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Study Objective: To determine whether there is a drug-drug interaction precluding the concomitant use of levetiracetam and high-dose methotrexate (HDMTX)., Design: Retrospective analysis., Setting: Large academic tertiary care medical center., Patients: Adult lymphoma patients who received HDMTX as a 4-h infusion with or without concomitant levetiracetam., Measurements and Main Results: Generalized estimating equations clustered on patient were used to assess each outcome. The primary outcome was the incidence of delayed MTX elimination (MTX level >1 µmol/L at 48 h). Secondary outcomes included incidence of acute kidney injury (AKI) and hospital length of stay (LOS). The 430 included patients receiving 1993 doses of HDMTX had a median (IQR) age of 66 (57.5, 72.6) years, 88 (20.5%) received concomitant levetiracetam with at least one dose of MTX, 267 (62.1%) were male, and 397 (92.3%) were Caucasian. HDMTX doses ranged from 1 to 8 g/m 2 . The most common lymphoma diagnoses were systemic diffuse large B-cell lymphoma (DLBCL; 58.5%) and systemic DLBCL with central nervous system (CNS) involvement (32.8%). Rates of delayed elimination with and without levetiracetam were 13.4% and 16.3%, respectively (OR = 0.80, 95% CI 0.47-1.34, p = 0.39). AKI occurred in 15.6% and 17.0% of patients with and without concomitant levetiracetam, respectively (OR = 0.83, 95% CI 0.52-1.33, p = 0.28). The median LOS with and without levetiracetam was 4.2 and 4.1 days, respectively (p = 0.039). On multivariable analyses, only age, body surface area, diagnosis of systemic DLBCL with CNS involvement, serum creatinine, hemoglobin, total bilirubin, and dose of HDMTX were associated with delayed elimination., Conclusions: High-dose methotrexate administered with concomitant levetiracetam was not associated with increased risk for delayed MTX elimination or AKI. These results support that levetiracetam and HDMTX are safe for coadministration., (© 2021 Pharmacotherapy Publications, Inc.)

MicroRNA-24-3p (miR-24-3p) has been implicated as a key promoter of chemotherapy resistance in numerous cancers. Meanwhile, cancer-associated fibroblasts (CAFs) can secret exosomes to transfer miRNAs, which mediate tumour development. However, little is known regarding the molecular mechanism of CAF-derived exosomal miR-24-3p in colon cancer (CC). Hence, this study intended to characterize the functional relevance of CAF-derived exosomal miR-24-3p in CC cell resistance to methotrexate (MTX). We identified differentially expressed HEPH, CDX2 and miR-24-3p in CC through bioinformatics analyses, and validated their expression in CC tissues and cells. The relationship among HEPH, CDX2 and miR-24-3p was verified using ChIP and dual-luciferase reporter gene assays. Exosomes were isolated from miR-24-3p inhibitor-treated CAFs (CAFs-exo/miR-24-3p inhibitor), which were used in combination with gain-of-function and loss-of-function experiments and MTX treatment. CCK-8, flow cytometry and colony formation assays were conducted to determine cell viability, apoptosis and colony formation, respectively. Based on the findings, CC tissues and cells presented with high expression of miR-24-3p and low expression of HEPH and CDX2. CDX2 was a target gene of miR-24-3p and could up-regulate HEPH. Under MTX treatment, overexpressed CDX2 or HEPH and down-regulated miR-24-3p reduced cell viability and colony formation and elevated cell apoptosis. Furthermore, miR-24-3p was transferred into CC cells via CAF-derived exosomes. CAF-derived exosomal miR-24-3p inhibitor diminished cell viability and colony formation and increased cell apoptosis in vitro and inhibited tumour growth in vivo under MTX treatment. Altogether, CAF-derived exosomal miR-24-3p accelerated resistance of CC cells to MTX by down-regulating CDX2/HEPH axis., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: It is known that γ-glutamyl hydrolase (GGH) is involved in the disposition of methotrexate (MTX), and GGH activity is regulated by DNA methylation in acute lymphoblastic leukemia (ALL) cells. The present study explores the methylation status of the GGH promoter in peripheral blood and its association with MTX levels and toxicities in Chinese children with ALL., Methods: Serum MTX concentrations were determined by fluorescence polarization immunoassay. Methylation quantification and genotyping for GGH rs3758149 and rs11545078 was performed by Sequenom MassARRAY in 50 pediatric patients with ALL., Results: Overall, the investigated region of the GGH promoter was in hypomethylated status. The methylation levels of cytosine phosphate guanine (CpG)&#95;7, CpG&#95;12, CpG&#95;17, and CpG&#95;20 were significantly higher in patients with B-cell ALL than other immunotypes (p<0.05). The methylation levels of CpG&#95;13.14, CpG&#95;17, and CpG&#95;19 showed a significant negative correlation with MTX C 24 hr (p<0.05). The methylation level of CpG&#95;8.9 correlated significantly with MTX C 42 hrs (p<0.05). The methylation level of CpG&#95;19 was significantly lower in patients with MTX toxicities (p<0.05)., Conclusions: The methylation levels of the GGH promoter might affect MTX exposure and toxicities. These findings provided reasonable explanations for the variability of MTX responses in patients with childhood ALL., (© 2020 Pharmacotherapy Publications, Inc.)

Aims: Rituximab is a chimeric IgG-1 monoclonal antibody that depletes B cells, aiding in the treatment of several conditions including autoimmune diseases. It is not licensed for use in children. This study aimed to quantify the B cell-related pharmacodynamics of rituximab in children with autoimmune disease., Methods: Routine electronic health record data were collected at a large paediatric tertiary hospital in London, UK. Dosing protocols were either 2 × 750 mg/m 2 intravenous infusions of rituximab on days 1 and 15, or 4 × 375 mg/m 2 infusions on days 1, 8, 15 and 22. Rituximab pharmacokinetics (PK) were not measured but CD19+ lymphocyte counts were taken before and after rituximab treatment. A dose-response model was constructed describing the life cycle of CD19+ lymphocytes, with rituximab assumed to increase the death rate. Rituximab effect was assumed to decay by first-order kinetics., Results: In total, 258 measurements of CD19+ lymphocyte counts were collected from 39 children with 8 autoimmune diseases. The elimination rate constant (% relative standard error) of rituximab effect decay was 0.036 (22.7%) days -1 and CD19+ turnover was 0.02 (41%) days -1 corresponding to half-lives of 19 and 35 days respectively. Rituximab increased CD19+ death rate 35-fold, with methotrexate and cyclophosphamide associated with further increases. Simulations suggested that a single infusion of 750 mg/m 2 provides similar 6-month suppression of CD19+ lymphocytes to current dosing., Conclusions: Rituximab pharmacodynamics (PD) in paediatric autoimmune diseases has been described. Compared with rituximab alone, the additional effect of methotrexate or cyclophosphamide was statistically significant but small., (© 2019 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

