Methotrexate Group Research Articles

Effects of antioxidant alfa-lipolic acid on polyphenol oxidase activity of acitretin- methotrexate combination in rat spleen tissue

Objectives: Acitretin, a lipophilic weak acid, is a slightly water-soluble vitamin A analog. Methotrexate is a folic acid analogue and an antimetabolite used for the treatment of many diseases.. Alpha lipoic acid is a nutritional coenzyme with important physiological effects in protein, fat and carbohydrate metabolism. Polyphenol oxidase is an oxidoreductase containing copper in its active site and oxidizes phenolic compounds. The aim of this study is to determine the effect of Alpha lipoic acid on the polyphenol oxidase enzyme activity in the spleen tissue in removing the damage caused by the free radicals produced by Acitretin and Methotrexate at the cellular level. Materials and Methods: Study groups were formed as Control , Alpha lipoic acid , Acitretin+Methotrexate and Acitretin+Methotrexate+Alpha lipoic acid groups. Rats who were starved for 24 hours were injected at the every morning. Acitretin, Methotrexate and Alpha Lipoic Acid were dissolved in 0.9% NaCl and given to rats by intraperitoneal injection. Rats were sacrificed by cervical dislocation on the 7th day after injection. After perfusion of the heart, the spleen was removed. Extracted spleen tissue samples were used for polyphenol oxidase enzyme activity determination. Results: Results: Compared with groups Control; While 18% inhibition was observed in the Alpha lipoic acid group, 20% inhibition was observed in the Acitretin + Methotrexate group. When Alpha lipoic acid was added to the Acitretin + Methotrexate group, while 19% activation was observed according to Control, 1% activation was observed compared to the Acitretin+Methotrexate group. Although there were differences between the groups, these differences were not statistically significant. Conclusion: The inhibition of PPO enzyme activity by ALA can be explained by the fact that while ALA alone acts as a radical scavenger that can regenerate substances with antioxidant properties in the spleen tissue, when given in combination, it acts as an antioxidant that triggers PPO activity.

Humanized Anti-CD25 Monoclonal Antibody Replacing MTX for Graft-Versus-Host Disease Prophylaxis in Haploidentical Hematopoietic Stem Cell Transplantation

Introduction Currently, a short course of methotrexate (MTX) remains the classical regimen for the prevention of graft-versus-host disease (GVHD) in haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Although MTX was widely applied for GVHD prophylaxis, it has been reported to be associated with significant toxicity and considerable side effects. We conducted a prospective, single-center, non-randomized controlled study to evaluate whether MTX can be safely removed from GVHD prophylaxis regimen after transplantation. Methods We prospectively reported and analyzed the data of 149 patients with hematologic malignancies who received haplo-HSCT from May 2022 to January 2023, and followed up until July 10, 2023. Patients were voluntarily enrolled in two groups according to different prevention programs of GVHD. A total of 82 patients were administrated with a combination of cyclosporine (CsA) or tacrolimus, mycophenolate mofetil (MMF), anti-human thymocyte immunoglobulin (ATG), MTX (15mg, +1d, 10mg, +3d, 10mg, +6d), and humanized anti-CD25 monoclonal antibody (Xenopax, 25mg, +0d, +4d) (single-dose anti-CD25 with MTX group). A total of 67 patients were given with CsA or tacrolimus, MMF, ATG and humanized anti-CD25 monoclonal antibody (Xenopax, 50mg, +0d, +4d) (double-dose anti-CD25 group). This study is registered with the Chinese Clinical Trial Registry (chictr.org.cn: ChiCTR2200060184). Results Compared with the single-dose anti-CD25 with MTX group, the median time to achieve neutrophil and platelet engraftment was shorten in double-dose anti-CD25 group (neutrophil: 9 days (7-20) vs 11 days (7-39), P=0.000; platelet: 9 days (7-30) vs 11 days (7-46), P=0.001). The incidence of total oral mucositis (OM) and grade 2-4 OM were significantly reduced in double-dose anti-CD25 group (total OM: 40.29% vs 63.41%, P=0.003; grade 2-4: 7.46% vs 32.05%, P=0.000). The median duration of OM was remarkably shorter in double-dose anti-CD25 group (5 days (2-18) vs 11 days (3-30), P=0.000). Lower cumulative incidence (CI) of grade II-IV aGVHD was observed in double-dose anti-CD25 group than in single-dose CD25 with MTX group (16.7 ± 4.6% vs 28.4 ± 5.0%, P=0.063). There was no statistical difference in two groups of the 100-day CI of grade III-IV aGVHD (6.1 ± 3.0% vs 13.6 ± 3.8%, P=0.123). Lower CI of cGVHD were occurred in double-dose anti-CD25 group (8.4 ± 4.1% vs 20.3 ± 5.1%, P=0.073). The CI of GVHD/relapse-free survival in double-dose anti-CD25 group was remarkably improved than that in single-dose CD25 with MTX group (73.9 ± 5.5% vs 55.6 ± 5.7%, P=0.044). No significant difference occurred in two groups of the overall CIs of CMV viremia, EBV viremia, overall survival, disease-free survival, relapse and treatment-related mortality. Conclusions Based on the efficacy and safety of the clinical trial results at our transplant center, replacing MTX with a single dose of humanized anti-CD25 monoclonal antibody is a promising GVHD prophylaxis for haplo-HSCT.

