|Year : 2018 | Volume
| Issue : 2 | Page : 85-92
Evaluation of the effects of TAK-242 and GIT-27 on methotrexate-induced liver injury
Bassim I Mohammad1, Bassim S Ahmed2, Alaa Fadhel Hassan3
1 Department of Pharmacology, University of Al-Qadisiyah, College of Pharmacy, Al Diwaniyah, Iraq
2 Department of Pathology and Forensic Medicine, Al-Mustansiriyah University, College of Medicine, Baghdad, Iraq
3 Department of Pharmacy, Al-Mahmoudiya Hospital, Baghdad, Iraq
|Date of Web Publication||26-Nov-2018|
Alaa Fadhel Hassan
Iraqi Ministry of Health, Al-Mahmoudiya General Hospital, Deptartment of Pharmacy, Baghdad
Source of Support: None, Conflict of Interest: None
Background: Methotrexate (MTX)-induced liver injury is a common problem that is described as increased level of hepatic biomarkers that is seen in 14%–25% of patients with inflammatory bowel disease and 49% of patients with rheumatoid arthritis or as idiosyncratic induced liver injury that is seen in 1% of patients with inflammatory bowel disease, or as fibrosis and cirrhosis in 17% of rheumatoid arthritis patients and 25% of psoriatic patients. This profile may rarely progress to acute liver failure. Aim: The aim is to study the effect of TAK-242 and GIT-27 on MTX-induced liver injury. Materials and Methods: Thirty-five Albino-Wistar rats were divided into five groups: the first group was maintained on distilled water, the second group was administered intraperitoneal (I.P.) dimethyl sulfoxide followed by oral MTX, the third group was administered oral MTX, the fourth group was administered I.P. TAK-242 followed by oral MTX, and the fifth group was administered I.P. GIT-27 followed by oral MTX. Results: The significant increase in markers of hepatic function, inflammatory and oxidative stress markers, as well as severe liver histopathologic change “steatosis” induced by methotrexate were alleviated in the animals pretreated with the drugs TAK-242 and GIT-27. With significant improvement in serum level of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bilirubin, interleukin-6, tumor necrosis factor-α, malondialdehyde and reduced glutathione; beside an improved histopathologic profile of moderate steatosis. Conclusion: This study suggests that both TAK-242 and GIT-27 protect against liver injury induced by MTX depending on their antagonism of the inflammatory Toll-like receptors 4 and 2/6.
Keywords: Drug-induced liver injury, GIT-27, Methotrexate, TAK-242, Toll-like receptors
|How to cite this article:|
Mohammad BI, Ahmed BS, Hassan AF. Evaluation of the effects of TAK-242 and GIT-27 on methotrexate-induced liver injury. Mustansiriya Med J 2018;17:85-92
|How to cite this URL:|
Mohammad BI, Ahmed BS, Hassan AF. Evaluation of the effects of TAK-242 and GIT-27 on methotrexate-induced liver injury. Mustansiriya Med J [serial online] 2018 [cited 2019 Jul 22];17:85-92. Available from: http://www.mmjonweb.org/text.asp?2018/17/2/85/246103
| Introduction|| |
Drug-induced liver injury (DILI) points to any liver injury caused by xenobiotics or chemicals including drugs or medicinal herbs, whether introduced in therapeutic doses or in overdose., DILI is the most common reason of drug withdrawal after preclinical or clinical studies for example (bromofenac and troglitazone), denied approval for example (ximelagatran), and cessation of development for example (fialuridine), also DILI is the most frequent reason of admission to hospital, liver transplantation, acute liver failure, and acute hepatitis., Methotrexate (MTX) is used to treat various cancers and neoplasms, rheumatoid arthritis-naïve patients, eczema, psoriasis,, inflammatory bowel disease, ulcerative colitis, and steroid-dependent Crohn's disease.,, Off-label MTX is indicated in acute graft versus host disease after allogeneic hematopoietic stem cell transplantation. In combination, it is used in tubal ectopic pregnancy with mifepristone,, narrowband ultraviolet phototherapy, and adalimumab or infliximab., MTX-induced liver injury present as hepatic fatty infiltration, fibrosis, and steatohepatitis. MTX increase cellular sensitization to free radicals (FR) leading to stimulation of immune system starting with hepatic satellite cells (HSCs) which result in leukocyte accumulation, neutrophils secretion of pro-inflammatory enzymes and cytokines like nuclear factor-κB (NF-κB) and tumor necrosis factor-α (TNF-α). This in turn cause more production of FR which leads to sinusoidal congestion, dilation, hepatic fatty vacuolation focal necrosis and portal inflammation which is the typical pattern of drug induced steatohepatitis produced by FR.,, This participation of immune system that results in the production of pro-inflammatory CKs is the link between MTX-induced toxicity and Toll-like receptor (TLR) pathways, which are the common participant receptors of the immune system, the activation of which is required for CK production.