We aimed to explore the mechanism of the KCNQ1OT1/miR-760/PPP1R1B axis acting to regulate methotrexate (MTX) resistance of colorectal cancer (CRC). Differentially expressed mRNAs and lncRNAs in MTX-sensitive CRC cell lines and MTX-resistant cell lines were determined through microarray analysis. Application of bioinformatics analysis was aimed to uncover the relationships among the lncRNAs/miRNAs/mRNAs, and to demonstrate the effects of cAMP signalling pathway in MTX-resistant CRC. The expression level of RNA and proteins was, respectively, detected using qRT-PCR and Western blot assays, whereas the dual-luciferase reporter gene assay was implemented to verify the targeted relationship. The influence of the lncRNA/miRNA/mRNA axis on biological functions of MTX-resistant cells and on the growth of tumours determined through both vitro and vivo experiments. LncRNA KCNQ1OT1 and PPP1R1B mRNA were overexpressed in MTX-resistant CRC tumour cells. KCNQ1OT1 functioned as a sponge of miR-760, which targeted PPP1R1B. Knockdown of KCNQ1OT1 enhanced chemosensitivity towards MTX through the sponging of miR-760. MiR-760 expressed at low levels targeted PPP1R1B in the activated cAMP signalling pathway under MTX treatment. Knockdown of KCNQ1OT1 dampened the proliferation of MTX-resistant (HT29/MTX) cells by regulating the miR-760/PPP1R1B axis, which also induced cell cycle arrest together with apoptosis. KCNQ1OT1 regulated the expression of PPP1R1B and the downstream genes CREB and CBP in the cAMP signalling pathway. MTX showed a suppressive function on CRC progression. KCNQ1OT1 enhanced the MTX resistance of CRC cells by regulating miR-760-mediated PPP1R1B expression via the cAMP signalling pathway., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Psoriasis has been related to a large number of cardiovascular risk factors such as hypertension, diabetes mellitus and arteriosclerosis. The increased carotid intima-media thickness (IMT) could be considered to be a marker of generalized arteriosclerosis., Objective: To assess the effect of systemic and biological drugs on psoriatic patients' carotid IMT., Methods: A prospective study was performed. We studied 53 patients with moderate and severe psoriasis from our psoriasis dermatological unit, analysing lipid and glucose metabolism and performing a carotid IMT sonography before introduction of systemic and biological drugs. After that, we performed an 8-month closely analytic and sonographic follow-up., Results: The IMT of the patients with psoriasis treated with biological drugs tended to decrease, although this occurrence was not statistically significant (P = 0.086). The subgroup analysis revealed that patients treated with methotrexate (P = 0.045) and anti-IL-12/23 (P = 0.010) presented a decrease in their IMT levels. This analysis also showed a decrease in glycaemia and insulin levels in patients treated with TNF-alpha inhibitors and ustekinumab., Conclusions: Our study suggests that the carotid IMT may benefit from treatment with biological drugs, particularly anti-IL-12/23 and methotrexate in patients suffering from moderate and severe psoriasis. However, larger longitudinal studies should be performed to fully confirm these results., (© 2018 European Academy of Dermatology and Venereology.)

Endometrial hyperplasia is a condition that may lead to the development of endometrial carcinoma. Initially, changes of the endometrium are caused by the estrogen's hyperstimulation that may lead to the development of an irregular bleeding and the infertility problems. Therapy of endometrial hyperplasia is limited to medical and surgical approaches. During the past decade, the new types of drugs were developed for the treatment of the endometrial hyperplasia. Here, for the first time, we investigated the cytotoxic effects of the various combinations of estrogen, raloxifene, and methotrexate in human ThESC cell line as a possible potential treatment of the endometrial hyperplasia. Our aim was to investigate and to determine the most efficient combination of investigated drugs in ThESC cells during 24-hr period using MTT assay, FACS analysis, and immunofluorescence staining. Our results demonstrated that the combination of raloxifene with methotrexate efficiently induced both the cytotoxicity and apoptosis in ThESC cells when compared to their single effect, as well as to the effect of combined treatment of raloxifene with estrogen. The application of the low doses of methotrexate combined with raloxifene offers all advantages of a potential beneficial antitumor match in cancer chemoprevention and therapy., (© 2017 John Wiley & Sons A/S.)

Interleukin (IL)-33 can function both as a conventional cytokine and as a nuclear factor regulating gene transcription. IL-33 expression is strongly upregulated in the nucleus of keratinocytes and serum of patients with psoriasis. However, the role of IL-33 in psoriasis is unclear, and IL-33 expression in the lesional psoriatic skin after conventional systemic treatments has not been investigated. In this study, we aimed to compare IL-33 mRNA in patients' lesional skin samples and IL-33 protein expression in patients' serum before and after treatment with methotrexate (MTX) and narrowband ultraviolet B (NB-UVB). IL-33 mRNA levels in lesional skin and IL-33 protein levels in serum were downregulated after treatment with MTX. Results revealed a significant decrease in IL-33 protein expression (P = 0.028). IL-33 expression increased after NB-UVB treatment. IL-33 production is associated with inflammatory skin in psoriasis, possibly through its cytokine function. However, high expression of IL-33 after NB-UVB treatment suggests the occurrence of unknown functions to alleviate psoriatic lesions without IL-33 involvement., (© 2017 Japanese Dermatological Association.)

CD34+ cell selection minimizes graft-versus-host disease (GVHD) after haploidentical donor stem cell transplant but is associated with slow immune recovery and infections. We report a Phase I/II study of prophylactic donor lymphocyte infusion (DLI) followed by methotrexate (MTX) GVHD prophylaxis after CD34-selected haploidentical donor transplant. A prophylactic DLI was given between day +30 and +42. Rituximab was given with DLI for the last 10 patients. The goal of the study was to determine a DLI dose that would result in a CD4+ cell count > 100/µL at Day +120 in ≥ 66% of patients with ≤ 33% grade II-III, ≤ 17% grade III, and no grade IV acute GVHD by Day +180. Thirty-five patients with malignant (n = 25) or nonmalignant disease (n = 10) were treated after CD34-selected haploidentical donor peripheral blood stem cell transplant. The DLI dose of 5 × 10 4 /kg met the CD4/GVHD goal with 67% of patients having CD4+ cells > 100/µL and 11% grade II-IV acute GVHD. The cumulative incidence of chronic GVHD was 16%. Fatal viral and fungal infections occurred in 11%. The 2 year estimated overall survival was 69% and the relapse rate was 14% for patients in remission at transplant. There was no effect of NK alloreactivity on relapse. Nine of ten patients at the target DLI dose cohort of 5 × 10 4 /kg are alive with median follow-up of 18 mos (range 6-29). Delayed prophylactic DLI and MTX was associated with promising outcomes at the target DLI dose. This trial was registered at clinicaltrials.gov, # NCT01027702., (© 2017 Wiley Periodicals, Inc.)

The Australasian Psoriasis Collaboration reviewed methotrexate (MTX) in the management of psoriasis in the Australian and New Zealand setting. The following comments are based on expert opinion and a literature review. Low-dose MTX (< 0.4 mg/kg per week) has a slow onset of action and has moderate to good efficacy, together with an acceptable safety profile. The mechanism of action is anti-inflammatory, rather than immunosuppressive. For pretreatment, consider testing full blood count (FBC), liver and renal function, non-fasting lipids, hepatitis serology, HbA1c and glucose. Body mass index and abdominal circumference should also be measured. Optional investigations in at-risk groups include an HIV test, a QuantiFERON-TB Gold test and a chest X-ray. In patients without complications, repeat the FBC at 2-4 weeks, then every 3-6 months and the liver/renal function test at 3 months and then every 6 months. There is little evidence that a MTX test dose is of value. Low-dose MTX rarely causes clinically significant hepatotoxicity in psoriasis. Most treatment-emergent liver toxicity is related to underlying metabolic syndrome and non-alcoholic fatty liver disease or non-alcoholic steatohepatitis. Alcohol itself is not contraindicated, but should be limited to < 20 gm/day. [Correction added on 6 January 2017, after first online publication: '20 mg/day' has been corrected to '20 gm/day'.] Although MTX is a potential teratogen post-conception, there is little evidence for this pre-conception. MTX does not affect the quality of sperm. There is no evidence that MTX reduces healing, so there is no specific need to stop MTX peri-surgery. MTX may be used in combination with cyclosporine, acitretin, prednisone and anti-tumour necrosis factor biologics., (© 2016 The Australasian College of Dermatologists.)