Navigating the Blood-Brain Barrier: An Anti-CD19 Modified-Exosome Delivery System for Precise Targeting of CNS Lymphoma

High-dose methotrexate (HDMTX) serves as the cornerstone of central nervous system lymphoma (CNSL) treatment, but its efficacy is limited due to low Blood-Brain Barrier (BBB) penetration (2% to 20%) and adverse effects like myelosuppression and hepatotoxicity. Thus, this study focuses on exosome-based drug delivery to enhance BBB permeability, reducing methotrexate (MTX) dosage while targeting CNSL specifically (Figure 1A). Considering the recipient's compatibility and reduced risk of adverse reactions or rejection, exosomes derived from human adipose tissue-derived stem cells (ADSCs) were selected as drug delivery vehicles. ADSCs-Exo underwent purification through ultracentrifugation and were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscope (TEM). CD19 is considered an excellent target for immunotherapy in DLBCL. To confer targeting capabilities, ADSCs were infected with a lentiviral vector encoding the anti-CD19 prior to the purification of exosomes. The anti-CD19 (patent number: ZL201811653085.8) was strongly expressed in ADSCs, integrated into ADSCs-derived exosomes, and localized on the external exosomal surface, as evidenced by western blots, colloidal gold immunoelectron microscopy, and flow cytometry. Based on the properties of MTX, we employed co-incubation to load MTX into anti-CD19-modified exosomes. The resulting exosomes, loaded with MTX, were then quantified for the encapsulated MTX using High Performance Liquid Chromatography. The anti-CD19-modified exosome-loaded methotrexate (anti-CD19-Exo-MTX) interacted with cerebrovascular endothelial cells and astrocytes of the BBB, leading to endocytosis and facilitating MTX transportation across the barrier. Additionally, anti-CD19-Exo-MTX crossed the BBB model without compromising its structure, as assessed by dextran's luminescent signal assay. Compared to drug-free formulations, anti-CD19-Exo-MTX exhibited enhanced BBB permeability, thereby improving its capability to inhibit CNSL cell proliferation. Liquid chromatography-tandem mass spectrometry identified and quantified proteins in exosomes, revealing significant enrichment of neuron projection and axon development-associated proteins. Subsequent neurite outgrowth assays, Nissl staining, and modified neurological severity scores confirmed the beneficial effects of ADSCs-Exo in mitigating MTX-induced neuron damage. To evaluate the efficacy of anti-CD19-Exo-MTX in vivo, intracranial orthotopic CNSL models in mice were established. Magnetic resonance imaging (MRI) analysis revealed a reduced disease burden in the anti-CD19-Exo-MTX group compared to both the MTX and Exo-MTX groups (Figure 1B), along with prolonged overall survival. Cerebrospinal fluid (CSF) drug concentration analysis demonstrated enhanced stability and longer-lasting drug levels for anti-CD19-Exo-MTX in the CSF, while showing no toxicity to vital organs, as evidenced by the absence of significant histological damage in the heart, liver, spleen, lung, and kidney tissues. To assess biodistribution and targeting of anti-CD19-Exo-MTX, exosomes were labeled with VivoTrack Dil (Fluorescence). Intracranial fluorescence signal was observed for anti-CD19-Exo-MTX, while Exo-MTX exhibited systemic distribution. Two hours post-injection, organs removal revealed weaker fluorescence signals in the liver and spleen for anti-CD19-Exo-MTX, confirming its specific targeting of MTX to CNSL in vivo. In conclusion, we developed anti-CD19-Exo-MTX, an exosome-based drug delivery platform, demonstrating enhanced BBB permeability and specific targeting for CNSL. This promising approach offers potential for effective CNSL treatment with reduced adverse effects.

Quantifying the Effect of Methotrexate on Adalimumab Response in Psoriasis by Pharmacokinetic–Pharmacodynamic Modeling

Previously, we showed that the combination of methotrexate and adalimumab treatment leads to less antidrug antibody development. In this study, we quantify the pharmacokinetics/pharmacodynamics (PK/PD) of adalimumab and evaluate the influence of methotrexate cotreatment. A population PK-PD model was developed using prospective data from 59 patients with psoriasis (baseline PASI= 12.6) receiving adalimumab over 49 weeks. Typical PK and PD parameters and their corresponding interpatient variability were estimated. We performed a covariate analysis to assess whether interpatient variability could be explained by addition of methotrexate and other covariates. In total, 330 PASIs, 252 adalimumab serum concentrations, and 247 antidrug antibody titers were available. Presence of antidrug antibodies (adalimumab group= 46.7%, adalimumab+ methotrexate group= 38.7%; P= .031) was correlated with increased adalimumab apparent clearance (P < .001). In the PD model, the use of concomitant methotrexate was borderline to significantly correlated with a decreased half-maximal inhibitory concentration (adalimumab concentration for which clinical response score is reduced by half; P < .10). On the basis of our PK-PD model, concomitant use of methotrexate indirectly increases adalimumab concentration, partially through less antidrug antibodies formation, which may result in better efficacy.

Apatinib plus chemotherapy for non-metastatic osteosarcoma: a retrospective cohort study.

Effective adjuvant therapy for osteosarcoma is necessary for improved outcomes. Previous studies demonstrated that apatinib plus doxorubicin-based chemotherapy may improve the efficacy of neoadjuvant therapy. This study aimed to clarify the effectiveness and safety of apatinib plus doxorubicin and cisplatin (AP) as neoadjuvant therapy for osteosarcoma. The clinical data of osteosarcoma patients who underwent neoadjuvant therapy and surgery between August 2016 and April 2022 were retrospectively collected and analyzed. Patients were divided into two groups: the apatinib plus AP (apatinib + AP) group and the methotrexate, doxorubicin, and cisplatin (MAP) group. This study included 42 patients with nonmetastatic osteosarcoma (19 and 23 patients in the apatinib + AP and MAP groups, respectively). The 1- and 2-year disease-free survival rates in the apatinib + AP group were higher than those in the MAP group, but the difference was not significant (P=0.165 and 0.283, respectively). Some adverse events were significantly more common in the apatinib + AP group than in the MAP group, including oral mucositis (grades 3 and 4) (52.6% vs. 17.4%, respectively, P=0.023), limb edema (47.4% vs. 17.4%, respectively, P=0.049), hand-foot syndrome (31.6% vs. 0%, respectively, P=0.005), proteinuria (26.3% vs. 0%, respectively, P=0.014), hypertension (21.1% vs. 0%, respectively, P=0.035), and hypothyroidism (21.1% vs. 0%, respectively, P=0.035). No drug-related deaths occurred. There was no statistically significant difference in the incidence of postoperative complications between the groups (P>0.05). The present study suggests that apatinib + AP may be a promising candidate for neoadjuvant therapy for osteosarcoma, warranting further validation in prospective randomized controlled clinical trials with long-term follow-up.