TAK-242 is also known as resatrovid. A cyclohexene derivative with chemical structure of Ethyl-(6R)-6-[N-(2-chloro-4-flurophenyl) sulfamoyl] cyclohex-1-ene-1-carboxylate is a selective inhibitor of TLR4 signal transduction pathway that interferes with Intracellular TLR/interleukin-receptor domain (TIR IC) adaptor molecules' interaction, thus preventing monocyte and macrophage (MQ) pro-inflammatory CK and nitrous oxide (NO) production both in vitro and in vivo., It was designed as a novel antisepsis agent, with anti-inflammatory action that protects against hypertension-related cardiac changes, cardiac apoptosis, and microinfarction after coronary microembolization, and also protects nerves against central nervous system ischemia/reperfusion (I/R) and traumatic injury. Furthermore, it ameliorates the low-grade inflammatory process accompanied by insulin resistance in diabetes.,, GIT-27 is a small isoxazoline compound (4,5-dihydro-3-phenyl-5-isoxazole acetic acid), also known as VGX-1027, which possesses very interesting immunomodulatory effect throughout antagonizing the action of ligand-stimulated TLR4 and TLR2/6, with preferable low toxicity and high efficacy.,, This drug has been developed for treating miscellaneous inflammatory disorders such as type 1 diabetes mellitus and decreasing diabetic neuropathy and pancreatic insulinitis,,, colitis, inflammatory bowel disease, pleurisy, modulation of systemic lupus erythematosus even at genetic level, and rheumatoid arthritis.,
Aim of the study
This study was performed to investigate whether treating the animals with TAK-242 and GIT-27 could reverse liver injuries induced by MTX or the tested drugs have a valuable hepatoprotective potential, especially considering that both drugs are anti-infl ammatory and immunomodulating agents.
| Materials and Methods|| |
Thirty-five male Albino-Wistar rats (4–6 months) (125–225 g) obtained from Kut technical Institute, University of Wasit, were maintained under nonspecific pathogen-free conditions under a constant temperature 24°C ± 3°C with 12:12 h light–dark cycle in wire-meshed cages (seven rats in each cage) with ad libitum access to water and regular rat diet. Animal handing and housing were preceded in accordance with the International Guidelines for the care and use of laboratory animals of the National Research Council.,, The animals were divided randomly into five groups as follows: control group: rats were kept on distilled water (D/W) throughout the treatment; vehicle pretreated group: rats were administered intraperitoneal (I.P.) dimethyl sulfoxide (DMSO) diluted with D/W 1:12.5 with a final concentration of 8% (the same concentration was used to dissolve both the drugs TAK-242 + GIT-27 according to their protocols and rats' weight) for 7 days, followed by 7 days of oral MTX 0.2 mg/kg; MTX group: rats left untreated for 7 days followed by 7 days of oral MTX 0.2 mg/kg (dependent on the adult dose for rheumatoid arthritis stated by the previous literature) that is diluted with D/W at a final concentration of 0.333 mg/ml and administered via rat oral gavage according to rat weight to stimulate DILI; TAK-242 pretreated group: animals were administered I.P. TAK-242 5 mg/kg for 7 days, which was dissolved with DMSO D/W at a final concentration of 17 mg/ml (DMSO solubility of ≥360 mg/ml according to the manufacturer) 1 h before its administration followed by 7 days of oral MTX 0.2 mg/kg; and GIT-27 pretreated group: rats were administered 4 I.P. challenge doses of GIT-27 25 mg/kg at 168, 120, 72, and 24 h, before starting treatment with oral MTX 0.2 mg/kg for 7 days. GIT-27 has been dissolved in DMSO D/W at a final concentration of 7 mg/ml (DMSO solubility ≥65 mg/ml according to the manufacturer) 1 h before its administration. After 24 h of the end of treatment, the rats were anesthetized with intramuscular (I.M.) ketamine 91 mg/kg–xylazine 9 mg/kg., Heart blood was obtained using direct needle puncture after sacrifice. Blood samples were allowed to be settled in 10 ml sterile labeled gel tubes and then centrifuged at 4000 rpm for 10 min at 25°C. The collected serum was stored in 2 ml Eppendorf tubes at −20°C until used for further analysis.,
Chemicals and drugs
DMSO was purchased as 99.5% solution (Central Drug House [P] Ltd., New Delhi, India) and MTX was purchased from a local pharmacy as 50 mg/5 ml injectable solution (KOÇAK pharma, Ístanbul, Turkey). TAK-242 was purchased as white crystalline powder (MedChemExpress, New Jersey, USA) and GIT-27 was supplied as off-white crystal (MedChemExpress, New Jersey, USA). Ketamine was purchased as 10% injectable solution (Alfasan woerden, JA Woerden, Holland) and xylazine as 20% injectable solution (Kepro, ZA Deventer, Holland).
