Interaction of nobiletin with methotrexate ameliorates 7-OH methotrexate-induced nephrotoxicity through endoplasmic reticulum stress-dependent PERK/CHOP signaling pathway.

Drug-induced nephrotoxicity is a frequent adverse event that contributes to acute kidney injury with tubular and/or glomerular lesions. Methotrexate (MTX) is a folate analog used against a myriad of malignancies and autoimmune diseases. Unfortunately, ambiguous renal toxicology limits its safe clinical usage. Based on our previous studies, 7-OH MTX as an overlooked oxidative metabolite of MTX was proposed to be the main culprit responsible for nephrotoxicity, while nobiletin, a naturally occurring polymethoxylated flavonoid screened from our prepared total phenolic extracts of Citrus aurantium L. (TPE-CA), was employed as a therapeutic agent for drug-drug interactions. According to the present study, nobiletin can ameliorate the renal accumulation of 7-OH MTX through the interaction with aldehyde oxidase. RNA-seq analysis revealed that 7-OH MTX was mainly related to protein processing in endoplasmic reticulum (ER) stress, with the PERK/CHOP pathway selected as the most significant for metabolic nephrotoxicity. Meanwhile, the cross-linked proteins and conducted signals were investigated by western blotting and further verified by GSK inhibition analyses. These results indicated that nobiletin protected renal function from MTX-induced nephrotoxicity by modulating metabolism and ameliorated the metabolic toxicity of 7-OH MTX on ER stress-induced PERK/CHOP conduction by maintaining Ca2+ homeostasis and reducing the production of reactive oxygen species.

Methotrexate and its therapeutic antagonists caffeine and theophylline, target a motogenic T-cell mechanism driven by thrombospondin-1 (TSP-1).

Methotrexate (MTX) is a widely used treatment for inflammatory diseases such as rheumatoid arthritis and psoriasis, based on the concept that it is immunosuppressive. Its mechanism of action, however, remains unclear, although it is thought to depend on adenosine. Caffeine and theophylline, which have several targets including adenosine receptors, have been shown to suppress the beneficial clinical effects of MTX. Here we show that MTX and caffeine and theophylline differentially affect a motogenic T-cell mechanism driven by endogenous thrombospondin-1 (TSP-1) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1). MTX stimulated TSP-1 expression and the motogenic TSP-1/TSP-1 receptor mechanism in primary human T cells, hence mimicking IL-2 and CXCL12, which similar to MTX, dampen inflammatory disease. SiRNA-mediated gene silencing of TSP-1 and LRP1 inhibited this stimulatory effect. Caffeine and theophylline inhibited the TSP-1/TSP-1 receptor mechanism by inhibiting LRP1 expression. These results indicate that the effect of MTX on T cells is immunoregulatory rather than immunosuppressive, and suggest a pathway dependent on TSP-1/TSP-1 receptor interactions for the regulation of immune responses.

Does daily folic acid supplementation reduce methotrexate efficacy?

Methotrexate is a mainstay treatment for autoimmune and inflammatory conditions in the field of Dermatology. However, in some patients, its use is associated with significant side effects and toxicity. Folate supplementation with either folic acid or folinic acid often mitigates side effects and reduces the incidence of systemic toxicity related to methotrexate. Although the value of methotrexate is clear, debate remains about folate supplementation. There is little agreement about the proper dosing or frequency of folate supplementation as many believe that daily folate supplementation can reduce methotrexate efficacy. Although daily use of folic acid does not appear to affect methotrexate efficacy, dosing of folinic acid close to methotrexate administration may hinder methotrexate efficacy. Therefore, folic acid should be used daily with methotrexate to ameliorate side effects, whereas folinic acid should only be used for methotrexate toxicity.

Curcumin and folic acid abrogated methotrexate induced vascular endothelial dysfunction.

Methotrexate, an antifolate drug widely used in rheumatoid arthritis, psoriasis, and cancer, is known to cause vascular endothelial dysfunction by causing hyperhomocysteinemia, direct injury to endothelium or by increasing the oxidative stress (raising levels of 7,8-dihydrobiopterin). Curcumin is a naturally occurring polyphenol with strong antioxidant and anti-inflammatory action and therapeutic spectra similar to that of methotrexate. This study was performed to evaluate the effects of curcumin on methotrexate induced vascular endothelial dysfunction and also compare its effect with that produced by folic acid (0.072 µg·g(-1)·day(-1), p.o., 2 weeks) per se and in combination. Male Wistar rats were exposed to methotrexate (0.35 mg·kg(-1)·day(-1), i.p.) for 2 weeks to induce endothelial dysfunction. Methotrexate exposure led to shedding of endothelium, decreased vascular reactivity, increased oxidative stress, decreased serum nitrite levels, and increase in aortic collagen deposition. Curcumin (200 mg·kg(-1)·day(-1) and 400 mg·kg(-1)·day(-1), p.o.) for 4 weeks prevented the increase in oxidative stress, decrease in serum nitrite, aortic collagen deposition, and also vascular reactivity. The effects were comparable with those produced by folic acid therapy. The study shows that curcumin, when concomitantly administered with methotrexate, abrogated its vascular side effects by preventing an increase in oxidative stress and abating any reduction in physiological nitric oxide levels.

