrease in the expression of numerous chemokine and T cell activation genes in the RA synovium. These effects strongly correlated with the molecular effects of MTX and RTX therapy on RA synovial tissue, but differed from the molecular changes induced by ADA (decreased expression of genes involved in cell proliferation). The molecular similarities between the effects of TCZ, RTX, and MTX therapies in the RA synovium indicate that B cell- and IL-6-dependent pathways play synergistic roles in the pathogenesis of the disease, in particular through activation of T cell responses. Moreover, these results open perspectives for the individualization of therapeutic decisions, based on a better knowledge of the synovial molecular effects of each type of RA therapy.

Methotrexate Normalizes Up‐Regulated Folate Pathway Genes in Rheumatoid Arthritis

The folate antagonist methotrexate (MTX) is an anchor drug in the treatment of rheumatoid arthritis (RA), but its mechanism of action with regard to the impact on folate metabolism remains elusive. The aim of the present study was to investigate the cellular pharmacologic impact of MTX on peripheral blood cells, by comparing MTX-treated RA patients to MTX-naive RA patients and healthy controls. Gene expression microarray data were used to investigate the expression of 17 folate pathway genes by peripheral blood cells from a cohort of 25 RA patients treated with MTX, 10 MTX-naive RA patients starting treatment with MTX, and 15 healthy controls (test cohort). Multiplex real-time polymerase chain reaction was used to validate the results in an independent cohort, consisting of 151 RA patients treated with MTX, 28 MTX-naive RA patients starting treatment with MTX, and 24 healthy controls (validation cohort). Multiple folate metabolism-related genes were consistently and significantly altered between the 3 groups in both cohorts. Concurrent with evidence of an immune-activation gene signature in MTX-naive RA patients, significant up-regulation of the folate-metabolizing enzymes γ-glutamyl hydrolase and dihydrofolate reductase, as well as the MTX/folate efflux transporters ABCC2 and ABCC5, was observed in the MTX-naive RA group compared to healthy controls. Strikingly, MTX treatment of RA patients normalized these differential gene expression levels to the levels observed in healthy controls. These results suggest that under inflammatory conditions, basal folate metabolism in the peripheral blood cells of RA patients is markedly up-regulated, and treatment with MTX restores folate metabolism to normal levels. Identification of this novel gene signature provides insight into the mechanism of action of MTX, thus paving the way for development of novel folate metabolism-targeted therapies.

The Importance of Determining the Drug Agent Responsible for Pancytopenia in Patients with Rheumatoid Arthritis and Additional Systemic Diseases

Methotrexate (MTX) is the most commonly used drug in the treatment of rheumatoid arthritis (RA). In MTXtreated RA patients, the prevalence of pancytopenia is nearly 3%. Several medications may be prescribed for RA patients with an additional systemic disease. This may result in bone marrow toxicity, making it difficult to know which drug induced the pancytopenic condition. Therefore, identifying the culprit agent through bone marrow aspiration and biopsy findings of the samples is critical for determining the best course of treatment for pancytopenia. Significantly increased amounts of eosinophilia in the bone marrow indicate that MTX is the culprit for the presentation of pancytopenia.

FRI0198 Expressions of immune response related genes were normalised after tocilizumab treatment in rheumatoid arthritis (RA) patients

BackgroundTocilizumab (TCZ), a humanised anti-interleukin (IL)6 receptor antibody, was shown to be therapeutically effective in RA patients but the biological effect of TCZ on the immune response was not fully...

Mature T/NK-cell lymphoproliferative disease and Epstein–Barr virus infection are more frequent in patients with rheumatoid arthritis treated with methotrexate

We retrospectively analyzed in 54 consecutively enrolled Japanese patients with rheumatoid arthritis (RA) and lymphoproliferative disease (LPD) relevant clinicopathological characteristics, in particular paying attention to treatment with methotrexate (MTX). Between the 28 patients treated with MTX (MTX-treated group) and the 26 who were not (non-MTX group), there was no difference in age, interval between onset of RA and LPD, and lymphoma stage. Immunohistochemical analysis showed that in the MTX-treated group, 15 (53 %) patients had mature B-cell LPD, eight (29 %) mature T/NK-cell LPD, and five (18 %) had Hodgkin lymphoma. In the non-MTX group, 22 (84 %) had mature B-cell LPD, 2 (8 %) had mature T/NK-cell LPD, and 2 (8 %) had Hodgkin lymphoma. The frequency of mature T/NK-cell LPD was significantly higher in the MTX-treated group (p < 0.05). Of the eight patients in the MTX-treated group with mature T/NK-cell LPD, two had large granular lymphocytic leukemia and the other six had a variety of different histological types with frequent CD8 but not CD56 expression. Epstein-Barr virus (EBV) infection was significantly higher in the MTX-treated group (p < 0.05); evidence of latent type II EBV infection was found in four of the eight patients with mature T/NK-cell LPD. Withdrawal of MTX led to complete remission in seven patients with mature T/NK-cell LPD irrespective of EBV infection. Our findings highlight that mature T/NK-cell LPD is a frequent complication in RA patients treated with MTX. EBV infection may play a role in the pathogenesis of T/NK-cell LPD, as well as B-cell LPD and Hodgkin lymphoma in MTX-treated RA patients.