Problem: Resistance to methotrexate is a leading clinical problem in gestational trophoblastic neoplasia (GTN), but there are limited laboratory models for this condition., Method of Study: We created isogenic trophoblastic cell lines resistant to methotrexate and compared these to the parent cell lines using gene expression microarrays and qRT-PCR followed by mechanistic studies using recombinant cytokines, pathway inhibitors, and patient sera., Results: Gene expression microarrays and focused analysis by qRT-PCR revealed methotrexate led to type I interferon upregulation, in particular interferon alpha 2 (IFNA2), and methotrexate resistance was associated with chronic low level increases in type I interferon expression. Recombinant IFNA2 imparted chemosensitive choriocarcinoma cells with partial resistance to methotrexate, while chemoresistant choriocarcinoma cells were uniquely sensitive to fludarabine, a STAT1 inhibitor. In pre-treatment patient sera, IFNA2 levels correlated with subsequent resistance to methotrexate chemotherapy., Conclusion: Methotrexate resistance is influenced by type I interferon signaling with prognostic and therapeutic implications for treating women with GTN., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Juvenile idiopathic arthritis (JIA) of the temporomandibular joint (TMJ) can cause severe growth disturbances of the craniomandibular system. Antigen-induced arthritis (AIA) of the rabbit TMJ is simulating the inflammatory process of the TMJ in JIA. The aim of this study was to investigate the effect of a systemic administration of methotrexate (MTX) on AIA in rabbits by means of three different histological staining methods., Methods: After sensitization, a bilateral arthritis of the TMJ was induced by an intra-articular administration of ovalbumin in 12 New Zealand white rabbits aged 10 weeks. From the 13th week of age, six of the 12 rabbits received weekly intramuscular injections of MTX, and the other six animals remained without therapy. Another six animals served as controls, receiving no treatment or intra-articular injections at all. After euthanasia at the age of 22 weeks, all TMJs were retrieved en bloc. Sagittal sections were cut and stained with haematoxylin-eosin (H-E), Safranin-O for the evaluation of the Mankin score and tartrate-resistant acid phosphatase (TRAP)., Results: In the arthritis group, a chronic inflammation with degeneration of the articular cartilage was visible. In the MTX group, the signs of cartilage degeneration were significantly reduced compared with the arthritis group. In contrast, the joints in the control group were inconspicuous. A correlation between the Mankin score and TRAP-positive cells could be found., Conclusions: Systemic administration of MTX seems to have a positive effect upon the inflammatory process in the rabbit TMJ but fails to eliminate the sign of arthritis completely., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aims: Infliximab, an anti-tumour necrosis factor-α monoclonal antibody, is indicated in rheumatoid arthritis (RA). Our objective was to evaluate the influence of the sources of infliximab pharmacokinetic variability in RA., Methods: Eighty-four patients treated with infliximab for RA were included in a prospective noncomparative study. They were analysed between two consecutive infliximab infusions. Infliximab concentrations were measured before the infusion, 2 h, 1 and 4 weeks after the infusion and immediately before the next infusion. Infliximab concentrations were described using a two-compartment population pharmacokinetic model., Results: The mean (interindividual standard deviation) estimated central volume of distribution was 2.3 l (36%) and systemic clearance was 0.019 l h(-1) (37%). The central volume of distribution increased with bodyweight; it was doubled between 50 and 90 kg. Systemic clearance increased with pre-infusion C-reactive protein concentration by 20%, varying from 3 to 14 mg l(-) 1, and was decreased by 30% when methotrexate was coadministered., Conclusions: The influence of methotrexate and inflammation on infliximab clearance suggests that individual adjustment of infliximab doses according to disease activity may be useful in RA., (© 2013 The British Pharmacological Society.)

Objective: 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., Methods: 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)., Results: 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., Conclusion: 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., (Copyright © 2013 by the American College of Rheumatology.)

Objective: To assess defects in expression of critical cell cycle checkpoint genes and proteins in patients with rheumatoid arthritis (RA) relative to presence or absence of methotrexate (MTX) treatment, and to investigate the role of JNK in induction of these genes by MTX., Methods: Flow cytometric analysis was used to quantify changes in levels of intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcription-polymerase chain reaction was used to identify changes in cell cycle checkpoint target genes., Results: RA patients expressed reduced baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and JNK total protein. The reduction in expression of mRNA for MAPK9, TP53, CDKN1A, and CDKN1B was greater in patients not receiving MTX than in those receiving low-dose MTX, with no difference in expression levels of CHEK2 and RANGAP1 mRNA between MTX-treated and non-MTX-treated patients. Further, JNK levels were inversely correlated with C-reactive protein levels in RA patients. In tissue culture, MTX induced expression of both p53 and p21 by JNK-2- and JNK-1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 were not induced by MTX. MTX also induced ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocked these MTX-mediated effects., Conclusion: Our findings support the notion that MTX restores some, but not all, of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells, via a JNK-dependent pathway., (Copyright © 2012 by the American College of Rheumatology.)

Methotrexate is a synthetic folic acid analogue valued for both its anti-proliferative and anti-inflammatory properties. Considered one of the original immune-modifying agents, it is used widely for the treatment of steroid-recalcitrant inflammatory diseases. While there are abundant studies documenting its efficacy in rheumatic diseases, the use of methotrexate for dermatological conditions, with the exception of psoriasis, has yet to be comprehensively explored. This two-part review firstly outlines current data concerning the pharmacology of methotrexate, including its mechanism of action, side-effect profile and recommended therapeutic approach, and, secondly, examines the emerging evidence for methotrexate's efficacy in a wide range of cutaneous disorders., (© 2011 The Authors. Australasian Journal of Dermatology © 2011 The Australasian College of Dermatologists.)

Aims: Infliximab, an anti-tumour necrosis factor α monoclonal antibody, has profoundly modified the treatment of several inflammatory diseases. The objective was to assess the influence of methotrexate on the variability of infliximab pharmacokinetics and concentration-effect relationship in axial ankylosing spondylitis (AAS) patients., Methods: Twenty-six patients with AAS were included in a prospective study. They were treated by infliximab 5 mg kg⁻¹ infusions at weeks 0, 2, 6, 12 and 18. Infliximab concentrations were measured before, and 2 and 4 h after each infusion, and at each intermediate visit (weeks 1, 3, 4, 5, 8, 10 and 14). Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) was measured at each visit. Infliximab pharmacokinetics was described using a two-compartment model with first-order distribution and elimination constants. A population approach was used. Infliximab pharmacodynamics was described using the area under the BASDAI curve., Results: A total of 507 blood samples and 329 BASDAI measurements were collected. The following pharmacokinetic parameters were obtained (interindividual coefficient of variation): volumes of distribution for the central compartment = 2.4 l (9.6%) and peripheral compartment = 1.8 l (26%), systemic clearance = 0.23 l day⁻¹ (22%) and intercompartment clearance = 2.3 l day⁻¹. Methotrexate influenced neither pharmacokinetic nor BASDAI variability., Conclusions: Using the present dosage, the clinical efficacy of infliximab is only weakly influenced by its serum concentrations. The results do not support the combination of methotrexate with infliximab in ankylosing spondylitis., (© 2011 The Authors. British Journal of Clinical Pharmacology © 2011 The British Pharmacological Society.)

Objective: To study the influence of antiinflammatory treatments, including methotrexate (MTX) and tumor necrosis factor (TNF) inhibitors, on antibody response following vaccination using a 7-valent conjugate pneumococcal vaccine in adult patients with established arthritis., Methods: Patients with rheumatoid arthritis (RA) or spondylarthropathy (SpA) (including psoriatic arthritis) were vaccinated (n = 505). All patients were stratified into 6 prespecified groups based on diagnosis and treatment (RA patients receiving MTX, RA patients receiving anti-TNF agents and MTX, RA patients receiving TNF inhibitors as monotherapy, SpA patients receiving anti-TNF agents and MTX, SpA patients receiving TNF inhibitors as monotherapy, and SpA patients receiving nonsteroidal antiinflammatory drugs [NSAIDs] and/or analgesics). SpA patients receiving only NSAIDs/analgesics served as a control group. All patients received 1 dose (0.5 ml) of vaccine intramuscularly. Levels of IgG antibodies against 23F and 6B serotypes were measured at vaccination and at 4-6 weeks following vaccination, using standardized enzyme-linked immunosorbent assays., Results: Positive antibody response was defined as an antibody response ratio (ARR) (i.e., ratio of post- to prevaccination antibody levels) of ≥2. The ARR differed significantly between the groups. A better ARR was seen among patients in the control group compared to those in groups treated with MTX or MTX in combination with TNF inhibitors. Among patients treated with TNF inhibitors as monotherapy, ARRs for both serotypes were lower numerically, but were not significantly different, compared to those in controls. Ongoing MTX treatment was predictive of reduced response (odds ratio 0.41 [95% confidence interval 0.24-0.68], P = 0.001). Higher age was associated with impaired positive antibody response. Concomitant prednisolone treatment elicited better positive antibody response in patients with RA., Conclusion: Treatment with MTX and higher age were predictive of an impaired antibody response to the 7-valent conjugate pneumococcal vaccine in this cohort of patients with chronic arthritis. TNF inhibitors did not significantly affect antibody responses., (Copyright © 2011 by the American College of Rheumatology.)