The ameliorative effects of cannabidiol on methotrexate-induced neuroinflammation and neuronal apoptosis via inhibiting endoplasmic reticulum and mitochondrial stress.

Methotrexate (MTX) is an antineoplastic agent and has neurotoxic effects. It exerts its toxic effect on the brain by triggering inflammation and apoptosis. Cannabidiol (CBD) is an agent known for its antioxidant, anti-inflammatory effects in various tissues. The aim of this study is to examine the protective effects of CBD treatment in various brain structures from MTX damage and to evaluate the effect of intracellular pathways involved in apoptosis. Thirty-two adult Wistar Albino female rats were divided into four groups as control, MTX (20 mg/kg intraperitoneally [i.p.]), MTX + CBD (0.1 mL of 5 mg/kg i.p.), and CBD (for 7 days, i.p.). At the end of the experiment, brain tissues collected for biochemical analyses as total oxidant status (TOS), total antioxidant status, oxidative stress index (OSI), histopathological and immunohistochemical analyses as tumor necrosis factor-α (TNF-α), serotonin, mammalian target of rapamycin (mTOR) staining, genetic analyses as caspase-9 (Cas-9), caspase-12 (Cas-12), C/EBP homologous protein (CHOP), and cytochrome-c (Cyt-c) gene expressions. In the histopathological and immunohistochemical evaluation, hyperemia, microhemorrhage, neuronal loss, and significant decreasing expressions of seratonin were observed in the cortex, hippocampus, and cerebellum regions in the MTX group. mTOR, TNF-α, Cas-9, Cas-12, CHOP, and Cyt-c expressions with TOS and OSI levels were increased in the cortex. It was observed that these findings were reversed after CBD application in all regions. MTX triggers neuronal apoptosis via endoplasmic reticulum and mitochondrial stress while destroying serotonergic neurons. The reversal of the pathological changes with CBD treatment proves that it has anti-inflammatory and antiapoptotic activity in brain.

Post-Transplant Cyclophosphamide, Tacrolimus or Cyclosporine a and Mycophenolate Mofetil Compared to Anti-Thymocyte Globulin tacrolimus or Cyclosporine a and Methotrexate Combinations As Graft- versus -Host Disease Prophylaxis Post Allogeneic Stem Cell Transplantation from Sibling and Unrelated Donors in Patients with Acute Myeloid Leukemia: a Study on Behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow

Background: Recently, the BMT CTN 1703 phase III study compared post-transplant cyclophosphamide with tacrolimus with mycophenolate mofetil (PTCy/TAC/MMF) to TAC /methotrexate (MTX) as graft- versus-host disease (GVHD) prophylaxis post allogeneic transplantation (HSCT), demonstrating a lower incidence of severe acute (a) GVHD and chronic (c) GVHD and better GVHD-free, relapse-free survival (GRFS). The control arm did not include anti-thymocyte globulin (ATG), used in many centers for GVHD prophylaxis. Methods: The study aim was to compare PTCy with TAC or cyclosporine A (CSA) and MMF (PTCy/TAC or CSA &amp; MMF) to ATG combined with TAC or CSA and MTX (ATG/TAC or CSA &amp; MTX) in acute myeloid leukemia (AML) patients (pts) undergoing HSCT from matched siblings (MSD) or 9-10/10 unrelated donor (UD) in first complete remission (CR1). Statistical tests included a multivariate analysis (MVA) adjusting for potential confounding factors using a Cox proportional-hazards regression model for main outcomes. . Results: 6050 pts met the inclusion criteria, 402 received PTCy/TAC or CSA &amp; MMF and 5648 received ATG/TAC or CSA &amp; MTX as GVHD prophylaxis. Median follow-up was 23.4 (IQR, 20.3-24.9) and 41.8 (IQR, 39.6-43.3) months (p&amp;lt;0.0001). The median year of the transplant was 2018 (2010-2020) and 2016 (2007-2020) (p&amp;lt;0.0001). Pts in the PTCy/TAC or CSA &amp; MMF group were younger, with a median age of 48.7 (range 18-5.6) versus 51.5 (8-77.8) years (p=0.024). The diagnosis was de novo AML in 84.1% vs 85.3% and secondary (s) AML in 15.9% vs. 14.7% (p=0.49). The cytogenetic risk was categorized as intermediate (70.9% vs. 67.1%), adverse (22.2% vs. 25.7 %), and favorable (6.9% vs. 7.2%) for pts in the PTCy/TAC or CSA &amp; MMF and ATG/TAC or CSA &amp; MTX groups, respectively (p=0.35) (data missing for 2214 pts). Karnofsky performance status (KPS) did not differ between the groups. There was a higher frequency of pt cytomegalovirus (CMV) seropositivity and female (F) donor to male (M) pt combination in the PTCy/TAC or CSA &amp; MMF group, 77.8% vs. 71.8% (p=0.009) and 18.4% vs. 14.4% (p=0.029). More pts in the PTCy/TAC or CSA &amp; MMF group received reduced intensity conditioning (RIC) 51.5% versus 41.1% in the ATG/TAC or CSA &amp; MTX group, respectively (p&amp;lt;0001). Day 60 neutrophil engraftment (ANC &amp;gt;0.5 x 10 9/L) was 98.7% vs. 98.6% (p=0.84). Day 180 incidence of a GVHD grade II-IV and III-IV was 21.2% vs. 20.4% (p=0.92) and 8.1% vs. 6% (p=0.1), in pts receiving PTCy/TAC or CSA &amp; MMF versus the ATG/TAC or CSA &amp; MTX GVHD prophylaxis, respectively. The 2-year (y) total and extensive chronic (c) GVHD were 33.7% vs. 30% (p=0.09) and 10.7 % vs. 11.2% (p=0.81), respectively. GVHD was the cause of death in 11.6% vs. 13.9% of pts who died. In the MVA, both aGVHD (grade II-IV or III-IV) and cGVHD (total or extensive) did not differ between the groups with hazard ratios (HRs) =1.15 (95% CI 0.86-1.53, p=0.35), HR=0.87 (95% CI 0.56-1.34, p=0.52), HR=0.91 (95% CI 0.7-1.18, p=0. 47 and HR=1.51 (95% CI 0.96-2.36, p=0.074). Two-y NRM was significantly lower in pts that received PTCy/TAC or CSA &amp; MMF versus ATG/TAC or CSA &amp; MTX for GVHD prophylaxis, HR=1.57 (95% CI 1.07-2.3, p=0.022). Other HSCT outcome parameters did not differ between the groups. The HR for 2-y RI was 0.99 (95% CI 0.77-127, p=0. 93). The HRs for 2-y leukemia-free survival (LFS), overall survival (OS), and GRFS were HR=1.15 (95% CI 0.94-1.42, p&amp;lt;0.18), HR=1.18 (95% CI 0.94-1.49, p=0.16) and HR=1.12 (95% CI 0.93-1.36, p=0.22), respectively. Donor type and conditioning regimen were poor prognostic factors for grade II-IV, III-IV aGVHD, and total and extensive cGVHD. For cGVHD, additional poor prognostic factors were F donor to M pt combination and pt CMV seropositivity. Poor prognostic factors for LFS, OS, and GRFS were 9/10 UD, age (by 10 y), sAML, adverse-risk cytogenetics, lower KPS, and pt CMV seropositivity. For NRM, factors were the same apart from cytogenetics risk which was not a prognostic factor. In addition, time from diagnosis to HSCT was a prognostic factor for NRM and RI. Other poor prognostic factors for RI were lower KPS and pt CMV seropositivity. Conclusions: In this registry-based retrospective analysis, comparing PTCy in combination with TAC or CSA and MMF to ATG in combination with TAC or CSA and MTX as GVHD prophylaxis, we observed a similar incidence and severity of both aGVHD and cGVHD. NRM was significantly lower with the PTCy-based GVHD prophylaxis, while all other transplant outcome parameters were similar.