Inhibition of JNK in synovium by treatment with golimumab in rheumatoid arthritis

The aim of this study was to investigate immunohistological changes in mitogen-activated protein kinases (MAPKs) in the synovium following treatment with golimumab, compared with methotrexate (MTX). We assessed synovial tissues for 13 different molecules to detect cytokine levels histologically from 10 methotrexate (MTX)-treated rheumatoid arthritis (RA) patients as controls and 10 golimumab plus MTX-treated RA patients. Synovium samples from both groups were assessed by hematoxylin and eosin (HE) staining and analyzed for expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), CD4 (T cells), CD8 (T cells), CD20 (B cells), CD68 (macrophages), receptor activator of nuclear (kappa) B ligand (RANKL), bromodeoxyuridine (BrdU), CD29 (β-1 integrin), phospho-p38 MAPK (Tyr180/Tyr182), phospho-p44/42 MAPK (ERK1/ERK2), and phospho-c-Jun N-terminal kinase (JNK), by an immunohistological examination. HE staining showed that there was a significant decrease in cell proliferation in the synovium in RA patients who received golimumab compared with the controls. TNF-α, IL-6, MMP3, BrdU, p38, and ERK were not seen at significant levels in either group. On the other hand, CD4, CD8, CD20, CD29, CD68, RANKL, and JNK were significantly decreased in the golimumab group compared with the control. Based on a histological analysis of the synovium, it appears that the efficacy of the treatment with golimumab may involve the inhibition of cell proliferation, with decreases in T cells, B cells, macrophages, β-1 integrin, RANKL, and JNK in the synovium, compared with MTX treatment, in RA.

Inhibition of MAP kinase in synovium by treatment with tocilizumab in rheumatoid arthritis

To investigate the histological changes of mitogen-activated protein kinase (MAPK) in synovium with tocilizumab in rheumatoid arthritis (RA), synovial tissue samples were assessed from ten methotrexate (MTX)-treated RA patients for control and ten tocilizumab with MTX-treated RA patients. The synovium was observed using hematoxylin and eosin (H&E) stain and analyzed immunohistochemically for the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), CD4 (T cell), CD20 (B cell), CD68 (macrophage), vascular endothelial growth factor (VEGF), CD29 (β-1 integrin), phospho-p38 MAPK (Tyr180/Tyr182), phospho-p44/42 MAPK [extracellular signal-regulated kinase (ERK) 1/ERK2], and phospho-c-Jun N-terminal kinase (JNK). H&E staining showed that there is a significant difference of cell proliferation; however, there is no change of hypervascularity in the synovium between both groups. An immunohistochemical examination showed that the decrease of CD29 (β-1 integrin) and JNK was found significant, while ERK was increased in the tocilizumab group. CD20, B-lymphocyte, was decreased in the tocilizumab group compared with the MTX group significantly. IL-6 was completely blocked in the patients who received tocilizumab. TNF-α was similarly expressed in the interstitial cells of synovium of patients in both groups. MMP-3 and CD68 were similarly expressed on the surface of synovium. VEGF was less expressed in both groups. These findings indicate that the inhibition of CD20, CD29, and JNK in MAPK may be involved in the efficacy of tocilizumab compared with MTX treatment in RA.