Objective: Low-dose methotrexate (MTX) is an effective therapy for rheumatoid arthritis (RA), yet its mechanism of action is incompletely understood. The aim of this study was to explore the induction of apoptosis by MTX., Methods: Flow cytometry was performed to assess changes in the levels of intracellular proteins, reactive oxygen species (ROS), and apoptosis. Quantitative polymerase chain reaction was performed to assess changes in the transcript levels of select target genes in response to MTX., Results: MTX did not directly induce apoptosis but rather "primed" cells for markedly increased sensitivity to apoptosis via either mitochondrial or death receptor pathways, by a JNK-dependent mechanism. Increased sensitivity to apoptosis was mediated, at least in part, by MTX-dependent production of ROS, JNK activation, and JNK-dependent induction of genes whose protein products promote apoptosis. Supplementation with tetrahydrobiopterin blocked these MTX-induced effects. Patients with RA who were receiving low-dose MTX therapy expressed elevated levels of the JNK target gene, jun., Conclusion: Our results support a model whereby MTX inhibits reduction of dihydrobiopterin to tetrahydrobiopterin, resulting in increased production of ROS, increased JNK activity, and increased sensitivity to apoptosis. The finding of increased jun levels in patients with RA receiving low-dose MTX supports the notion that this pathway is activated by MTX in vivo and may contribute to the efficacy of MTX in inflammatory disease., (Copyright © 2011 by the American College of Rheumatology.)

Methotrexate (MTX) an antifolate drug and leucovorin its antidote, are used in the treatment of both neoplastic and non-neoplastic diseases in young women. We hypothesize that MTX treatment might comprise a deleterious effect on fast proliferating reproductive cells, an unavoidable and unwanted side effect. MTX given dose dependently to rats for 20 days prevented vaginal cyclicity and caused a reduction in serum progesterone and estradiol. External morphology of reproductive tract displayed thinning of organs and reduction in their weights. To reveal mechanism of MTX action, we examined the histology of ovary, oviduct, uterus, cervix and vagina. Results suggested that in a dose-dependent fashion MTX restrained preantral and antral follicular growth in ovary. Epithelium and stroma of oviduct, uterus, cervix and vagina were disrupted and lost their normal structures. Such alterations in ovarian function raised serum follicle stimulating hormone, luteinizing hormonal profiles. Expression of steroidogenic acute regulatory protein and P450 cholesterol side chain cleavage gene, which are both essential for steroidogenesis, markedly decreased in ovary upon MTX treatment. Total RNA, DNA and protein concentrations, glucose 6 phosphate dehydrogenase, lactate dehydrogenase and alkaline phosphatase enzyme activities in ovary were distinctly altered. Leucovorin supplementation and withdrawal of the treatment, improved MTX caused effects partially. These results for the first time indicate that the malfunction of female reproductive organs by MTX treatment in young women is not only correlated to the disrupted circulating levels of hormones and histoarchitecture of tissues but also discrepancies in steroidogenic genes and hormone regulated enzyme activities in ovary.

Objective: Prior research demonstrates that hydroxychloroquine (HCQ) lowers glycosylated hemoglobin (HbA(1c) ) in diabetes patients without rheumatic disease. We examined medical records of patients with diabetes mellitus (DM) and concomitant rheumatic illness to measure changes in HbA(1c) after starting HCQ or methotrexate (MTX)., Methods: We used electronic medical records to identify patients beginning treatment with either HCQ or MTX who had a diagnosis of DM (or a pretreatment HbA(1c) value of ≥7%) and at least 1 HbA(1c) measurement both before and within 12 months after initiation of treatment. Using a structured medical record abstraction, we examined rheumatic disease diagnosis, cumulative steroid use, duration (months) between drug initiation and lowest HbA(1c) value, a change in DM medication, body mass index (BMI), age, and sex. Adjusted linear regression models determined changes in HbA(1c) from pretreatment values to the lowest posttreatment values within 12 months., Results: We identified 45 patients taking HCQ and 37 patients taking MTX who met the inclusion criteria. Rheumatoid arthritis had been diagnosed in approximately half of the patients in each group. Age, sex, and mean pretreatment HbA(1c) levels were similar across groups. The mean BMI of those taking HCQ (35.4 kg/m(2) ) was slightly higher than that of those taking MTX (32.2 kg/m(2) ) (P = 0.13). Glucocorticoid use appeared more common in those taking MTX (46%) than in those taking HCQ (29%) (P = 0.17). The mean reduction in HbA(1c) from pretreatment values to the lowest posttreatment values was 0.66% (95% confidence interval [95% CI] 0.26, 1.05) in those taking HCQ compared with 0.11% (95% CI -0.18, 0.40) in those taking MTX. In fully adjusted analyses, the reduction in HbA(1c) among those taking HCQ was 0.54% greater than the reduction among those taking MTX (P = 0.041)., Conclusion: HCQ initiation was associated with a significantly greater reduction in HbA(1c) as compared with MTX initiation among diabetes patients with rheumatic disease., (Copyright © 2010 by the American College of Rheumatology.)

The Chinese Hamster Ovary production cell line development process using methotrexate (MTX) amplification is well studied and commonly used for biopharmaceutical processes. However, successful MTX amplification varies from clone to clone and suggested reasons include vector fragmentation during the transfection process and genomic rearrangement of the Chinese Hamster Ovary chromosomes. Here, we elucidated the vector integration patterns of 40 transfected single-cell clones by Southern blotting and showed that vector fragmentation occurs at a significant level in our experiment. This concurs with MTX amplification studies implying that single-cell cloning is necessary to ensure a successful amplification process. Truncations at the ends of the integrated vectors were also observed, whereas gross DNA insertions were not detected in our data. This suggests that end deletions are common, whereas insertion events are rare in animal cells.

Objective: To investigate A(1), A(2A), A(2B), and A(3) adenosine receptors in lymphocytes and neutrophils from patients with early rheumatoid arthritis (ERA) as well as from RA patients treated with methotrexate (MTX) or anti-tumor necrosis factor alpha (anti-TNFalpha), as compared with those in age-matched healthy controls, and to examine correlations between the status and functionality of adenosine receptors and TNFalpha release and NF-kappaB activation., Methods: Adenosine receptors were analyzed by saturation binding assays and Western blot analyses. We investigated the potency of typical A(2A) and A(3) agonists in the production of cAMP in control subjects, ERA patients, and RA patients treated with MTX or anti-TNFalpha. In a separate cohort of RA patients, TNFalpha release and NF-kappaB activation were evaluated in plasma and nuclear extracts, respectively., Results: In ERA patients, we found a high density and altered functionality of A(2A) and A(3) receptors. The binding and functional parameters of A(2A) and A(3) receptors normalized after anti-TNFalpha, but not MTX, treatment. TNFalpha release was increased in ERA patients and in MTX-treated RA patients, whereas in anti-TNFalpha-treated RA patients, release was comparable to that in the controls. NF-kappaB activation was elevated in ERA patients and in MTX-treated RA patients. Anti-TNFalpha treatment mediated decreased levels of NF-kappaB activation., Conclusion: A(2A) and A(3) receptor up-regulation in ERA patients and in MTX-treated RA patients was associated with high levels of TNFalpha and NF-kappaB activation. Treatment with anti-TNFalpha normalized A(2A) and A(3) receptor expression and functionality. This new evidence of A(2A) and A(3) receptor involvement opens the possibility of exploiting their potential role in human diseases characterized by a marked inflammatory component.

As the cure rates for haematological malignancies have improved, the exploration of the balance between efficacy and side effects has become a major research target. The antifolate methotrexate is widely used in the treatment of acute lymphoblastic leukaemia, non-Hodgkin lymphoma, and osteosarcoma. Even when given identical methotrexate doses, patients vary significantly in their response and pattern of toxicities. This diversity can, to some extent, be linked to sequence variations in genes involved in drug absorption, metabolism, excretion, cellular transport, and effector targets or target pathways. In the coming years pharmacogenomics is expected to change our approaches to individualised therapy with methotrexate. However, genetic polymorphisms affect the pharmacokinetics and dynamics of all the drugs a patient receive as well as the normal tissues tolerance to a given drug exposure. Thus, although high-throughput techniques will allow mapping of tens of thousands of genetic polymorphisms in one run, it will be a major challenge to dissect out which of these have the strongest impact on efficacy and toxicity and hence should be the targets for intervention. This paper discusses the pharmacology of methotrexate and reviews studies on haematological malignancies that have attempted to predict the risk of toxicity by specific clinical or genetic features.