MMF Is Associated with Worse OS in CMV Seropositive AML Patients Undergoing MUD HCT with Calcineurin-Inhibitor-Based Gvhd Prophylaxis

Background: Calcineurin-inhibitor (CNI) with mycophenolate mofetil (MMF) or methotrexate (MTX) is commonly used as graft-versus-host disease (GVHD) prophylaxis for HLA-matched hematopoietic cell transplantation (HCT) but MMF, which targets both T- and B-lymphocytes, is associated with higher risk for cytomegalovirus (CMV) reactivation [ Blood Adv.2023;7(8):1394-1403]. We investigated whether overall survival (OS) differed with MMF- vs MTX-based GVHD prophylaxis according to recipient CMV serostatus and underling HCT disease indication. Methods: We evaluated 3105 patients with acute myeloid leukemia (AML, n=1572), acute lymphoblastic leukemia (ALL, n=453) or myelodysplastic neoplasms (MDS, n=1080) who underwent HLA-matched unrelated donor (MUD) peripheral blood (PB) HCT from 2008-17 using a publicly available Center for International Blood and Marrow Transplant Research (CIBMTR) dataset. Four groups were compared: MTX/ CMV+ (n=1527), MTX/ CMV- (n=916), MMF/ CMV+ (n=395), and MMF/ CMV- (n=267). All patients also received tacrolimus; none received post-transplant cyclophosphamide (PTCy). Primary outcome was OS. Results: The median (interquartile range) age was 57 (45-64), 56 (45-64), 62 (54-68), and 61 (50-67) years in the respective 4 groups. Within each group, most patients had AML or MDS. Disease status was mostly early or intermediate (51-63%). About half had HCT-CI &amp;gt; 2. In vivo T cell depletion was used in 41%, 65%, 48% and 52%, respectively. More patients in the MTX groups received myeloablative conditioning (58% and 55%, respectively), while reduced intensity/non-myeloablative conditioning was more common in the MMF groups (73% and 65%, respectively). In multivariate models stratified by disease, we noted significantly worse OS in CMV+ patients who received MMF, but this effect was restricted to AML. Among AML patients, OS was only 31% (95% confidence interval [CI] 25-37) among the MMF/CMV+ group, but was higher and not significantly different for the MMF/CMV- (54%, 95% CI 45-63) and MTX/CMV+ (51%, 95% CI 48-55) groups compared to the MTX/CMV- group (58%, 95% CI 53-63) [ Figure 1, Table 1] In ALL, GVHD prophylaxis type/CMV serostatus had no significant association with OS. In MDS, CMV+ patients had worse OS (HR 1.2, 95% CI 1.01-1.4, p=0.03) than CMV- patients irrespective of GVHD prophylaxis (MMF/CMV+ vs MTX/CMV+: HR=1.1, 95% CI 0.8-1.4, p=0.5). Donor CMV serostatus had no impact on these associations. Data on CMV reactivation and causes of death were not available; however, our data suggest that the inferior OS in AML was driven mostly by higher non-relapse mortality and some increase in relapse (data not shown). To evaluate how the use of PTCy prophylaxis might impact the interaction between MMF and recipient CMV+ serostatus, we next analyzed a separate CIBMTR MUD PB-HCT cohort that received PTCy/CNI/MMF prophylaxis (N=242; AML, n=136, ALL, n=42; MDS, n=64) and assessed the effect of CMV serostatus on OS. A majority (95%) received HCT from 2015-18. Our results showed that CMV+ recipients had a significantly worse OS than CMV- recipients, but only in AML (HR 2.7, 95% CI 1.1-6.4, p=0.03) and not in ALL (HR 0.4, 95% CI 0.1-1.5, p=0.2), or MDS (HR 0.7, 95% CI 0.3-1.5, p=0.3). As all patients in this cohort received PTCy/CNI/MMF, we were unable to assess the independent impact of MMF and CMV serostatus in this setting. Nevertheless, these data suggest similar trends as seen in the CNI cohort (without PTCy), where the inferior OS in the MMF/CMV+ group was noted only in AML patients. Conclusion: Our data suggest that the use of MMF with CNI-based prophylaxis (without PTCy) should be avoided in AML CMV+ patients. Why this adverse effect of CMV seropositivity and MMF prophylaxis is restricted to AML patients, and not seen in ALL, and whether the same applies to PTCy-prophylaxis with/without MMF, needs further investigation but could be due to CMV-induced expansion of NK cells [ Béziat. Blood. 2013; Foley. Blood. 2012] which mediates alloreactivity against AML but not ALL [ Ruggeri Blood 1999] and is suppressed by MMF more than MTX [ Ohata. BBMT. 2011].