Comparable efficacy with varying dosages of glucarpidase in pediatric oncology patients.

BACKGROUND: Glucarpidase rapidly reduces methotrexate plasma concentrations in patients experiencing methotrexate-induced renal dysfunction. Debate exists regarding the role of glucarpidase in therapy given its high cost. The use of reduced-dose glucarpidase has been reported, and may allow more institutions to supply this drug to their patients. This report explores the relationship between glucarpidase dosage and patient outcomes in pediatric oncology patients. METHODS: The authors evaluated data from 26 patients who received glucarpidase after high-dose methotrexate. Decrease in plasma methotrexate concentrations and time to renal recovery were evaluated for an association with glucarpidase dosage, which ranged from 13 to 90 units/kg. RESULTS: No significant relationship was found between glucarpidase dosage (units/kg) and percent decrease in methotrexate plasma concentrations measured by TDx (P > 0.1) or HPLC (P > 0.5). Patients who received glucarpidase dosages <50 units/kg had a median percent reduction in methotrexate plasma concentration of 99.4% (range, 98-100) measured by HPLC compared to a median percent reduction of 99.4% (range, 77.2-100) in patients who received ≥50 units/kg. Time to SCr recovery was not related to glucarpidase dosage (P > 0.8). CONCLUSIONS: The efficacy of glucarpidase in the treatment of HDMTX-induced kidney injury was not dosage-dependent in this retrospective analysis of pediatric oncology patients.

Water channel in the binding site of a high affinity anti-methotrexate antibody.

In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 107 M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻5 s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

Molecular Docking Studies of Naringenin and its Protective Efficacy against Methotrexate Induced Oxidative Tissue Injury.

BACKGROUND: Although Methotrexate (MTX) possesses a wide clinical spectrum of activity, its toxic side effects on normal cells and drug resistance often hamper its successful outcome. Naringenin (NG) is one of the promising bioactive flavonoids that are extensively found in grapes, citrus fruits, and fruit arils of Pithecellobium dulce. OBJECTIVE: Only a few experimental in vivo studies on the efficacy of NG against chemotherapeutic drugs have been carried out. Aiming to fill this gap, the present study was carried out to characterize and identify its possible therapeutic targets and also to explore its protective efficacy against MTX-induced tissue damage. METHODS: Oxidative stress was induced in mice with MTX (20 mg/kg B.wt), and animals were orally administered with 10 mg/kg B.wt NG for 10 consecutive days. On day 11, all animals were sacrificed, and hematological and serum biochemical parameters were analyzed. The anti-oxidant efficacy of NG against MTX was evaluated by quantifying tissue superoxide dismutase (SOD), glutatione peroxidase (GPx), reduced glutathione (GSH) and catalase along with oxidative stress markers [malondialdehyde (MDA) and nitric oxide (NO)]. Further, the histopathological analysis was performed to confirm the protective efficacy of FPD. In silico docking studies were also performed to exploring anti-oxidant enzyme-based targets. RESULTS: Our results showed that concurrent administration of NG counteracted oxidative stress induced by MTX, as evidenced by increased expression of anti-oxidant markers, decreased expression of renal and hepatotoxicity serum marker enzymes (p <0.05). A molecular docking study was performed using Auto dock vina to understand the mechanism of ligand binding (S-NG and R-NG)with anti-oxidant enzymes. The binding affinity of S-NG with catalase, GPx, ALP, and SGPT was -10.1, -7.1, -7.1, and -7.3 kcal/mol, respectively, whereas for R-NG was -10.8, -7.1, -7.6, and -7.4 kcal/mol, respectively. Further, histopathological analysis affirmed the protective efficacy of NG against MTX-induced hepatic and renal toxicities. CONCLUSION: Treatment with NG significantly reduced MTX-induced pancytopenia, renal, and hepatic toxicity.