Estimation of serum level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALPL) (hepatocellular markers), Interleukin-6 (IL-6), TNF-α (inflammatory markers), and level of lipid peroxide (LPO) was done via sandwich-enzyme-linked immunosorbent assay (ELISA) kits. Hepatobiliary bilirubin (Bb) and malondialdehyde (MDA) were measured by competitive ELISA kits, while total serum protein (TSP) and reduced glutathione (GSH) were measured via assay kits and their content was estimated depending on Equations 1 and 2.,, All kits were purchased from Elabscience, Georgia, USA, and were performed according to the manufacturers' procedure.
where the term OD refers to the optical densities measured by the spectrophotometer of the sample, the blank, and the standards for both Equations 1 and 2.
Tissue sample collection and histopathological study
A cut was done to the rat's abdomen using a sharp scissor, and the liver was dissected out immediately. Liver tissue samples were fixed in containers with 30 ml of 10% formalin and then stored until they were processed. Liver sectioning and embedding was done according to the traditional processing procedure (paraffin-embedded method) described by Bancroft and Stevens to prepare liver tissue for microscope evaluation, then the tissue was stained with hematoxylin and eosin (H and E), Liver structure evaluation after MTX-induced injury was done utilizing the histological scoring system for nonalcoholic fatty liver disease (NAFLD) (NAS score) which comprehend three main changes in the liver: steatosis (S), lobular inflammation (L), and ballooning of hepatocytes (B). Total NAS score represents the sum of scores for steatosis, lobular inflammation, and ballooning (S + L + B) and ranges from 0 to 8.
Statistical analysis was done using International Business Machines Corp. [IBM] SPSS v20 package for windows 8, New York, USA. The resulted data were presented as mean (x̄) ± standard deviation (S.D)., Statistical differences among groups of data were determined using ANOVA test followed by least significant difference test. Pearson's correlation was measured to estimate the correlation among the measured markers.,, P < 0.05 was considered statistically significant.,
| Results|| |
This study involved 35 male Albino-Wistar rats; there was no loss in sample because of death or any other causes as shown in [Figure 1].
Methotrexate effect on markers of hepatic function and inflammatory and oxidative stress
In comparison with the control group, treating rats with 0.2 mg/kg MTX only for 1 week resulted in a significant increase in the serum level of hepatocellular and hepatobiliary markers. ALT, AST, ALPL, and Bb were increased significantly (P < 0.05), while TSP was decreased significantly (P < 0.05). Furthermore, there was a significant increase in serum level of IL-6 and TNF-α, as well as LPO and MDA (P < 0.05), while GSH was decreased significantly [P < 0.05, [Table 1]].
|Table 1: Changes in serum level of biochemical markers between rats treated with MTX and control group for 14 days, (n=7 each group)|
Click here to view
Correlation coefficient among study markers
The measured inflammatory and oxidative stress markers were analyzed for association with hepatic function tests to clarify treatment effect throughout alteration in their level, which was found to be significant. It seems that decrement of serum GSH was associated with a statistically significant increase in serum ALT and ALPL. GSH level showed moderate negative correlation with both ALT (R = −0.421) and ALPL (R = −0.356), while increment in both serum LPO and MDA was associated with a statistically significant increase in serum ALPL; both showed a moderate positive correlation with a value of (R = 0.384) between LPO and ALPL and (R = 0.381) between MDA and ALPL.
Effect of dimethyl sulfoxide on chemical parameters
DMSO pretreated rats showed nonsignificant changes (P > 0.05) in serum level of hepatic function markers and serum level of inflammatory and oxidative stress markers similar to those treated with MTX (data not shown).
TAK-242 and GIT-27 pretreatment effect on markers of hepatic function and inflammatory and oxidative stress
This study displays that the two pretreated rats' groups with TAK-242 and GIT-27 in comparison with hepatotoxic MTX-treated group resulted in the following changes in markers of hepatic function [Table 2].
|Table 2: Serum liver enzymes changes among rats treated with MTX, TAK-242 and GIT-27 (pre-treatment groups) for 14 days, (n=7 each group)|
Click here to view
Pretreatment with TAK-242 caused a significant decrease (P < 0.05) in serum level of ALT (by a mean decrease of 18.80 ng/ml), AST (by a mean decrease of 3.55 ng/ml), ALPL (by a mean decrease of 5.79 ng/ml), and Bb (by a mean decrease of 0.60 mcg/ml) and a significant increase (P < 0.05) in TSP (by a mean increase of 430.13 mcg/ml). It also causes a significant decrease (P < 0.05) in serum level of the inflammatory markers IL-6 (by a mean decrease of 20.65 pg/ml) and TNF-α (by a mean decrease of 45.79 pg/ml), decrease in the oxidative stress markers MDA (by a mean decrease of 21.66 ng/ml), and significant increase (P < 0.05) in GSH (by a mean increase of 0.07 mg/ml), while decrement in LPO level is still nonsignificant (P > 0.05, by a mean decrease of 10.00 ng/ml).