Dimethyl Fumarate Attenuates Methotrexate Hepatotoxicity in Mice Via the Nrf2/HO-1/Anti-Apoptotic Signaling Pathway

Background: Methotrexate (MTX), a folate antagonist used to treat cancer and inflammatory diseases, is known to generate reactive oxygen species. Objectives: The research investigates the impact of dimethyl fumarate (DMF), a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, on an MTX-induced mouse hepatotoxicity model. Methods: Forty-two mice were divided into 6 groups: Control, MTX, DMF 120, and 3 groups of MTX co-treated with DMF 30, 60, and 120 mg/kg. Dimethyl fumarate was gavaged once daily for 10 days. On the fifth day, the animals received MTX 20 mg/kg intraperitoneally. On the eleventh day, the animals were sacrificed, and serum and liver samples were collected to assess the level of oxidative/anti-oxidative and apoptotic/anti-apoptotic markers. Results: Dimethyl fumarate prevented the increase of liver function enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) induced by MTX (P &lt; 0.001). It prevented the increase in AST and ALT levels, indicating liver recovery (P &lt; 0.001). Furthermore, DMF restored antioxidant markers superoxide dismutase, catalase, glutathione peroxidase, and total thiol while reducing the level of thiobarbituric acid reactive substances (P &lt; 0.001). Dimethyl fumarate also downregulated hepatic mRNA expression of caspase 3 and upregulated Bcl-2, heme oxygenase 1, and Nrf2 genes in MTX co-treated DMF groups. Conclusions: Dimethyl fumarate alleviates oxidative stress and apoptosis, which may be achieved by the Nrf2/HO-1 pathway. Therefore, DMF may be clinically effective in preventing or treating MTX-induced hepatotoxicity.

Pinostrobin, a dietary bioflavonoid exerts antioxidant, anti-inflammatory, and anti-apoptotic protective effects against methotrexate-induced ovarian toxicity in rats

This study investigated the protective activities of pinostrobin (PIN) against methotrexate (MTX)-induced ovarian toxicity. Female rats were administered with PIN (50 mg/kg) for 4 weeks, while MTX was administered from weeks 2–4 of PIN treatment. Serum hormonal profiles, ovarian oxidative stress, inflammatory and apoptotic biomarkers as well as ovarian histomorphometry were evaluated. MTX administration elicited profound deficit in serum progesterone and estrogen (E2) levels, while luteinizing hormone (LH) and follicle stimulating hormone (FSH) were significantly increased. Additionally, MTX administration was associated with significant increases in ovarian malondialdehyde, nitric oxide, NF-кB, TNF-α, IL-6, IL-1β, iNOS and caspase-3 activity, as well as notable reduction in the activities of glutathione peroxidase, catalase and superoxide dismutase as well as the level of glutathione. Whereas, treatment with PIN significantly decreased serum levels of FSH and LH, as well as ovarian levels of NO, MDA, caspase 3, NF-κB, IL-1β, IL-6, TNF-α and iNOS. PIN also significantly upregulated GSH, GPx, CAT and SOD in the ovarian tissues as well as increased serum E2 and progesterone levels compared to the MTX group. Furthermore, PIN significantly restored altered ovarian histoarchitecture in the treated group. These findings suggests that PIN exerts protective effects against MTX-triggered ovarian damages.

Gallic Acid Protects Against Methotrexate-Induced Intestinal Mucositis; Oxidative Stress, Histopathology and Inflammatory Status

Background: Gastrointestinal (GI) mucositis is one of the serious side effects of methotrexate (MTX) treatment. It is known that oxidative stress plays an important role in drug-induced side effects. Objectives: The present study aimed to assess the effect of gallic acid (GA) against MTX-induced intestinal mucositis in male Wistar rats. Methods: Twenty-eight adult male Wistar rats were randomly divided into 4 groups (n = 7), including (1) control group; (2) GA group (gallic acid: 30 mg/kg/day, orally); (3) MTX group [20 mg/kg, intra peritoneal (IP)]; and (4) (MTX + GA) group (MTX: 20 mg/kg, IP and gallic acid: 30 mg/kg/day, orally). Then amounts of malondialdehyde (MDA), nitric oxide (NO), glutathione peroxidase (GPx), glutathione (GSH), superoxide dismutase (SOD), interleukin 2 (IL-2) and interleukin 6 (IL-6) were analyzed in serum samples and then the histopathological examinations of the duodenum and jejunum of animals groups. Results: The results showed that treatment with GA significantly reduced the MTX-induced elevation of serum MDA (P &lt; 0.001), NO (P &lt; 0.001), IL-2 (P &lt; 0.001) and IL-6 (P &lt; 0.001) contents and increased MTX-induced reduction in GSH (P &lt; 0.001) content, GPx (P &lt; 0.001) and SOD (P &lt; 0.001) activity. In addition, the histopathological results showed that MTX leads to intestinal tissue damage, and gallic acid can remarkably improve the pathological changes. Conclusions: Our results indicate that gallic acid can mitigate oxidative stress and pro-inflammatory parameters and also moderately prevent histopathological damage of the small intestine of rats exposed to MTX.