The antiepileptic drugs phenobarbital and carbamazepine reduce transport of methotrexate in rat choroid plexus by down-regulation of the reduced folate carrier.

Intrathecal methotrexate (MTX) has been associated with severe neurotoxicity. Because carrier-associated removal of MTX from the cerebrospinal fluid (CSF) into blood remains undefined, we determined the expression and function of MTX transporters in rat choroid plexus (CP). MTX neurotoxicity usually manifests as seizures requiring therapy with antiepileptic drugs (AEDs) such as phenobarbital (PB). Because we have demonstrated that PB reduces activity of MTX influx carrier reduced folate carrier (Rfc1) in liver, we investigated the influence of the AEDs PB, carbamazepine (CBZ), or gabapentin on Rfc1-mediated MTX transport in CP. Reverse transcriptase-polymerase chain reaction and Western blot analysis showed similar expression of the MTX influx carrier Rfc1 and organic anion transporter 3 or efflux transporter multidrug resistance-associated protein 1 (Mrp1) and breast cancer resistance protein (Bcrp) in rat CP tissue and choroidal epithelial Z310 cells. Confocal microscopy revealed subcellular localization of Rfc1 and Bcrp at the apical and of Mrp1 at the basolateral CP membrane. Uptake, efflux, and inhibition studies indicated MTX transport activity of Rfc1, Mrp1, and Bcrp. PB and CBZ but not gabapentin significantly inhibited Rfc1-mediated uptake of MTX in CP cells. Studies on the regulatory mechanism showed that PB significantly inhibited Rfc1 translation but did not alter carrier gene expression. Altogether, removal of intrathecal MTX across the blood-CSF barrier may be achieved through Rfc1-mediated uptake from the CSF followed by MTX extrusion into blood, particularly via Mrp1. Antiepileptic treatment with PB or CBZ causes post-transcriptional down-regulation of Rfc1 activity in CP. This mechanism may result in enhanced MTX toxicity in patients with cancer who are receiving intrathecal MTX chemotherapy by reduced CSF clearance of the drug.

Adenosine abolishes MTX-induced suppression of osteoclastogenesis and inflammatory bone destruction in adjuvant-induced arthritis.

Methotrexate (MTX) is widely utilized for the treatment of patients with rheumatoid arthritis (RA); however, recent observation of the MTX-resistant patients proposed some difficulty in MTX-dependent therapeutic approach for RA. To access cellular events related to MTX resistance in RA in respect to inflammatory bone destruction, we investigated on an involvement of the potent inflammatory mediator adenosine in the regulation of osteoclastogenesis and inflammatory bone destruction. In rats with adjuvant-induced arthritis (AA rats), MTX efficiently suppressed bone destruction when it was administrated within 3 days after adjuvant injection, while it could not suppress inflammatory bone destruction if MTX was injected at the time of onset of inflammation (at day 10 after adjuvant injection). Time-course change in the level of plasma adenosine of AA rats was estimated by use of high-performance liquid chromatography and elucidated that adenosine level was markedly elevated till 10 days after adjuvant injection. In vitro bone marrow culture system for evaluating osteoclastogenesis, MTX markedly suppressed osteoclastogenesis in a stromal cell-dependent manner. This MTX-induced suppression of osteoclastogenesis was abrogated by the addition of adenosine. MTX suppressed the expression of mRNA for the receptor activator NF-κB ligand (RANKL), but it did not suppress the expression of osteoprotegerin (OPG). The addition of MTX and adenosine together markedly suppressed the level of OPG expression. Abolishment of MTX action by adenosine was significantly blocked by MRS1754, a highly selective antagonist for the A(2b) adenosine receptor (A(2b)AR), but not by caffeine, an antagonist for A1, A(2a), A3 AR (A1AR, A(2a)AR, and A3AR), which suggests that adenosine acts through A(2b)AR. Immunohistochemical studies showed abundant expression of A(2b)AR in cells localized in the bone-bone marrow boundary of the distal tibia in AA rats but not in control rats. When adenosine was injected in the ankle joints of MTX-treated AA rats, the suppressive effects of MTX on bone destruction was abolished. The current data therefore suggest that upregulation of adenosine production abolished the suppressive effect of MTX on osteoclastic bone destruction. Involvement of the adenosine-A(2b)AR system may explain MTX resistance in RA.

Efficacy of vitamin E in protection against methotrexate induced placental injury in albino rats.