Pre-treatment with GIT-27 causes significant (P < 0.05) decrease in serum level of AST (by mean decrease of 3.51 ng/ml), ALPL (by mean decrease of 5.01 ng/ml), and Bb (by mean decrease of 0.56 mcg/ml). Also significant decrease (P < 0.05) in serum level of inflammatory markers IL-6 (by mean decrease of 27.06 ng/ml) and TNF-α (by mean decrease of 47.06 pg/ml) and significant decrease in the oxidative stress markers MDA (by mean decrease of 19.84 ng/ml) and significant increase (P < 0.05) in GSH (by mean increase of 0.05 mg/ml) whilst it causes nonsignificant decrement in serum level of ALT (by mean decrease of 12.11 ng/ml), and LPO (by mean decrease of 10.87 ng/ml), and nonsignificant (P > 0.05) increase in TSP (by mean increase of 348.17 mcg/ml).
Treatment effects on liver histopathological findings
The histopathological findings from DILI were graded as mild, moderate, and severe[Table 3]. They were examined in five treatment groups, each containing seven rats. According to NAFLD component scoring system, [Figure 2] shows no liver abnormality in hepatic architecture and normal lobular rearrangement in control group animals, while the highest grading score (severe) was found in MTX-treated animals as shown in [Figure 3] and [Figure 4], respectively, and groups pretreated with the drugsTAK-242 and GIT-27 [Figure 5] and [Figure 6] show lower grade scores with moderate hepatic changes. The lowest grading scores were seen in TAK-242 pretreated animals. Note that pretreatment with vehicle (DMSO) shows the same changes as seen in MTX-treated animals (data not shown).
|Table 3: The assessment of liver injury according to NAFLD histopathological grading scores among the treatment groups MTX, TAK-242 and GIT-27 (pre-treatment groups) for 14 days, (n=7)|
Click here to view
|Figure 2: Liver section of normal control rats (no abnormality) showing normal lobular rearrangement (H and E, ×100)|
Click here to view
|Figure 3: Liver section of methotrexate-treated rat (moderate-to-severe steatosis) showing hepatocyte degeneration and microvesicular and macrovesicular fat vacuoles connecting and opening onto each other forming fatty cystic chains (H and E, ×100)|
Click here to view
|Figure 4: Liver section of methotrexate-treated rat (moderate-to-severe steatosis) showing hepatocyte fatty degeneration with moderate inflammatory cell infiltration (H and E, ×100)|
Click here to view
|Figure 5: Liver section of TAK-242 pretreated rats (moderate) showing hepatocyte fatty degeneration. No inflammatory cells shown (H and E, ×100)|
Click here to view
|Figure 6: Liver section of GIT-27 pretreated rats (moderate) showing hepatocyte degeneration and microvesicular and macrovesicular fatty cysts. No inflammatory cells shown (H and E, ×100)|
Click here to view
| Discussion|| |
MTX-induced liver injury is proposed to be resultant from its effect on de novo synthesis of folate, hepatic drug metabolism and accumulation, oxidative stress, inflammation, and apoptosis., Treatment of the animals with 0.2 mg/kg oral MTX for 7 days resulted in significant increase in serum levels of ALT, AST, ALPL, and Bb with significant decrease in TSP, in accordance with previous studies.,,,, Serum transaminase elevation is assumed to be a mark of hepatocytes damage since they are considered intracellularly concentrated enzymes. This is proposed to be a result of MTX metabolism which proceeds primarily in the liver with concurrent reactive oxygen species (ROS) production and its intracellular retention as polyglutamate (MTX-PG), which causes rapid folate depletion due to the proliferative nature of hepatocytes.,, The scenarios associated with ROS production would change hepatocellular biological membranes, affecting their permeability and structural proteins causes leak and thus high level of hepatic enzymes., While ALPL and Bb serum elevation would reflect MTX hepatobiliary injury since they are concentrated in both liver spleen, reduction of TSP is proposed to be resultant of MTX possible renal tubular injury that leads to loss of proteins as well as formation of protein adducts with the FRs.,
Oxidative stress is proposed to be a major contributor of MTX-induced liver Injury., FRs are generated first during MTX hepatic metabolism to 7-hydroxy metabolite, second from MTX and MTX-PG metabolite-mediated consumption of cellular antioxidants leading to mitochondrial dysfunction, third by MTX increasing plasma cysteine level producing superoxide and reactive nitrogen species., In accordance with previous studies, MTX-significant elevation of pleotropic inflammatory CKs IL-6 and TNF-a,, would suggest an oxidative stress-induced inflammation throughout activation of neutrophil, monocyte, and leucocyte accumulation in the hepatic tissue resulted from MTX-induced liver injury., The increment in the serum level of TNF-α would result from MTX-induced imbalance in TNF-α/NF-κB and hepatocyte inflammation, while IL-6 high serum level is proposed to be induced by MTX-stimulated IL-1β secretion.,