Mutations in the MSH2 gene predispose to a number of tumourigenic conditions, including hereditary non-polyposis colon cancer (HNPCC). MSH2 encodes a protein in the mismatch repair (MMR) pathway which is involved in the removal of mispairs originating during replication or from damaged DNA. To identify new therapeutic strategies for the treatment of cancer arising from MMR deficiency, we screened a small molecule library encompassing previously utilized drugs and drug-like molecules to identify agents selectively lethal to cells lacking functional MSH2. This approach identified the drug methotrexate as being highly selective for cells with MSH2 deficiency. Methotrexate treatment caused the accumulation of potentially lethal 8-hydroxy-2'-deoxyguanosine (8-OHdG) oxidative DNA lesions in both MSH2 deficient and proficient cells. In MSH2 proficient cells, these lesions were rapidly cleared, while in MSH2 deficient cells 8-OHdG lesions persisted, potentially explaining the selectivity of methotrexate. Short interfering (si)RNA mediated silencing of the target of methotrexate, dihydrofolate reductase (DHFR), was also selective for MSH2 deficiency and also caused an accumulation of 8-OHdG. This suggested that the ability of methotrexate to modulate folate synthesis via inhibition of DHFR, may explain MSH2 selectivity. Consistent with this hypothesis, addition of folic acid to culture media substantially rescued the lethal phenotype caused by methotrexate. While methotrexate has been used for many years as a cancer therapy, our observations suggest that this drug may have particular utility for the treatment of a subset of patients with tumours characterized by MSH2 mutations.

Objective: To determine whether multidrug-resistance efflux transporters are expressed on immune effector cells in synovial tissue from patients with rheumatoid arthritis (RA) and compromise the efficacy of methotrexate (MTX) and leflunomide (LEF)., Methods: Synovial tissue biopsy samples obtained from RA patients before treatment and 4 months after starting treatment with MTX (n = 17) or LEF (n = 13) were examined by immunohistochemical staining and digital image analysis for the expression of the drug efflux transporters P-glycoprotein, multidrug resistance-associated protein 1 (MRP-1) through MRP-5, MRP-8, MRP-9, and breast cancer resistance protein (BCRP), and the relationship to clinical efficacy of MTX and LEF was assessed., Results: BCRP expression was observed in all RA synovial biopsy samples, both pretreatment and posttreatment, but not in control noninflammatory synovial tissue samples from orthopedic patients. BCRP expression was found both in the intimal lining layer and on macrophages and endothelial cells in the synovial sublining. Total numbers of macrophages in RA patients decreased upon treatment; in biopsy samples with persistently high macrophage counts, 2-fold higher BCRP expression was observed. Furthermore, median BCRP expression was significantly increased (3-fold) in nonresponders to disease-modifying antirheumatic drugs (DMARDs) compared with responders to DMARDs (P = 0.048). Low expression of MRP-1 was found on synovial macrophages, along with moderate expression in T cell areas of synovial biopsy specimens from one-third of the RA patients., Conclusion: These findings show that the drug resistance-related proteins BCRP and MRP-1 are expressed on inflammatory cells in RA synovial tissue. Since MTX is a substrate for both BCRP and MRP-1, and LEF is a high-affinity substrate for BCRP, these transporters may contribute to reduced therapeutic efficacy of these DMARDs.

Objective: To determine whether methotrexate (MTX) can overcome the atherogenic effects of cyclooxygenase 2 (COX-2) inhibitors and interferon-gamma (IFNgamma), both of which suppress cholesterol efflux protein and promote foam cell transformation in human THP-1 monocyte/macrophages., Methods: Message and protein levels of the reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFNgamma, with and without MTX. Foam cell transformation of lipid-laden THP-1 macrophages was detected with oil red O staining and light microscopy., Results: MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean +/- SEM 112.8 +/- 13.1% for NS398 plus MTX versus 71.1 +/- 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor-mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A2A receptor-specific antagonist ZM241385. MTX also prevented NS398 and IFNgamma from increasing transformation of lipid-laden THP-1 macrophages into foam cells., Conclusion: This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall.

Methotrexate is a folic acid analog pioneered for use in inflammatory diseases by dermatologists, and used successfully for over 40 years for a wide variety of cutaneous diseases. In addition to its antiproliferative properties, methotrexate has other more recently recognized anti-inflammatory properties related to its effects on adenosine. Further research concerning its mechanism of action and genetic enzymatic variations suggest future possibilities for maximizing therapy and predicting adverse events. In this review the present authors will explore methotrexate's pharmacokinetics, mode of administration, dosing guidelines, side effect profile, and medication interactions. In addition, the present authors hope to offer practical guidelines for dose initiation and adjustment, and to summarize new research on its mechanism of action and implications for future therapy.

Objective: Evidence from in vitro, in vivo, and clinical studies indicates that adenosine mediates, at least in part, the antiinflammatory effects of methotrexate (MTX), although the biochemical events involved have not been fully elucidated. This study was undertaken to investigate whether MTX exerts antiinflammatory effects in mice that lack ecto-5'-nucleotidase (ecto-5'-NT) (CD73) and are unable to convert AMP to adenosine extracellularly, in order to determine whether adenosine is generated intracellularly and transported into the extracellular space or is generated from the extracellular dephosphorylation of AMP to adenosine., Methods: Male CD73 gene-deficient mice and age-matched wild-type mice received intraperitoneal injections of saline or MTX (1 mg/kg/week) for 5 weeks. Air pouches were induced on the back by subcutaneous injection of air; 6 days later, inflammation was induced by injection of carrageenan., Results: Fewer leukocytes, but higher levels of tumor necrosis factor alpha (TNFalpha), accumulated in the air pouches of vehicle-treated CD73-deficient mice compared with those of wild-type mice. As expected, MTX treatment reduced the number of leukocytes and TNFalpha levels in the exudates and increased exudate adenosine concentrations in wild-type mice. In contrast, MTX did not reduce exudate leukocyte counts or TNFalpha levels or increase exudate adenosine levels in CD73-deficient mice., Conclusion: These results demonstrate that the antiinflammatory actions of MTX are mediated, at least in part, by increased release of adenine nucleotides that are hydrolyzed extracellularly to adenosine via an ecto-5'-NT-dependent pathway.

Background: The mechanism of the action of methotrexate (MTX) in the treatment of psoriasis has not been completely elucidated., Objective: To assess the effect of MTX on the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, activation molecule CD69 and T-cell phenotype in skin specimens from patients with psoriasis., Methods: We performed an immunohistochemical analysis of the expression of T-cell phenotype and cell adhesion/activation molecules in skin biopsies from patients with psoriasis treated with a fixed dose of MTX (12.5 mg/week). To determine data on the epidermal/dermal T-cell infiltration we carried out a manual quantification., Results: Skin samples prior to therapy showed a moderate to severe inflammatory infiltrate, mainly due to T lymphocytes with a helper/inducer (CD4) phenotype. Most of these cells also expressed ICAM-1 and VCAM-1. Blood vessels showed expression of E-selectin and VCAM-1, and keratinocytes were positive for ICAM-1 staining. The cell infiltrate was reduced after therapy, as well as the expression of cell adhesion molecules. However, we also noted the persistence of the T lymphocyte phenotype CD8(+), expressing the CD69 activation molecule, after the MTX treatment., Conclusions: MTX downregulates the expression of some adhesion molecules, a phenomenon that may contribute to its anti-inflammatory therapeutic effect in psoriasis. The infiltrating T cells post-treatment have an activated cytotoxic phenotype, which may suggest a pathogenic role in the continuation and/or recurrence of psoriasis.