Clinical effect of different immunosuppressive treatment regimens in children with ocular myasthenia gravis: a retrospective analysis

To investigate the clinical effect of different immunosuppressive treatment regimens in children with ocular myasthenia gravis (OMG). A retrospective analysis was conducted on 130 children with OMG who were treated in the Department of Neurology, Jiangxi Children's Hospital, from February 2018 to February 2023. According to the treatment regimen, they were divided into four groups: glucocorticoid (GC) group (n=29), mycophenolate mofetil (MMF) group (GC+MMF; n=33), methotrexate (MTX) group (GC+MTX; n=30), and tacrolimus (FK506) group (GC+FK506; n=38). Treatment outcomes and adverse reactions were compared among the groups. After 3 months of treatment, the FK506 group had significantly lower scores of Myasthenia Gravis Quantitative Scale and Myasthenia Gravis-Specific Activities of Daily Living than the other three groups (P<0.05). After 3 months of treatment, the FK506 group had a significantly lower dose of prednisone than the GC group, and after 6 and 9 months of treatment, the MMF, MTX, and FK506 groups had a significantly lower dose of prednisone than the GC group (P<0.05). After 12 months of treatment, the MMF, MTX, and FK506 groups had a significantly lower incidence rate of GC-related adverse reactions than the GC group (P<0.05). For children with OMG, the addition of various immunosuppressants can reduce the dosage of GC and adverse reactions. Among them, FK506 shows superior efficacy compared to other immunosuppressants in the early treatment of OMG.

Effect of P-glycoprotein inhibitor (Carvedilol) on pharmacological and hematological effects of its substrate (methotrexate) on pregnant and lactating rat mothers

Sixty albino rats (40 female rats and 20 males) were allocated into four dosing groups administered orally carvedilol (T1) 0.72mg/kg, methotrexate (T2) 0.36mg/kg, combined doses (T3) carvedilol + methotrexate and control group (Distilled water) for 2 months in male and 2 weeks in female rats before mating and after copulation and approval of pregnancy, dosing continued in female groups during pregnancy and lactation periods. Half of the animal groups were euthanized one day before parturation to determine P-gp concentration in the placenta and liver of pregnant mothers. In contrast, the other half was left for parturition and lactation to study the effects of carvedilol (P-gp inhibitor) on methotrexate (P-gp substrate) when given alone and in combination with some pharmacological, hematological, and biochemical parameters in lactating mothers and pups at the end of lactation. The pharmacological results showed the highest significant antinociceptive responses both in the early and late phase in the T3 (CV+MTX) followed by the T2 (MTX) group. In contrast, the T1 (Cv) animal group showed nearly resembling results as that of the control group in the early and late phases of the formalin test. The written test also recorded the same result as the Formlin test, indicating a significantly higher analgesic effect in T3 followed by T2 in comparison to T1 and control groups. The TNF alpha results supported the anti-inflammatory effect, recording a significant decline mainly in the T3 and T2 groups. The hematological results recorded significantly more reduction in WBC levels in mother and their pups in comparison with T1 and control groups. The serum electrolyte levels (Na+, K+, Ca+2) recorded in all treated groups and pups showed a significant increase in potassium levels in the T1 and T2 mother rat groups. The P-glycoprotein (P-gp) concentration measured in liver and placenta of euthanized pregnant mother before delivery recorded potent inhibition of P-gp in T1 &amp; T3 groups which might altered pharmacokinetic and pharmacodynamic effects of substrate by carvedilol that considered a potent P-gp inhibitor drugs, this might explain our results of increase in pharmacological effect of methotrexate and their side effect on electrolyte concentration and hematology in combined group of CV+ MTX. Keywords: Carvedilol, Methotrexate, p-gp inhibitor, Anti-inflammatory, Hematology, Electrolyte.

Methotrexate to treat hand osteoarthritis with synovitis (METHODS): an Australian, multisite, parallel-group, double-blind, randomised, placebo-controlled trial

Hand osteoarthritis is a disabling condition with few effective therapies. Hand osteoarthritis with synovitis is a common inflammatory phenotype associated with pain. We aimed to examine the efficacy and safety of methotrexate at 6 months in participants with hand osteoarthritis and synovitis. In this multisite, parallel-group, double-blind, randomised, placebo-controlled trial, participants (aged 40-75 years) with hand osteoarthritis (Kellgren and Lawrence grade ≥2 in at least one joint) and MRI-detected synovitis of grade 1 or more were recruited from the community in Melbourne, Hobart, Adelaide, and Perth, Australia. Participants were randomly assigned (1:1) using block randomisation, stratified by study site and self-reported sex, to receive methotrexate 20 mg or identical placebo orally once weekly for 6 months. The primary outcome was pain reduction (measured with a 100 mm visual analogue scale; VAS) in the study hand at 6 months assessed in the intention-to-treat population. Safety outcomes were assessed in all randomly assigned participants. This trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN12617000877381). Between Nov 22, 2017, and Nov 8, 2021, of 202 participants who were assessed for eligibility, 97 (48%) were randomly assigned to receive methotrexate (n=50) or placebo (n=47). 68 (70%) of 97 participants were female and 29 (30%) were male. 42 (84%) of 50 participants in the methotrexate group and 40 (85%) of 47 in the placebo group provided primary outcome data. The mean change in VAS pain at 6 months was -15·2 mm (SD 24·0) in the methotrexate group and -7·7 mm (25·3) in the placebo group, with a mean between-group difference of -9·9 (95% CI -19·3 to -0·6; p=0·037) and an effect size (standardised mean difference) of 0·45 (0·03 to 0·87). Adverse events occurred in 31 (62%) of 50 participants in the methotrexate group and 28 (60%) of 47 participants in the placebo group. Treatment of hand osteoarthritis and synovitis with 20 mg methotrexate for 6 months had a moderate but potentially clinically meaningful effect on reducing pain, providing proof of concept that methotrexate might have a role in the management of hand osteoarthritis with an inflammatory phenotype. National Health and Medical Research Council of Australia.