In accordance with the previous studies, MTX-induced oxidative stress was reflected by a significant increase in the level of two sensitive products of lipid peroxidation (MDA and LPO) besides a significant decrease in the level of the antioxidant GSH.,, This was assumed to result from and yet stimulated by MTX-induced FR which attacks cellular lipid bilayer membrane, increasing the content of unsaturated fatty acid; disturbing surface negative charges, membrane permeability, and fluidity; as well as sensitizing the membranous proteins to oxidative damage; also a high MDA would alkylate mitochondrial enzymes rendering them inactive.,, The significant decrease in serum GSH level would be attributed to MTX-induced oxidant/antioxidant imbalance due to increasing rate of FR-macromolecular structure adduct formation, MTX-PG formation, and cellular accumulation, thus directly interacting and increasing the consumption of NADPH which maintains the reduced state of GSH.,, MTX-induced steatosis and inflammation is observed in histological section of treated animals possibly attributed to MTX increase in both oxidative and nitrosative stress which stimulates inflammatory response by activating Kupffer cells and MQ.,,
In agreement with previous studies, pretreatment with TAK-242 first significantly decreases serum level of ALT and AST, TNF-α, IL-6 and MDA and also significantly improved serum level of GSH.,,,,, This is further confirmed by the histopathological assessment of this study which reveal decreasing in the severity of drug-induced steatohepatitis (DISH) from severe to moderate. TAK-242 blockade of TLR4 is reported to decrease the serum level of hepatic transaminase and hepatocyte damage after acetaminophen-induced liver injury and bile duct ligation in animal models with significant modulation in liver histopathologic alteration seen after I/R injury in Rats' model.,, TAK-242 inhibited both TLR4–myeloid differential 88 (MyD88)-dependent TIR domain containing adaptor protein (TIRAP/Mal) and MyD88 independent TNF receptor-associated factor 6 (TRAF6) signaling pathways. This result in the induction of NF-κB, expression of co-receptor proteins lymphocyte antigen 96 (MD2), cluster of differentiation 14 (CD14) as well as activator protein-1 (AP-1) which stimulate inflammatory immune response and release of CK as TNF-a and IL-6,, as well as stimulate mitogen activated protein kinase (MAPK) and c-Jun phosphorylation which are involved in ROS generation., While TAK-242 effect on oxidative stress is also proposed to be due to the downregulation of TLR4 itself since it is expected to be upregulated by oxidative stress through release of intracellular and extracellular damage-associated molecular patterns (DAMPs) that function as “alarmins” after been recognized by TLRs., This is further confirmed by total relief of inflammation assessed by histological examination of the pretreated animals after exposure to methotrexate. This action of TAK-242 was described in both murine and cellular-human models irrespective of the kind of stimuli.,
In agreement with previous studies, GIT-27 caused a significant decrease in serum TNF-α and IL-6,,, and also a significant decrease in serum AST, ALPL and Bb as well as almost borderline significant decrement in ALT. This could be attributed to its anti-inflammatory action, so its nonselective inhibition of TLR would decrease inflammation as well as the resultant ROS generation which leads to hepatocyte injury and death., This effect on ROS is also achieved in this study by GIT-27 significant decrease in serum level of MDA 1st and improvement of GSH level 2nd. This is consistent with another study which revealed that GIT-27 treatment in murine diabetic model improved the associated hepatic steatosis and induced restoration of smaller size fat vacuoles. This hepatic alteration was almost exactly achieved in this study confirmed by GIT-27 reduction of hepatic histopathological changes to moderate grade after exposure to methotrexate; though not to the mild grade, this could be due to the short course of pretreatment of the study in comparison with the same study. Furthermore, nonsignificant improvement of GIT-27 in serum level of LPO could be attributed to this moderation of but not the total amelioration of DISH mentioned before. Accordingly, the significant decrement of pro-inflammatory CK in GIT-27 pretreated rats is attributed to its inhibition of TLR4-, TLR6-, and rather TLR2-activation by the DAMPs resulted from damaged cells that would increase in NF-κB and p38 MAPK especially by TLR4, but unlike TAK-242, it has no effect on AP-1 or c-Jun singling cascade., This inhibition would decrease MQ activation, migration to the site of inflammation, as well as secretion of pro-inflammatory CK such as TNF-α and IL-6; this is further confirmed by the decrease in inflammatory cell infiltration to a few cell grading seen in histopathologic evaluation.,,
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Pandit A, Sachdeva T, Bafna P. Drug-induced hepatotoxicity: A review. Journal of Applied Pharmaceutical Science 2012;2:233-4.
Singh R, Kumar S, Rana AC, Sharma N. Different models of hepatotoxicity and related liver disease: A review. Int Res J Pharm 2012;3.
Kleiner DE. The histopathological evaluation of drug-induced liver injury. Histopathology 2017;70:81-93.