Methotrexate (MXT) is a chemotherapeutic drug that has been used in a wide range of clinical practices. Unfortunately, the administration of MXT during pregnancy may induce abortion, fetal deformities, and intrauterine growth retardation. Vitamin E is an antioxidant agent that can ameliorate free radical damage. The current work aimed to shed more light on the possible protective effect of vitamin E against MXT induced placental toxicity and to determine the possible mechanisms; biochemically, histologically, and immunohistochemically. Four groups were used: control pregnant, Vitamin E (VIT E) pregnant, Methotrexate (MXT) pregnant, and Vitamin E Methotrexate (VIT E-MXT) pregnant. The placental tissues were processed for light, immunohistochemical, and electron microscopic study. Other samples were obtained for biochemical study; the placental oxidant/antioxidant status was evaluated. The results showed that MXT caused various placental morphological changes in the form of distorted chorionic projection with an accumulation of hemosiderin granules in the trophoblastic cells. Maternal blood vessels showed a homogenous acidophilic material Edema of the extra-embryonic fetal membranes was noticed. A significant decreased in placental weight as well as increase in the oxidative and inflammatory markers were detected. Increased COX2 and decreased eNOS expressions were observed in the MXT group if compared to the control group. VIT E significantly restored the normal histological and immunohistochemical appearance, placental weight, and oxidant/antioxidant balance. It could be concluded the biochemical, morphological, and morphometric findings suggested that vitamin E coadministration is promising in attenuating the placental toxic effect of methotrexate. In this study, VIT E decreased the inflammatory and oxidative stress effect of methotrexate on the placental tissue by enhancing the level of eNOS.

Melatonin rescued methotrexate-induced spatial deficit and hyperhomocysteinemia and increased asymmetric dimethylarginine in plasma and dorsal hippocampus in developing rats.

AIMS: With the improvement of the survival rates in children acute lymphoblastic leukemia (ALL), some children ALL survivors show impaired cognitive function. Methotrexate (MTX), an essential component in ALL treatment, has been reported to be related to neurologic sequelae and to increased oxidative stress through its interactions with enzymes in the folate pathway. Asymmetric dimethylarginine (ADMA) is the main endogenous inhibitor of nitric oxide synthase, and increased ADMA may result from increased oxidants. Melatonin is an antioxidant; however, its role in MTX neuropathy is not well studied. We developed a rat model mimicking child ALL treatment to explore peripheral and central homocysteine and ADMA regulation after MTX and found potential treatment choice. MAIN METHODS: Preweaning male Sprague-Dawley rats were used in this study. Experiment 1 evaluated spatial performance in rats with intrathecal (IT) MTX, intraperitoneal (IP) MTX, or combined IT and IP MTX, protocols mimicking ALL treatment in children. Experiment 2 focused on rats with combined IT and IP MTX, evaluating spatial performance and plasma and dorsal hippocampal homocysteine and ADMA levels, their regulation, and the protective effect of melatonin. KEY FINDINGS: Combined IT and IP MTX treatment caused in spatial deficits in developing rats, and melatonin restored the spatial performance. Alterations in peripheral and central homocysteine and ADMA concentrations and their regulation were found and could be alleviated by melatonin treatment. SIGNIFICANCES: Combined IP and IT MTX treatment caused spatial deficits in developing rats. Melatonin could restore spatial performance through alleviating the effects on the imbalance of oxidative stress.

Alleviation of Liver Dysfunction, Oxidative Stress and Inflammation Underlies the Protective Effect of Ferulic Acid in Methotrexate-Induced Hepatotoxicity.

INTRODUCTION: In multiple studies, involvement of oxidative stress in the pathogenesis of methotrexate (MTX)-mediated liver damage has been confirmed. Use of many drugs has been examined experimentally in order to prevent or diminish oxidative stress. However, no study has yet examined the effects of ferulic acid (FA) on MTX-induced liver damage. This study aimed at evaluating the effects of FA on protection against liver damage induced by MTX in mice. MATERIALS AND METHODS: In this the mice were divided into five groups in a random manner: I) control; II) MTX (20 mg/kg); III and IV) FA (50 and 100 mg/kg) + MTX; and V) FA (100 mg/kg), and we measured serum factors, oxidative stress and inflammatory factors. RESULTS: In the MTX group, accumulation of inflammatory cells, accumulation of red blood cell (RBC), and nuclear pyknosis (NP) were detected in the liver. In line with the histological data, the levels of nitric oxide (NO), malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-α increased (TNF-α), whereas the reduced glutathione (GSH), catalase (CAT), total antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) content reduced in the MTX group. However, FA ameliorated these hazardous effects in the antioxidant and anti-inflammatory systems in MTX-treated groups. CONCLUSION: Based on our findings, oxidative stress impairment and MTX-induced liver damage were ameliorated following FA pretreatment at both histological and biochemical levels. Therefore, FA can be effectively used in abrogation of MTX-induced toxicity.