Vitamin D: An effective therapy against methotrexateinduced cardiotoxicity

Purpose: To determine the potential cardioprotective effect of vitamin D (VD) against methotrexate (MTX) induced cardiotoxicity.Methods: A total of thirty-five (35) rats were randomly assigned to five equal groups. Control groupreceived no treatment; MTX group received intraperitoneal (IP) injection of MTX in a single dose of 20 mg/kg on day 8; VD group received 200 IU/kg VD daily dissolved in sunflower oil orally; sunflower oil (SO) group received 1 mL/kg/day SO orally; MTX + VD group received a single dose of MTX (20 mg/kg, IP) on day 8 and VD (200 IU/kg, orally) for 21 days. Myocardial tissue samples were harvested and used for clinical chemistry, histopathological, and ultrastructural evaluation.Results: Histopathological damage in MTX group was more severe than in control group under both light and electron microscopy. Expression of transient receptor potential melastatin 2 (TRPM2) and caspase-3 markers was significantly higher in MTX group (p &lt; 0.05). Glutathione peroxidase (GSH-Px) enzyme activity in cardiac tissue was lower in MTX group, whereas malondialdehyde (MDA) levels increased significantly (p &lt; 0.05). In MTX + VD group, VD treatment alleviated histopathological damage and significantly lowered TRPM2 and caspase-3 expressions (p &lt; 0.05). Vitamin D also reduced tissue MDA levels, and increased GSH-Px activity albeit non-significantly (p &gt; 0.05),Conclusion: These findings suggest that VD exerts an ameliorative effect on MTX-induced cardiotoxicity in rats. Therefore, TRPM2 channel affords a novel therapeutic approach for treatment of diseases related to chemotherapy-induced oxidative stress.

Exosomes derived from mesenchymal stem cells primed with disease-condition-serum improved therapeutic efficacy in a mouse rheumatoid arthritis model via enhanced TGF-β1 production

BackgroundsRheumatoid arthritis (RA) is a chronic and systemic autoimmune disease characterized by synovial inflammation-mediated progressive destruction of the cartilage and bone, resulting in reduced quality of life. We primed human telomerase reverse transcriptase-overexpressing immortalized human adipose tissue-derived mesenchymal stem cells (iMSCs) with serum derived from a non-human primate RA model and studied the immunomodulatory ability of exosomes obtained from primed iMSCs.MethodsAfter immunophenotyping, nanoparticle tracking analysis, and in vitro functional tests, Dulbecco’s phosphate-buffered saline (dPBS, Group C), exosomes derived from the supernatant of iMSCs (Exo-FBS, Group E), exosomes derived from the supernatant of iMSCs primed with RA serum (Exo-RA, Group F), and methotrexate (Group M) were administered in collagen-induced arthritis (CIA) model mice. dPBS was administered to the normal (N) group for comparison (n = 10/group).ResultsExo-RA had a significantly higher number of exosomes compared to Exo-FBS when measured with nanoparticle tracking analysis or exosome marker CD81, and Transforming growth factor-β1 amounts were significantly higher in Exo-RA than in Exo-FBS. When Exo-FBS or Exo-RA was administered to the collagen-induced arthritis model, serum interleukin (IL)-4 and the proportion of Th2 (CD4+CD25+GATA3+) and M2 (CD11c − CD206+ of CD45+CD64+) cells were significantly increased compared to the control group. Furthermore, Exo-RA could alleviate cartilage damage by significantly lowering the concentrations of proinflammatory cytokines such as tumor necrosis factor-α, keratinocyte chemoattractant, and IL-12p70.ConclusionExosomes derived from disease-condition-serum-primed iMSCs ameliorated cartilage damage in a RA model by enhancing TGF-β1 production, inducing Th2 and M2 polarization and lowering proinflammatory cytokines, TNF-α, KC, and IL-12p70 in the host. Patient-derived serum can be used as an iMSC priming strategy in iMSC-derived exosome treatment of RA.

Expectant management of tubal pregnancies with human chorionic gonadotropin up to 2000 mIU/mL.

To describe outcomes of expectant management (EM) versus methotrexate (MTX) treatment in tubal pregnancies with pretreatment human chorionic gonadotropin (hCG) less than 2000 mIU/mL. This retrospective cohort from two tertiary hospitals included women with confirmed tubal pregnancies and pretreatment hCG <2000 mIU/mL. Exclusion criteria were unrecorded pregnancy site, unconfirmed diagnosis, and surgical treatment upon diagnosis. The primary outcome was eventual rate of surgical treatment. Between December 2009 and June 2021, 545 of 2114 (25.8%) women diagnosed with a tubal pregnancy met our inclusion criteria. We compared women who underwent EM (N = 201) with women who received MTX (N = 344). All women in the EM group had a declining trend of hCG. The MTX group had higher pretreatment hCG and higher rates of yolk sac or embryo presence on ultrasound. Eventual surgical treatment rate was higher in the MTX group compared with the EM group (39 [11.3%] vs. 9 [4.5%], P = 0.006), with no difference in the treatment failure rate or tubal rupture rate. In a subgroup analysis of women with pretreatment hCG between 1000 and 2000 mIU/mL, eventual surgical treatment, treatment failure, and tubal rupture rates did not differ between groups. Logistic regression analysis revealed that eventual surgical treatment was independently associated with hCG levels less than 1000 mIU/mL (adjusted odds ratio [aOR] 0.28, 95% confidence interval [CI] 0.14-0.56) and endometriosis (aOR 9.20, 95% CI 3.55-23.81). Expectant management of tubal pregnancies with pretreatment hCG levels less than 2000 mIU/mL and even between 1000 and 2000 mIU/mL and with a declining trend of hCG demonstrated lower or comparable rates of eventual surgical treatment, when compared with MTX treatment.