Objective: To evaluate the contribution of metabolites (methotrexate [MTX] and folate polyglutamate [PG] levels) and pharmacogenetic biomarkers in the folate pathway to the effects of MTX in patients with rheumatoid arthritis not previously treated with this antifolate., Methods: Forty-eight MTX-naive adult patients were enrolled in a prospective longitudinal study. MTX therapy was initiated at 7.5 mg/week and was increased every 4-6 weeks until a therapeutic response was achieved. Response was assessed using the Disease Activity Score in 28 joints (DAS28). Red blood cell (RBC) MTX and folate PG levels were measured with 9 common polymorphisms in the folate pathway. Statistical analyses consisted of generalized linear models and multivariate regressions., Results: After 6 months of therapy, the median weekly MTX dosage was 17.5 mg and the median decrease in the DAS28 was 2.0. There was a large interpatient variability in RBC MTXPG levels (median 35 nmoles/liter [interquartile range 28-51] at month 6). Patients with a lesser decrease in the DAS28 (fewer improvements) had lower RBC MTXPG levels (P < 0.05) despite the higher MTX dose administered (P < 0.05). RBC folate PG levels decreased significantly during treatment, and a lesser decrease in RBC folate PGs was associated with a lesser decrease in the DAS28 (P < 0.05). Primary side effects were gastrointestinal and neurologic in nature. Risk genotypes associated with toxicity were in gamma-glutamyl hydrolase (-401CC), 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (347GG), methylenetetrahydrofolate reductase (1298AC/CC), methionine synthase (2756AA), and methionine synthase reductase (66GG)., Conclusion: RBC MTXPG levels are a useful means by which to monitor therapy. The genetic associations presented generate hypotheses, and confirmation in independent cohorts is warranted.

We performed a comparative analysis on CHO cells with varying recombinant monoclonal antibody production rates (qAb) and investigated the regulation of MAb production. Two families of CHO cells each composed of one parental and two progeny cell lines, generated by stepwise increases in methotrexate (MTX) concentration, were studied. The MAb heavy chain (HC) and light (LC) gene copy numbers and mRNA levels were quantitated by Southern and northern blotting in midexponential growth phase. We observed 2- to 3-fold amplification in gene copy numbers in high producing cell lines when compared with parental cell lines. However, the mRNA levels in the high producers were 5- to 7-fold higher, correlating well with the 5- to 7-fold increase in qAb. These results were confirmed by real-time qPCR analysis. Our study reveals that the MTX-mediated MAb overexpression results from both an increase in the gene copy number and more efficient transcription of each gene copy. We also observed that the HC to LC gene copy ratio may affect the protein yield in MAb expression systems.

Objective: To investigate the effects of combination therapy with M40403 and methotrexate (MTX) on collagen-induced arthritis (CIA) in rats., Methods: CIA was elicited in Lewis rats that had been assigned to different experimental groups, and the rats were treated daily, starting at the onset of arthritis (day 26), with M40403 2 mg/kg intraperitoneally, MTX 0.15 mg/kg orally, or combination therapy (M40403 2 mg/kg plus MTX 0.015 mg/kg)., Results: The histopathologic features of CIA in type II collagen-challenged rats included erosion of the articular cartilage and bone resorption. Treatment of rats with MTX 0.15 mg/kg orally delayed the development of clinical signs (days 26-35) and improved histologic status in the knee and paw, as clearly demonstrated by a significant reduction in erosion of the articular cartilage at the joint margins and subchondral bone resorption. Furthermore, radiographic evidence of protection against bone resorption and soft tissue swelling was apparent in the tibiotarsal joints of rats treated with MTX 0.15 mg/kg daily. Furthermore, combination therapy with M40403 2 mg/kg plus MTX 0.015 mg/kg exerted significant protection against the development of arthritis, similar to that observed with MTX alone at a dose of 0.15 mg/kg. In contrast, no significant protection was observed in animals treated with M40403 2 mg/kg alone or with MTX 0.015 mg/kg alone., Conclusion: This study provides the first evidence that M40403, a potent superoxide dismutase mimetic, exerts a significant synergistic effect with MTX in rats with CIA.

Objective: To evaluate whether microbubble-enhanced ultrasound (US) treatment promotes the delivery of methotrexate (MTX) into synovial cells and the enhanced antiinflammatory effects of intraarticular MTX therapy in a rabbit arthritis model., Methods: Arthritis was induced in both knees of 53 rabbits by immunization with ovalbumin. MTX including a microbubble agent was then injected into the left and right knee joints, and the right knees were exposed to US (MTX+/US+ group), while the left knees were not (MTX+/US- group). The knee joints were evaluated histologically in 7 rabbits at 5 time points up to day 56. Quantitative gene expression of interleukin-1beta (IL-1beta) in synovial tissue was measured on days 7 and 28. Eight rabbits were used for the measurement of MTX concentration in synovial tissue 12 hours after treatment. To evaluate the effect of microbubble-enhanced US treatment in the absence of MTX, only the microbubble agent was injected into the left and right knee joints of 10 rabbits with or without US exposure, and these animals were evaluated histologically on days 7 and 28., Results: The MTX concentration in synovial tissue was significantly higher in the MTX+/US+ group than in the MTX+/US- group. Synovial inflammation was less prominent in the MTX+/US+ group compared with the MTX+/US- group, judging from the results of the histologic evaluation and the gene expression levels of IL-1beta in synovial tissue. It also appeared that microbubble-enhanced US exposure itself did not affect inflammation., Conclusion: Microbubble-enhanced US exposure promoted the uptake of MTX into synovial cells, which resulted in enhancement of the antiinflammatory effects of the intraarticular MTX injection. These results suggest that application of this technique may have clinical benefit.

Objective: Low-dose methotrexate (MTX), a mainstay in the treatment of rheumatoid arthritis, is effective in only 60-70% of patients, a finding mirrored by poor antiinflammatory efficacy in some animal models, most notably collagen-induced arthritis. To determine whether genetic factors or the model itself is responsible for the poor response to MTX, we directly compared the responses of 4 inbred mouse strains to MTX in the air-pouch model of acute inflammation., Methods: The exudate leukocyte count and adenosine concentration were determined in inbred mice treated with MTX (0.75 mg/kg intraperitoneally every week for 4 weeks) or vehicle 4 hours after injection of carrageenan into the air pouch using previously described methods. Quantitative trait locus mapping was performed using an in silico, or computer-based, method to identify loci potentially associated with each phenotype., Results: MTX significantly reduced the exudate leukocyte count in C57BL/6J and BALB/cJ mice, but not DBA/1J (the strain used in the collagen-induced arthritis model) or DBA/2J mice. In a parallel manner, MTX increased adenosine concentration in inflammatory exudates of C57BL/6J and BALB/cJ mice, but not DBA/1J or DBA/2J mice. Antiinflammatory and adenosine responses to MTX in DBA/1J x C57BL/6J F(1) and F(2) offspring were most consistent with single genetic loci being responsible for each phenotype. In silico mapping identified partially overlapping loci containing candidate genes involved in both responses., Conclusion: Genetic factors contribute to the antiinflammatory efficacy of MTX, and a single locus involved in MTX-induced adenosine up-regulation is likely responsible for the observed resistance to MTX in DBA/1J mice.

Structural data are reported to 2.5 A resolution for the first full analysis of the methotrexate-resistant Leu22Arg (L22R) variant of mouse dihydrofolate reductase (mDHFR) crystallized as a ternary complex with methotrexate (MTX) and the cofactor NADPH. These results are compared with the MTX and NADPH ternary complexes of L22R human DHFR (hDHFR) and those of mouse and human wild-type DHFR enzymes. The conformation of mDHFR Arg22 is such that it makes hydrogen-bonding contacts with Asp21, Trp24 and a structural water molecule, observations which were not made in the L22R hDHFR ternary complex. These data show that there is little difference between the structures of the wild type and L22R variant for either mouse or human DHFR; however, there are significant differences between the species. Comparison of these structures reveals that the active site of mDHFR is larger than that in the hDHFR structure. In mDHFR, the position of MTX is shifted 0.6 A toward helix C (residues 59-65), which in turn is shifted 1.2 A away from the active site relative to that observed in the hDHFR ternary complexes. In the L22R variant mDHFR structure, MTX makes shorter contacts to the conserved residues Ile7, Val115 and Tyr121 than in the L22R variant human DHFR structure. These contacts are comparable in both wild-type enzymes. The unexpected results from this comparison of the mouse and human DHFR complexes bound with the same ligand and cofactor illustrate the importance of detailed study of several species of enzyme, even when there is a high sequence homology between them. These data suggest that the differences in binding interactions of the L22R variant are in agreement with the weaker binding affinity for MTX in the variant enzymes; the larger size of the binding site in mDHFR supports the observation that the binding affinity of MTX for L22R mDHFR is significantly weaker than that of the L22R hDHFR enzyme.