Decrease of CD68 and MMP-3 expression in synovium by treatment of adalimumab for rheumatoid arthritis

In order to investigate the histological change in the secondary non-responder cases of adalimumab compared with methotrexate (MTX) treatment, we performed immunohistochemical examination of synovial tissue by seven different molecules to detect expression patterns of cytokines. We histologically assessed synovial tissues from five MTX-treated rheumatoid arthritis (RA) patients as controls and five adalimumab plus MTX-treated RA patients after arthroscopic synovectomy in the knee joints. The synovium of both groups were assessed by hematoxylin and eosin (H&E) and analyzed the positive expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), B-cell precursor and mature B-cell transmembrane protein, CD20, macrophage marker, CD68, vascular endothelial growth factor (VEGF), receptor activator of nuclear (kappa) B ligand (RANKL) by immunohistochemical examination. H&E staining showed the increase of vascular and cell proliferations in the synovium of the RA patients who received adalimumab compared with the controls. TNF-α, IL-6 and CD20 were not significantly different in either group. On the other hand, MMP-3 and CD68 showed a significant decrease in the adalimumab group compared with controls (P < 0.05). VEGF and RANKL were weakly positive in both groups. Based on the histological analysis of synovium, the effect attenuation of adalimumab may be involved in vascular and cell proliferations; however, there was inhibition of the expression of CD68 and MMP-3 in synovium. These findings indicate CD68 and MMP-3 may have key roles in the mechanism of efficacy of adalimumab.

Histological analysis of synovium by treatment of etanercept for rheumatoid arthritis

In order to investigate the histological change in effect attenuation cases of etanercept compared with methotrexate (MTX), we performed immunohistochemical examination by seven different molecules. We histologically assessed synovial tissue from five MTX-treated rheumatoid arthritis (RA) patients as control and six etanercept and MTX-treated RA patients after synovectomy by arthroscopy. The synovium of both groups were assessed by hematoxylin and eosin (HE) and we also analysed the expression of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), B-cell precursors and mature B-cell transmembrane protein, CD20, macrophage marker, CD68, bromodeoxyuridine (BrdU) and vascular endothelial growth factor (VEGF) by immunohistochemistry. HE staining showed vascular and cell proliferations of the synovium of the RA patients who received etanercept compared with the control MTX group. TNF-alpha and IL-6 were expressed in both groups.MMP-3 and CD68 expressed less significantly in the etanercept group compared with the control (P < 0.05). CD20 strongly expressed in the etanercept group significantly (P < 0.05). BrdU expressed in the synovium in the etanercept group significantly (P < 0.05). VEGF was not found overall in both group. Based on the histological change of synovium, treatment by etanercept may be involved in vascular and cell proliferations with inhibition of the expression of CD68 and MMP-3 in synovium of RA patients. These findings indicate immunohistochemical change of synovium with etanercept is one of the mechanism of efficacy of etanercept.

HLA-G 14-bp polymorphism regulates the methotrexate response in rheumatoid arthritis

Methotrexate (MTX) represents the antirheumatic drug mainly used in rheumatoid arthritis (RA). HLA-G antigens are inducible nonclassical major histocompatibility complex class Ib molecules important for maintaining anti-inflammatory conditions. The HLA-G gene is characterized by a deletion/insertion polymorphism of 14 bp that controls specific mRNA stability and protein levels. It has been reported that MTX therapy mediates an increase of interleukin-10-producing cells. This cytokine up-regulates HLA-G expression. For this, we tested the hypothesis of an MTX-mediated HLA-G production and the possible relationship with the HLA-G 14-bp polymorphism. Peripheral blood mononuclear cells from healthy individuals and non-MTX-treated RA patients were activated with different MTX concentrations, and soluble HLA-G (sHLA-G) and interleukin-10 production was investigated by specific immunoenzymatic assay. HLA-G 14-bp polymorphism genotyping was performed in healthy individuals and RA patients, defined as 'responders' and 'nonresponders' to the MTX therapy. MTX activation induces the production of sHLA-G molecules. A significant association was observed between the highest sHLA-G1 concentrations and the -14/-14 bp genotype. The analysis of the HLA-G 14-bp polymorphism in MTX-treated RA patients has confirmed an increase of the -14/-14 bp genotype in the responder group (chi=6.12, P=0.02; chi test) (odds ratio=2.46 (95% confidence interval, 1.26-4.84) P=0.009; logistic regression model). Our results propose that the MTX induces the production of the anti-inflammatory sHLA-G molecules that concur with the therapy response. Furthermore, the association between -14/-14 bp genotype and MTX clinical outcome proposes this polymorphism as a therapy marker in the early phases of the disease.

Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A3 adenosine receptor expression

Methotrexate (MTX) exerts an anti-inflammatory effect via its metabolite adenosine, which activates adenosine receptors. The A3 adenosine receptor (A3AR) was found to be highly expressed in inflammatory tissues and peripheral blood mononuclear cells (PBMCs) of rats with adjuvant-induced arthritis (AIA). CF101 (IB-MECA), an A3AR agonist, was previously found to inhibit the clinical and pathological manifestations of AIA. The aim of the present study was to examine the effect of MTX on A3AR expression level and the efficacy of combined treatment with CF101 and MTX in AIA rats. AIA rats were treated with MTX, CF101, or both agents combined. A3AR mRNA, protein expression and exhibition were tested in paw and PBMC extracts from AIA rats utilizing immunohistochemistry staining, RT-PCR and Western blot analysis. A3AR level was tested in PBMC extracts from patients chronically treated with MTX and healthy individuals. The effect of CF101, MTX and combined treatment on A3AR expression level was also tested in PHA-stimulated PBMCs from healthy individuals and from MTX-treated patients with rheumatoid arthritis (RA). Combined treatment with CF101 and MTX resulted in an additive anti-inflammatory effect in AIA rats. MTX induced A2AAR and A3AR over-expression in paw cells from treated animals. Moreover, increased A3AR expression level was detected in PBMCs from MTX-treated RA patients compared with cells from healthy individuals. MTX also increased the protein expression level of PHA-stimulated PBMCs from healthy individuals. The increase in A3AR level was counteracted in vitro by adenosine deaminase and mimicked in vivo by dipyridamole, demonstrating that receptor over-expression was mediated by adenosine. In conclusion, the data presented here indicate that MTX induces increased A3AR expression and exhibition, thereby potentiating the inhibitory effect of CF101 and supporting combined use of these drugs to treat RA.

Reduction of the efficacy of methotrexate by the use of folic acid: Post hoc analysis from two randomized controlled studies

To examine the effect of folic acid on the efficacy of methotrexate (MTX) treatment in rheumatoid arthritis (RA) at 12 months in 2 phase III randomized controlled trials (RCTs) of leflunomide in which MTX was used as a comparator. Analyses were restricted to patients randomized to receive MTX who had rheumatoid factor data. The US study recruited 482 patients with active RA; 179 received at least 1 dose of MTX, and all were mandated to receive 1 mg of oral folic acid once or twice daily. The multinational European study recruited 999 patients with active RA; 489 received at least 1 dose of MTX, and oral folic acid was not required, although 50 received folate after developing an adverse event. Because of similar entry criteria for both studies, the data for patients with available primary outcome data at week 52 were pooled (n = 668), and the patients were grouped by folic acid use (n = 225) and nonuse (n = 443). To account for the significant between-study differences in the MTX groups, baseline covariates were adjusted using propensity scores so that folic acid users could be matched with nonusers. This allowed for a comparison of differences in American College of Rheumatology (ACR) 20% improvement criteria at week 52. At study entry, non-folic acid users had a significantly lower mean body weight, shorter mean RA duration, and higher mean disease activity (measured by joint counts, patient's and physician's global assessments, and acute-phase reactant levels). The mean MTX dosage at week 52 was similar in the 2 RCTs. Using propensity score matching techniques, the proportion of patients achieving an ACR 20% response at week 52 averaged 17% higher in the non-folic acid group than in the folic acid group (range 15-21%). Similarly, the proportion of patients achieving ACR 50% and ACR 70% responses averaged 14% (range 12-16%) and 12% (range 9-14%) higher, respectively, in the non-folic acid group. Adverse events were reported in 93% of US study patients and 94% of the multinational study patients. Elevated liver transaminase levels (above the upper limit of normal) were reported in 29% of the US study patients (majority receiving folic acid) and 62% of the multinational study patients (majority not receiving folic acid). After using propensity scores to adjust for differences in the baseline characteristics of folic acid users and non-folic acid users, 9-21% fewer MTX-treated RA patients taking folic acid had ACR 20%, 50%, or 70% improvement at 52 weeks compared with those who did not receive folic acid in the 2 phase III RA clinical trials. As a post hoc analysis, the results of this data analysis should be considered "hypothesis generating" and an impetus for future studies regarding the effects of folic acid on the efficacy of MTX in RA.

Polymorphisms in the methylenetetrahydrofolate reductase gene were associated with both the efficacy and the toxicity of methotrexate used for the treatment of rheumatoid arthritis, as evidenced by single locus and haplotype analyses.