Higher Odds of Adverse Cutaneous Reactions in Patients With Dermatomyositis Treated With Hydroxychloroquine Compared With Methotrexate.

Hydroxychloroquine (HCQ) use for the treatment of dermatomyositis (DM) has been associated with adverse cutaneous reactions. We applied a new user, active comparator, retrospective design to assess differences in adverse cutaneous reactions or hospitalizations between HCQ and methotrexate (MTX) use among patients with DM. We used a national network of data from insurance registries (TriNetX), enrolling patients with two International Classification of Diseases (ICD) codes for DM separated by 6 months or more who had a prescription for either (but not both) HCQ or MTX on or after DM diagnosis. Outcomes were adverse cutaneous reactions (ICD codes) or hospital admission (Current Procedural Terminology (CPT) codes) within 4 months from the prescription dispense date. Logistic regression was used to produce adjusted odds ratios (aORs) and 95% confidence intervals (CIs) comparing outcomes in the HCQ group (n=1364) and the MTX group (n=1400), adjusted for age at first DM diagnosis, year of birth, sex, and time from DM diagnosis to first prescription. Overall, we found no significant difference in odds of hospitalization in those taking HCQ (aOR 1.05; 95% CI: 0.79-1.39) compared with those on MTX. Patients with DM on HCQ had 30% higher odds of adverse cutaneous reaction diagnosis compared with patients on MTX (aOR 1.30; 95% CI: 1.02-1.59). Age at DM diagnosis was an effect modifier of this association, with higher odds of adverse cutaneous reaction among patients taking HCQ who were younger at diagnosis. Compared with MTX use, HCQ use, especially in younger patients, may result in higher odds of adverse cutaneous reactions.

Prevention of Radiographic Progression in Higher-Risk Patients with Rheumatoid Arthritis Using Filgotinib in PhaseIII Studies: Narrative Review of PostHoc Analyses.

Filgotinib is an oral preferential Janus kinase1 inhibitor that demonstrated significant reductions in radiographic progression, with an acceptable tolerability and safety profile, vs placebo in patients with rheumatoid arthritis (RA) and an inadequate response to methotrexate (MTX-IR; FINCH1) and vs MTX in MTX-naïve patients with RA (FINCH3). International treatment guidelines identify multiple poor prognostic factors (PPFs) associated with worse disease outcomes among patients with RA. However, questions remain both about the clinical utility of considering PPFs and about which PPFs should drive treatment decisions. Additionally, the role of radiographic findings in clinical practice continues to be discussed and to evolve. This review examines radiographic results from posthoc analyses of phase3 trials of filgotinib that examined subgroups with 4PPFs or with baseline estimated rapid radiographic progression (e-RRP). In MTX groups, there were trends toward greater progression among patients with 4PPFs or e-RRP, suggesting these subgroups may comprise a higher-risk population. Results show general consistency for the efficacy of filgotinib 200mg plus MTX vs placebo plus MTX/MTX monotherapy on radiographic assessments, including change from baseline in modified total Sharp score and proportions without radiographic progression, even among MTX-IR or MTX-naïve patients with 4PPFs or e-RRP who may be at higher risk of bone damage. Multivariate analysis identified multiple factors associated with baseline e-RRP status. This summary of the current understanding of benefits associated with filgotinib on radiographic progression and the relevance of baseline factors to these benefits may help inform treatment decisions for patients facing high risk of radiographic progression.

Ovarian reserve in children with juvenile idiopathic arthritis using biologic disease-modifying anti-rheumatic drugs.

The aim of the study is to assess the effect of juvenile idiopathic arthritis (JIA) and biologic disease-modifying anti-rheumatic drugs (bDMARDs) on ovarian reserve in children. A cross-sectional study was performed from March 2021 to March 2022 and included 81 patients with JIA and 49 healthy children. Serum anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol levels were analyzed using electrochemiluminescence methods. The mean of current age (13.5 ± 3.2 vs. 14.4 ± 2.4years), height standard deviation score (SDS) (- 0.35 ± 1.18 vs. - 0.44 ± 0.94), body mass index SDS (0.12 ± 1.33 vs. 0.25 ± 1.28), and the median weight SDS (- 0.13 (- 2.27-3.23) vs. - 0.52 (- 3.4-3.3)) were similar in JIA patients and controls (p > 0.05). Patients with JIA were divided into two groups according to their treatment regimens: treated with methotrexate (MTX) (biologic naive) (n = 32) and treated with MTX plus bDMARDs (n = 49). No significant differences were detected between the 3 groups regarding menarche age, menstrual cycle length, and flow duration (for all p > 0.05). The median serum concentration of AMH was 2.94 (1.12-7.88) ng/ml in the control group, 3.02 (0.36-8.54) ng/ml in the biologic naïve group, and 3.01 (0.99-8.26) ng/ml in the MTX plus bDMARD group. There were no significant differences between 3 groups according to serum AMH, FSH, LH, and estradiol levels (p > 0.05). Biologic DMARDs are reassuring in terms of ovarian reserve in girls with JIA and demonstrate that AMH is unaffected by treatment. Prospective studies with larger sample sizes are needed to confirm our findings and to evaluate the impact on the future fertility of patients. Key Points • Although biologic disease-modifying anti-rheumatic drugs (bDMARDs) are being game-changing treatment options in juvenile idiopathic arthritis, their effect on fertility and ovarian reserve is one of the most discussed issues. • In addition to treatment used, autoimmune diseases might also have a negative effect on fertility. • In this cross-sectional study, we found that anti-Mullerian hormone level of patients who were on bDMARDs, patients who were on methotrexate, and healthy controls were similar. • Our results suggest that bDMARDs are reassuring in terms of ovarian reserve in girls with JIA and demonstrate that AMH is unaffected by treatment.