The structure of Leishmania major pteridine reductase (PTR1) in complex with NADPH and the inhibitor 2,4,6-triaminoquinazoline (TAQ) has been solved in a new crystal form by molecular replacement and refined to 2.6 A resolution. The inhibitor mimics a fragment, the pterin head group, of the archetypal antifolate drug methotrexate (MTX) and exploits similar chemical features to bind in the PTR1 active site. Despite being a much smaller molecule, TAQ displays a similar inhibition constant to that of MTX. PTR1 is a target for the development of improved therapies for infections caused by trypanosomatid parasites and this analysis provides information to assist the structure-based development of novel enzyme inhibitors.

Objective: Methotrexate is one of the most commonly used disease-modifying antirheumatic drugs in the management of psoriatic arthritis (PsA). Despite the differences between the inflammation in PsA and rheumatoid arthritis (RA), the effects of methotrexate on the synovium have been described solely in RA. In this study, we sought to determine the effects of methotrexate on the inflammatory infiltrate and on cytokine and metalloproteinase gene expression in the synovium of PsA patients., Methods: Ten patients with PsA (median duration 18 months) underwent arthroscopy and synovial biopsy of an inflamed knee before and after clinical improvement induced by methotrexate. Immunohistologic analysis was performed using antibodies to CD3, CD4, CD8, CD68, factor VIII, vascular cell adhesion molecule, E-selectin, and intercellular adhesion molecule (ICAM). Matrix metalloproteinase 3 (MMP-3) and tissue inhibitor of metalloproteinases 1 (TIMP-1) messenger RNA (mRNA) were quantified by competitive reverse transcription-polymerase chain reaction (RT-PCR). Interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, interferon-gamma (IFNgamma), and tumor necrosis factor alpha (TNFalpha) mRNA expression was quantified by real-time PCR., Results: Patients received a median methotrexate dosage of 13.75 mg/week (range 7.5-15) for a median of 11.5 months (range 7-14 months). The Ritchie Articular Index, swollen joint count, and Disease Activity Score were significantly reduced. There was a decrease in all immunohistologic staining, although this was statistically significant only for CD3, CD4, CD8, CD68, E-selectin, and ICAM. Despite clinical improvement in all patients, there was a residual T cell infiltrate in all synovial biopsy tissues. The synovial lining layer thickness, but not hypervascularity, was significantly reduced. There was also a significant reduction in MMP-3, but not TIMP-1, expression. Before treatment, PsA synovium was characterized by a predominant expression of the proinflammatory cytokines IL-15, IFNgamma, IL-1beta, and TNFalpha and the antiinflammatory cytokine IL-10. Methotrexate reduced synovial IL-1alpha, IL-1beta, IL-8, IL-10, IL-15, IFNgamma, and TNFalpha mRNA expression, but the effect was significant only for IL-8., Conclusion: Methotrexate produced a clinical response in PsA by reducing, but not abolishing, the inflammatory infiltrate, adhesion molecule expression, and MMP-3 and proinflammatory cytokine gene expression, particularly IL-8, in the synovium. Methotrexate did not reduce hypervascularity, which is a prominent differentiating feature of PsA synovium., (Copyright 2004 American College of Rheumatology)

A current challenge is to define the biological characteristics of colon tumor cells resistant to chemotherapy. Distinct sub-populations of mucus-secreting cells were previously obtained from the colon cancer cell line HT-29 after long-term treatment with the anti-cancer drugs, 5-fluorouracil (5-FU) and methotrexate (MTX). Since mucins are increasingly implicated as playing a role in carcinogenesis, we studied the pattern of mucin expression in two HT-29 clones of mucus-secreting and two clones of enterocyte-like phenotype which differ in their capacity to resist to 5-FU and/or MTX. The expression of both transmembrane (MUC1, MUC3, MUC4) and secreted gel-forming (MUC2, MUC5AC, MUC5B, MUC6) mucins in clones was studied by northern and/or western blotting. The four HT-29 clones showed three cellular phenotypes: (1) The mucus-secreting clone HT29-5F12 consists of unpolarized cells with mucus secretions that have anti-colonic mucin immunoreactivity, and mainly expresses MUC2 and is resistant to 5-FU and sensitive to MTX; (2) The mucus-secreting clone HT29-5M21 forms a monolayer of polarized cells with strong anti-gastric mucin immunoreactivity and mainly expresses MUC5AC and MUC5B and is resistant to MTX and sensitive to 5-FU; (3) The two enterocyte-like clones, HT29-5F7 and HT29-5M12 are resistant to both MTX and 5-FU and express mainly MUC1 and MUC5B, respectively. These clones which originate from a same colorectal tumour and display different patterns of mucin expression as well as differing resistance to MTX and 5-FU will make useful in vitro models for studying the potential role of mucins or other biological markers in drug resistance pathways.

Aim: To investigate whether chronic administration of medium doses of methotrexate (MTX) causes suppression of skeletal growth in young mice and to determine whether folinic acid supplementation could reverse this effect., Methods: Four equal groups of Balb/c young male mice (6 animals in each group; mean body weight 11.9 +/- 0.25 g, in their rapid growth phase) were subjected to the following drug treatment for a period of 3 wk. Group 1 was given intraperitoneal MTX (3.5 mg kg(-1) body weight) every second day. Group 2 received folinic acid (7.0 mg kg(-1) body weight) intraperitoneally every second day. Group 3 was given both drugs (MTX every second day and folinic acid 8 h post-MTX injection). Group 4 was injected with physiological saline every other day to serve as a control group. Total body weight of the animals in each group was monitored every second day for the entire study period. The animals were sacrificed, the bilateral femurs and tibias of each animal were harvested and X-rays of the bones were taken. The length of each femur and tibia was measured using a micrometer. Measurements from the radiographs were also recorded using image analysis software. The MTX concentrations in the plasma and the folate levels in erythrocytes were determined. The heights of the distal femoral and the proximal tibial growth plate for each animal were measured on histological tissue sections., Results: Mean lengths of both the tibia and femur of animals were compared in the four treatment groups. A significant decrease in the mean lengths (one-way ANOVA, p < 0.005) was observed in the group receiving MTX alone. Similarly, there was a significant decrease (p < 0.001) in the height of the femoral and tibial growth plate in this group when compared with the other groups. The main effect of MTX seemed to be on the hypertrophic proliferative zone of chondrocytes in the growth plate. Furthermore, animals in this MTX-treated group also showed increased levels of MTX in plasma and low levels of erythrocyte folate., Conclusion: These data show that chronic administration of MTX induces suppression of skeletal growth in mice, possibly through the inhibition of the pathway of de novo DNA synthesis. Folinic acid treatment following MTX administration appears to reverse this growth inhibition. Based on these observations, children suffering from juvenile rheumatoid arthritis, osteosarcoma or acute lymphoblastic leukaemia and receiving MTX over long periods of time could be at risk of short-term suppression of skeletal growth. If this is the case, it is possible that they could benefit from dietary supplementation with folinic acid.

Objective: To test the hypothesis that rats with adjuvant-induced arthritis (AIA) need more metabolic energy to maintain body weight than healthy control rats or rats with AIA treated with methotrexate (MTX)., Methods: Rat AIA was induced by Mycobacterium butyricum injection at the base of the tail. The MTX-treated group was injected with MTX (1.0 mg/kg/week) in phosphate buffered saline. Negative controls (i.e., disease-free) and the MTX-treated group were pair-fed with positive controls (i.e., untreated AIA rats) to ensure equal mean body weights., Results: An additional 0.85 gm of food per day per rat was needed by the positive control group and 0.54 gm per day by the MTX-treated group to maintain a body weight comparable with that of the negative control group during days 5-15 post-adjuvant injection. During days 15-34 post-adjuvant injection an additional 1.4 gm of food per day per rat was needed by the positive controls and 0.62 gm by the MTX-treated group., Conclusion: The results of this study indicate that adjuvant arthritis has a metabolic cost, which increases substantially as the disease becomes clinically apparent (days 15-34). MTX treatment does not completely eliminate the caloric cost of the disease. During days 5-15 post-adjuvant injection, an average of 6% of the total calories eaten by the positive controls was metabolized to support subclinical inflammation and other physiologic processes of this disease. During the active phase of the disease (i.e., clinical inflammation), this value increased to 18% of total calories.