5,10-Methylenetetrahydrofolate reductase (MTHFR), a key enzyme involved in folate metabolism, has two common polymorphisms that affect enzyme activity. The objective of this study was to examine whether there was a correlation between the genotype or haplotype of the MTHFR gene and the efficacy or toxicity of methotrexate (MTX) in the treatment of rheumatoid arthritis. MTX-treated rheumatoid arthritis patients (n = 106) were selected from outpatient clinics and used for a retrospective study to examine the correlation between genotypes or haplotypes concerning polymorphisms of the MTHFR gene, and the efficacy or toxicity of MTX. Estimation of the haplotype frequencies was performed by maximum likelihood estimation based on expectation maximization algorithm. Single locus analysis examining each locus separately showed that patients with 1298C were receiving significantly lower doses of MTX compared to patients without [P < 0.05, relative risk (RR) = 2.18, 95% confidence interval (CI) 1.17-4.06], while a higher rate of overall MTX toxicity was observed in patients with 677T than those without (P < 0.05, RR = 1.25, 95% CI 1.05-1.49). An estimation of haplotype frequencies showed that there was no 677T-1298C haplotype in the population. Posterior distribution of the diplotype configuration for each individual was concentrated on a single configuration. Patients with the 677C-1298C haplotype were receiving lower doses of MTX than those without (P < 0.05, RR = 2.14, 95% CI 1.13-4.07), while subjects with 677T-1298A had a higher frequency of side-effects from MTX (P < 0.05, RR = 1.42, 95% CI 1.11-1.82). Both single locus and haplotype analyses suggest that polymorphisms within the MTHFR gene are associated with both the efficacy and toxicity of MTX in rheumatoid arthritis patients. Pharmacokinetic studies are necessary to prove the association.

Usefulness of the American College of Rheumatology recommendations for liver biopsy in methotrexate-treated rheumatoid arthritis patients.

To test the usefulness and cost savings resulting from application of the new American College of Rheumatology (ACR) guidelines for assessing the risk for the development of clinically significant liver disease in rheumatoid arthritis (RA) patients treated with methotrexate (MTX). One-hundred twelve MTX-treated RA patients were prospectively followed up for MTX hepatotoxicity and underwent liver biopsies according to modified guidelines of the Psoriatic Task Force (PTF). All biopsies were graded according to the Roenigk classification. The new ACR recommendations were then retrospectively applied to test their usefulness and cost-effectiveness in this cohort. Based on the PTF guidelines, 66 patients underwent liver biopsies; a total of 110 liver biopsies were performed. Two patients had biopsy-related complications. Five patients were found to have Roenigk grade IIIB or IV histologic abnormalities. The total cost for this group was $111,380. Applying the new ACR criteria, only 15 patients would have undergone liver biopsies; there would have been a total of 18 biopsies, with no complications. Four of the 5 patients with Roenigk grade IIIB or IV liver abnormalities would have been identified. One patient with insulin-dependent diabetes mellitus (IDDM) who was found to have cirrhosis (Roenigk grade IV) on liver biopsy as a result of use of the PTF guidelines would have been missed with use of the ACR guidelines. The total cost for the group receiving biopsies based on the ACR guidelines would have been $16,956. Overall, the new ACR guidelines had 80% sensitivity and 82% specificity and resulted in a cost savings of $1,430 per patient. The new ACR guidelines on MTX monitoring and biopsy surveillance appear to be clinically useful and result in considerable cost savings. However, 1 IDDM patient with significant liver histologic abnormalities would have been missed. We suggest that IDDM be added to the ACR guidelines as a risk factor for MTX hepatotoxicity.

Methotrexate therapy in rheumatoid arthritis patients diminishes lectin-induced mononuclear cell proliferation

Methotrexate (MTX) is an anti-folate drug used in cancer chemotherapy because of its anti-proliferative effects. However, it is unclear whether the anti-proliferative effects of MTX contribute to the efficacy of low-dose MTX in the treatment of rheumatoid arthritis (RA). To date, either no change or a paradoxical increase in lectin-induced proliferation has been observed in cultures of peripheral blood mononuclear cells (PBMC) from MTX-treated RA patients (RA + MTX). In these earlier studies, high folate-containing media and tritiated thymidine (3H-TdR) were used. Our studies were designed to test the hypothesis that the use of a culture medium with a low folate content along with tritiated deoxyuridine (3H-UdR) permits detection of diminished phytohemagglutinin (PHA)-induced proliferative responses of PBMC from RA + MTX. The data demonstrate decreased PHA-induced cellular proliferation of cultured PBMC from RA + MTX compared with controls. When comparing the PBMC proliferative responses in high vs low folate medium, a significantly greater increase (P less than 0.05) in proliferation occurs in the cells from RA + MTX cultured in the high folate medium. This suggests that an in vivo folate-deficient state of the cells from RA + MTX may be corrected in vitro when a high folate medium is used in culture. We conclude that the use of 3H-UdR and a medium containing folate within the normal range of plasma folate levels eliminates artifacts associated with the use of high folate medium and 3H-TdR, which obscures the anti-proliferative effect of MTX in RA patients.