Alempijevic T, Zec S, Milosavljevic T. Drug-induced liver injury: Do we know everything? World J Hepatol 2017;9:491-502.
Campbell JM, Bateman E, Stephenson MD, Bowen JM, Keefe DM, Peters MD, et al.
Methotrexate-induced toxicity pharmacogenetics: An umbrella review of systematic reviews and meta-analyses. Cancer Chemother Pharmacol 2016;78:27-39.
Bianchi G, Caporali R, Todoerti M, Mattana P. Methotrexate and rheumatoid arthritis: Current evidence regarding subcutaneous vs. oral routes of administration. Adv Ther 2016;33:369-78.
Carrascosa JM, de la Cueva P, Ara M, Puig L, Bordas X, Carretero G, et al
. Methotrexate in moderate to severe psoriasis: Review of the literature and expert recommendations. Actas Dermosifiliogr 2016;107:194-206.
Coskun M, Steenholdt C, de Boer NK, Nielsen OH. Pharmacology and optimization of thiopurines and methotrexate in inflammatory bowel disease. Clin Pharmacokinet 2016;55:257-74.
Gabbani T, Deiana S, Lunardi S, Manetti N, Annese V. Safety profile of methotrexate in inflammatory bowel disease. Expert Opin Drug Saf 2016;15:1427-37.
Yee J, Orchard D. Monitoring recommendations for oral azathioprine, methotrexate and cyclosporin in a paediatric dermatology clinic and literature review. Australas J Dermatol 2018;59:31-40.
Levȇque D, Becker G, Toussaint E, Fronecker LM, Pillard C. Clinical pharmacokinetics of methotrexate in oncology. Int J Pharmacokinet 2017;2:137-47.
Wan S, Xiang Y, Fang W, Huang D. The effect of methotrexate in combination with mifepristone on ectopic pregnancy: A meta-analysis. Int J Clin Exp Med 2016;9:14990-5003.
Yang C, Cai J, Geng Y, Gao Y. Multiple-dose and double-dose versus single-dose administration of methotrexate for the treatment of ectopic pregnancy: A systematic review and meta-analysis. Reprod Biomed Online 2017;34:383-91.
Quetglas EG, Armuzzi A, Wigge S, Fiorino G, Barnscheid L, Froelich M, et al.
Review article: The pharmacokinetics and pharmacodynamics of drugs used in inflammatory bowel disease treatment. Eur J Clin Pharmacol 2015;71:773-99.
Miele L, Liguori A, Marrone G, Biolato M, Araneo C, Vaccaro FG, et al.
Fatty liver and drugs: The two sides of the same coin. Eur Rev Med Pharmacol Sci 2017;21:86-94.
Khokhar A, Qayyum A, Khan MW. Protective effect of melatonin against methotrexate induced hepatotoxicity in mice. Pak Armed Forces Med J 2017;67:126-30.
Cure E, Kirbas A, Tumkaya L, Cure MC, Kalkan Y, Yilmaz A, et al.
Protective effect of infliximab on methotrexate-induced liver injury in rats: Unexpected drug interaction. J Cancer Res Ther 2015;11:164-9.
Hussey SE, Liang H, Costford SR, Klip A, DeFronzo RA, Sanchez-Avila A, et al.
TAK-242, a small-molecule inhibitor of toll-like receptor 4 signalling, unveils similarities and differences in lipopolysaccharide- and lipid-induced inflammation and insulin resistance in muscle cells. Biosci Rep 2012;33:37-47.
Zhang Y, Peng W, Ao X, Dai H, Yuan L, Huang X, et al.
TAK-242, a toll-like receptor 4 antagonist, protects against aldosterone-induced cardiac and renal injury. PLoS One 2015;10:e0142456.
Gárate I, García-Bueno B, Madrigal JL, Caso JR, Alou L, Gómez-Lus ML, et al.
Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress. J Neuroinflammation 2014;11:8.
Wang XT, Lu YX, Sun YH, He WK, Liang JB, Li L, et al.
TAK-242 protects against apoptosis in coronary microembolization-induced myocardial injury in rats by suppressing TLR4/NF-κB signaling pathway. Cell Physiol Biochem 2017;41:1675-83.
Oya S, Yokoyama Y, Kokuryo T, Uno M, Yamauchi K, Nagino M. Inhibition of toll-like receptor 4 suppresses liver injury induced by biliary obstruction and subsequent intraportal lipopolysaccharide injection. Am J Physiol -Gastrointest Liver Physiol 2013;306:G244-G252.
Hadi N, Jabber H. Potential activity of GIT27 against renal ischemia reperfusion injury: An experimental study in male rats. Pathophysiol Cell Injury J 2016;5:87-99.
Stosic-Grujicic S, Cvetkovic I, Mangano K, Fresta M, Maksimovic-Ivanic D, Harhaji L, et al.