Objective: To analyze S-100 protein expression, in the form of myeloid-related protein 8 (MRP8), MRP14, and the heterodimer MRP8/MRP14, in psoriatic arthritis (PsA) patients compared with rheumatoid arthritis (RA) and spondylarthropathy (SpA) patients, and to determine the effect of methotrexate (MTX) on the MRP antigen expression in PsA patients., Methods: Serum, synovial fluid (SF), and synovium (taken at arthroscopy) samples were obtained from PsA (before and after MTX treatment), RA, and SpA patients. Concentrations of MRP8/MRP14 in serum and SF were measured by enzyme-linked immunosorbent assay. Expression of MRP8, MRP14, and MRP8/MRP14 in synovium was determined by immunohistochemistry., Results: MRP8, MRP14, and MRP8/MRP14 levels were increased in serum, SF, and synovium from PsA, RA, and SpA patients. In all 3 groups, paired samples of serum and SF showed significantly higher MRP8/MRP14 levels in SF (mean +/- SD 15310 +/- 16999 ng/ml [median 11400]) than in serum (908 +/- 679 ng/ml [median 695]) (P = 0.0001). MRP8/MRP14 levels in serum correlated with systemic parameters of disease activity (erythrocyte sedimentation rate [ESR] r = 0.55, P = 0.005; C-reactive protein [CRP] level r = 0.55, P = 0.005), whereas levels in SF correlated with local parameters of disease activity (white blood cell count r = 0.45, P = 0.01; acute-phase serum amyloid A level r = 0.32, P = 0.03). MRP expression was significantly higher in the synovial sublining layer (SLL) of PsA patients compared with RA and SpA patients. MRP antigens were predominantly expressed in perivascular areas of the SLL in PsA patients. Following MTX treatment, MRP expression in serum and synovium from PsA patients was significantly reduced. Serum levels of MRP were more sensitive to the effects of MTX than were the ESR, CRP, or clinical joint scores., Conclusion: MRP levels in serum and SF correlate with local and systemic inflammation and are equally increased in PsA, RA, and SpA patients. In contrast, MRP8, MRP14, and MRP8/MRP14 expression in the SLL of PsA patients is increased, particularly in perivascular regions, compared with that in RA and SpA patients, suggesting a central role of MRP proteins in transendothelial migration of leukocytes in PsA. Moreover, MRP expression is reduced following MTX treatment. MRP proteins may represent a novel therapeutic target in inflammatory arthritis.

A population-based multicentre study for childhood acute lymphoblastic leukemia (ALL) was conducted in Hong Kong from 1993 to 1997. One hundred and forty-five newly diagnosed ALL patients were treated by the HKALL 93 protocol. Patients were stratified into three risk groups according to age, presenting white cell count, immunophenotyping and cytogenetic study. The patients received the same induction and early and late intensification at week 5 and week 20. Fifty-eight standard risk (SR) patients received regular intrathecal methotrexate as CNS preventive therapy, while 49 intermediate risk (IR) patients received high dose intravenous methotrexate and regular intrathecal methotrexate. Thirty-eight high risk (HR) patients were treated with prophylactic cranial irradiation and an additional intensification block at week 35. The induction remission rate was 97.2% with 2% induction death. Two patients died during first complete remission. Relapse occurred in 20.7, 42.9 and 42.1% of SR, IR and HR patients respectively. By multivariate logistic regression, age> or =10 years and white cell count> or =100 x 10(9)/l were the two significant variables accounting for mortality. The 5-year overall and event-free survival of the whole group was 81.3 and 62.6% respectively. According to risk groups, the event-free survival was 79, 49 and 61% for SR, IR and HR patients respectively, while the overall survival was 96, 73 and 68% for SR, IR and HR patients respectively. In conclusion, the treatment protocol had low treatment-related mortality but was associated with a rather high relapse rate, especially in IR patients. Salvage therapy achieved sustained second remission in some patients. More intensive treatment especially a late intensification is required to improve the outcome., (Copyright 2002 John Wiley & Sons, Ltd.)

Objective: Low-dose weekly methotrexate therapy remains a mainstay in the treatment of inflammatory arthritis. Results of previous studies demonstrated that adenosine, acting at one or more of its receptors, mediates the antiinflammatory effects of methotrexate in animal models of both acute and chronic inflammation. We therefore sought to establish which receptor(s) is involved in the modulation of acute inflammation by methotrexate and its nonpolyglutamated analog MX-68 (N-[[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]-carbonyl]-L-homoglutamic acid)., Methods: We studied the effects of low-dose methotrexate (0.75 mg/kg intraperitoneally [IP] every week for 5 weeks), MX-68 (2 mg/kg IP 2 days and 1 hour before induction of inflammation), dexamethasone (1.5 mg/kg IP 1 hour before induction of inflammation), or vehicle control on acute inflammation in an air-pouch model in A(2A) and A(3) receptor knockout mice., Results: Low-dose weekly methotrexate treatment increased the adenosine concentration in the exudates of all mice studied and reduced leukocyte and tumor necrosis factor alpha accumulation in the exudates of wild-type mice, but not in those of A(2A) or A(3) receptor knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was equally effective at suppressing leukocyte accumulation in A(2A) knockout, A(3) knockout, and wild-type mice, indicating that the lack of response was specific for methotrexate and MX-68., Conclusion: These findings confirm that adenosine, acting at A(2A) and A(3) receptors, is a potent regulator of inflammation. Moreover, these results provide strong evidence that adenosine, acting at either or both of these receptors, mediates the antiinflammatory effects of methotrexate and its analog MX-68.

The utilization of dicistronic mRNA expression vectors, containing the gene of interest upstream of an amplifiable marker gene, has shown success in rapidly, efficiently and reproducibly obtaining stable cell lines that express high levels of the protein of interest. For this reason, human thyroid-stimulating hormone (hTSH), a heterodimeric glycoprotein composed of non-covalently linked alpha- and beta-subunits, was expressed in Chinese hamster ovary (CHO) cells using a system based on dicistronic expression vectors. These contained the genes of interest and the amplifiable gene markers dihydrofolate reductase (DHFR) and adenosine deaminase (ADA), separated by an internal ribosome entry site isolated from the encephalomyocarditis virus. After the cells (CHO-DHFR-) had been co-transfected with the expression vectors and submitted to gene amplification in culture medium containing stepwise increments of methotrexate, it was possible to isolate clones that presented a secretion level of up to 7.2+/-1.3 microg/10(6) cells per day, the highest ever reported for the expression of this glycoprotein hormone. A second treatment, involving the utilization of deoxycoformycin, directed to amplify the ADA marker gene, provided a clone with an additional 2-3-fold increase in hTSH secretion, reaching a secretion level of 17.8+/-7.6 microg/10(6) cells per day. Cell culture and hTSH production in a hollow-fibre bioreactor were set up in order to carry out a preliminary physico-chemical, immunological and biological characterization of this hormone in comparison with pituitary-extracted hTSH (from the National Institute of Diabetes and Digestive and Kidney Diseases) and the only recombinant hTSH now available (Thyrogen). The availability of recombinant hTSH is very important in the diagnosis and therapy of thyroid carcinoma, via stimulation of radioiodine uptake.

Efficiency of viral promoters and various cell lines in directing high-level expression of human erythropoietin (Epo) was investigated. To investigate the effects of various viral promoters and cell lines on the Epo expression level, genomic Epo with the 5' and 3' untranslated regions (UTRs) deleted was cloned next to the simian virus 40 early promoter, cytomegalovirus early promoter or SRalpha promoter. These expression vectors were transfected into COS-7, BHK-21 and Chinese hamster ovary (CHO)/dhfr(-) cells, respectively. The COS-7 cells transfected with the vector containing the SRalpha promoter showed the highest expression level ( approximately 103 IU/ml) at 72 h post-transfection. For the development of Epo-producing stable cell lines, BHK-21 and CHO/dhfr(-) cells transfected with the 5',3'-UTR-deleted genomic Epo under the control of the SRalpha promoter were cultured with media containing zeocin. Several clones of zeocin-resistant BHK-21 and CHO/dhfr(-) cells were cultured in the presence of methotrexate (MTX). A BHK-21 clone selected in the presence of 500 nM MTX expressed and secreted approximately 490 IU/ml Epo into the medium. A CHO/dhfr(-) clone selected in the presence of 20 nM MTX expressed and secreted approximately 45 IU/ml Epo into the medium. Southern-blot analysis indicated that enhancement of Epo expression in the MTX-resistant stable cells might be related to the amplification of gene copy number.