Investigation on inhibitory effect of folic acid on methotrexate-induced epithelial-mesenchymal transition focusing on dihydrofolate reductase.

Use of methotrexate (MTX) can induce serious adverse lung reactions, such as pulmonary fibrosis. Recently, we demonstrated that the epithelial-mesenchymal transition (EMT), which triggers pulmonary fibrosis, was induced by MTX, and folic acid (FA) suppressed MTX-induced EMT in A549 cells. In this study, the role of dihydrofolate reductase (DHFR), a target of MTX, in FA-mediated inhibition of MTX-induced EMT was evaluated. The inhibitory effects of FA and tetrahydrofolate (THF), a metabolite of FA produced by DHFR, on MTX-induced increases in mRNA expression of α-SMA, an EMT marker, were compared. The IC50 values of FA and THF for DHFR were 103.3 and 19.4 µM, respectively. In contrast, DHFR knockdown did not alter the mRNA expression of α-SMA. Notably, the inhibitory effect of FA, but not THF, on MTX-induced EMT was blunted in DHFR knockdown cells. These results suggested that DHFR may not directly contribute to MTX-induced EMT, but may contribute to suppression of MTX-induced EMT via production of THF in A549 cells.

Efficacy of curcumin on prevention of drug-induced nephrotoxicity: A review of animal studies.

Drug-induced nephrotoxicity is a frequent serious adverse effect, contributing to morbidity and increased healthcare utilization. Prevention or reversal is key. Curcumin has useful biological features that include antioxidant, anti-inflammatory, and anticancer properties. This review covers aspects of curcumin in relation to prevention of drug-induced nephrotoxicity: dosage and schedule, effect on kidney biomarkers and histological changes, and mechanisms of curcumin's protective effects. Despite success in some animal models, human studies and clinical administration of curcumin for nephroprotection remains limited due to difficulty in achieving therapeutic levels following oral administration and in determining the optimal dosing schedule. Lack of sufficient evidence from animal studies, coupled with low systemic bioavailability, continues to limit the utilization of curcumin in addressing and controlling drug-induced nephrotoxicity. Therefore, human studies are required to fully assess and validate the therapeutic potential of curcumin.

Spirulina ameliorates methotrexate hepatotoxicity via antioxidant, immune stimulation, and proinflammatory cytokines and apoptotic proteins modulation.

AIMS: Methotrexate (MTX) is an efficient cytotoxic drug used against various carcinogenic, inflammatory and autoimmune diseases; however, the hepatotoxicity of MTX limits its use. Therefore, the present study aimed to evaluate the potential hepatoprotective and immune-stimulant effect of Spirulina platensis (SP) against MTX acute toxicity. MAIN METHODS: Thirty-two male Wistar rats were randomly allocated into the following four groups (n = 8): control, SP (500 mg/kg bwt, oral gavage daily for 21 days), MTX (20 mg/kg bwt, single ip injection), and MTX+SP. Hepatic and splenic histoarchitecture, leukocyte counts and serum immunoglobulins were evaluated. Hepatic oxidant/antioxidant status, proinflammatory cytokines (tumor necrosis factor-α, and interleukin 6), and pro-apoptotic proteins (caspase 3 and Bax) immunoexpression were assessed. KEY FINDINGS: MTX induced extensive hepatic necrosis and vacuolation, and sever lymphoid depletion in splenic white pulp with increased levels of serum transaminases, lactate dehydrogenase, and hepatic malondialdehyde, tumor necrosis factor-α and interleukin 6; and number of caspase 3- and Bax-positive hepatocytes. A significant decrease in leukocyte counts, serum immunoglobulins (IgA, IgM and IgG) level, and hepatic antioxidant enzymes (GSH, GPx, SOD, and CAT) was also detected. Pretreatment with SP resulted in significant improvements in hepatic and splenic histologic architecture, as well as restoring liver enzymes and reduction of lipid peroxidation product, proinflammatory cytokines, and caspase 3 and Bax immunoexpression. Additionally, a significant increase in antioxidant enzymes, serum immunoglobulins, and total leukocyte counts was demonstrated. SIGNIFICANCE: SP possesses promising antioxidant, anti-inflammatory, anti-apoptotic and immune stimulatory properties against MTX-induced hepatotoxicity and immunosuppression.