Apotent immunomodulatory compound, (S, R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid, prevents spontaneous and accelerated forms of autoimmune diabetes in NOD mice and inhibits the immunoinflammatory diabetes induced by multiple low doses of streptozotocin in CBA/H mice. J Pharmacol Exp Ther 2007;320:1038-49.
Min HS, Kim JE, Lee MH, Song HK, Lee MJ, Lee JE, et al.
Effects of toll-like receptor antagonist 4,5-dihydro-3-phenyl-5-isoxasole acetic acid on the progression of kidney disease in mice on a high-fat diet. Kidney Res Clin Pract 2014;33:33-44.
Saurus P, Kuusela S, Dumont V, Lehtonen E, Fogarty CL, Lassenius MI, et al.
Cyclin-dependent kinase 2 protects podocytes from apoptosis. Sci Rep 2016;6:21664.
Saurus P, Kuusela S, Lehtonen E, Hyvönen ME, Ristola M, Fogarty CL, et al.
Podocyte apoptosis is prevented by blocking the toll-like receptor pathway. Cell Death Dis 2015;6:e1752.
Fagone P, Muthumani K, Mangano K, Magro G, Meroni PL, Kim JJ, et al.
VGX-1027 modulates genes involved in lipopolysaccharide-induced toll-like receptor 4 activation and in a murine model of systemic lupus erythematosus. Immunology 2014;142:594-602.
Mangano K, Sardesai N, D'Alcamo M, Libra M, Malaguarnera L, Donia M, et al. In vitro
inhibition of enterobacteria-reactive CD4+CD25- T cells and suppression of immunoinflammatory colitis in mice by the novel immunomodulatory agent VGX-1027. Eur J Pharmacol 2008;586:313-21.
Olayinka ET, Ore A, Adeyemo OA, Ola OS. Ameliorative effect of gallic acid on methotrexate-induced hepatotoxicity in rat. J Xenobiotics 2016;6:6092.
Yucel Y, Oguz E, Kocarslan S, Tatli F, Gozeneli O, Seker A, et al.
The effects of lycopene on methotrexate-induced liver injury in rats. Bratisl Lek Listy 2017;118:212-6.
Zhao Y, Xin Y, Gao J, Teng RY, Chu HC. Analgesic effect of TAK-242 on neuropathic pain in rats. Int J Clin Exp Med 2015;8:11202-7.
Yousif NG, Mohammad BI, Al-Khalidy SA. Effect of N-acetyl cysteine and TAK-242 on sepsis induced myocardial injury: Down regulation of MMP-2 pathway in mice. Pathophysiol Cell Injury J 2016;5:111-25.
Hawk CT, Leary ST, Morris TH. Formulary for Laboratory Animals. 3rd
ed. 2121 State Avenue, Ames, Iowa 50014, USA. Blackwell Publishing; 2005.
IQ 3Rs Leadership Group – Contract Research Organization Working Group Recommended Dose Volumes for Common Laboratory Animals; 2016.
Matsunaga N, Tsuchimori N, Matsumoto T, Ii M. TAK-242 (resatorvid), a small-molecule inhibitor of toll-like receptor (TLR) 4 signaling, binds selectively to TLR4 and interferes with interactions between TLR4 and its adaptor molecules. Mol Pharmacol 2011;79:34-41.
Tunali-Akbay T, Sehirli O, Ercan F, Sener G. Resveratrol protects against methotrexate-induced hepatic injury in rats. J Pharm Pharm Sci 2010;13:303-10.
Suvarna SK, Layton C, Bancroft JD. Bancroft's Theory and Practice of Histological Techniques. 7th
Ed. British Library Cataloguing in Publication Data. Churchill Livingstone Elsevier; 2013.
Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al.
Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313-21.
Hadi NR, Al-Amran FG, Swadi A. Metformin ameliorates methotrexate-induced hepatotoxicity. J Pharmacol Pharmacother 2012;3:248-53.
] [Full text]
Tawfik MK. Combination of coenzyme Q10 with methotrexate suppresses Freund's complete adjuvant-induced synovial inflammation with reduced hepatotoxicity in rats: Effect on oxidative stress and inflammation. Int Immunopharmacol 2015;24:80-7.
David AV, Satyanarayana N, Parasuraman S, Bharathi S, Arulmoli R. Ameliorative effect of quercetin in methotrexate induced toxicity in Sprague-Dawley rats: A histological study. Indian J Pharm Educ Res 2016;50:S200-8.
Almansour MI, Jarrar YB, Aloyaidy KA, Jarrar BM. Ameliorative effect of propolis against hepatorenal alterations induced by methotrexate: Morphological study. Int J Morphol 2017;35:756-64.
Walker TM, Rhodes PC, Westmoreland C. The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures. Toxicol In Vitro
Ali N, Rashid S, Nafees S, Hasan SK, Sultana S. Beneficial effects of chrysin against methotrexate-induced hepatotoxicity via attenuation of oxidative stress and apoptosis. Mol Cell Biochem 2014;385:215-23.
Kose E, Sapmaz HI, Sarihan E, Vardi N, Turkoz Y, Ekinci N, et al.
Beneficial effects of montelukast against methotrexate-induced liver toxicity: A biochemical and histological study. ScientificWorldJournal 2012;2012:987508.
El-Sheikh AA, Morsy MA, Abdalla AM, Hamouda AH, Alhaider IA. Mechanisms of thymoquinone hepatorenal protection in methotrexate-induced toxicity in rats. Mediators Inflamm 2015;2015:859383.
Abo-Haded HM, Elkablawy MA, Al-Johani Z, Al-Ahmadi O, El-Agamy DS. Hepatoprotective effect of sitagliptin against methotrexate induced liver toxicity. PLoS One 2017;12:e0174295.
Darwish SF, El-Bakly WM, Arafa HM, El-Demerdash E. Targeting TNF-α and NF-κB activation by bee venom: Role in suppressing adjuvant induced arthritis and methotrexate hepatotoxicity in rats. PLoS One 2013;8:e79284.
Çakır T, Özkan E, Dulundu E, Topaloğlu Ü, Şehirli AÖ, Ercan F, et al.
Caffeic acid phenethyl ester (CAPE) prevents methotrexate-induced hepatorenal oxidative injury in rats. J Pharm Pharmacol 2011;63:1566-71.
Tabassum H, Parvez S, Pasha ST, Banerjee BD, Raisuddin S. Protective effect of lipoic acid against methotrexate-induced oxidative stress in liver mitochondria. Food Chem Toxicol 2010;48:1973-9.
Yokoi T, Yokoyama Y, Kokuryo T, Yamaguchi J, Nagino M. Inhibition of toll-like receptor 4 ameliorates experimental postischemic injury in the cholestatic liver through inhibition of high-mobility group box protein b1 (HMGB1) signaling. Surgery 2018;163:270-6.
Salama M, Elgamal M, Abdelaziz A, Ellithy M, Magdy D, Ali L, et al.
Toll-like receptor 4 blocker as potential therapy for acetaminophen-induced organ failure in mice. Exp Ther Med 2015;10:241-6.
Sha T, Iizawa Y, Ii M. Combination of imipenem and TAK-242, a toll-like receptor 4 signal transduction inhibitor, improves survival in a murine model of polymicrobial sepsis. Shock 2011;35:205-9.
Yu P, Cheng X, Du Y, Huang L, Dong R. TAK-242 can be the potential agents for preventing invasion and metastasis of hepatocellular carcinoma. Med Hypotheses 2013;81:653-5.
Wei CB, Tao K, Jiang R, Zhou LD, Zhang QH, Yuan CS, et al.
Quercetin protects mouse liver against triptolide-induced hepatic injury by restoring Th17/Treg balance through tim-3 and TLR4-myD88-NF-κB pathway. Int Immunopharmacol 2017;53:73-82.
Shao Z, Jiao B, Liu T, Cheng Y, Liu H, Liu Y, et al.
TAK-242 treatment ameliorates liver ischemia/reperfusion injury by inhibiting TLR4 signaling pathway in a swine model of maastricht-category-III cardiac death. Biomed Pharmacother 2016;84:495-501.
Wen Z, Ji X, Tang J, Lin G, Xiao L, Liang C, et al.
Positive feedback regulation between transglutaminase 2 and toll-like receptor 4 signaling in hepatic stellate cells correlates with liver fibrosis post Schistosoma japonicum
infection. Front Immunol 2017;8:1808.
Khan MA, Farahvash A, Douda DN, Licht JC, Grasemann H, Sweezey N, et al.
JNK activation turns on LPS- and gram-negative bacteria-induced NADPH oxidase-dependent suicidal NETosis. Sci Rep 2017;7:3409.
Lin A, Wang G, Zhao H, Zhang Y, Han Q, Zhang C, et al.
TLR4 signaling promotes a COX-2/PGE2/STAT3 positive feedback loop in hepatocellular carcinoma (HCC) cells. Oncoimmunology 2016;5:e1074376.
HO SS. Immune-mediated drug induced liver injury: A multidisciplinary approach. Library/The Sydney Scholarship Repository/Postgraduate Theses/Sydney Digital Theses. Faculty of Pharmacy: The University of Sydney;2015.
Takashima K, Matsunaga N, Yoshimatsu M, Hazeki K, Kaisho T, Uekata M, et al.
Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model. Br J Pharmacol 2009;157:1250-62.
Cha JJ, Hyun YY, Lee MH, Kim JE, Nam DH, Song HK, et al.
Renal protective effects of toll-like receptor 4 signaling blockade in type 2 diabetic mice. Endocrinology 2013;154:2144-55.
Laird MD, Shields JS, Sukumari-Ramesh S, Kimbler DE, Fessler RD, Shakir B, et al.
High mobility group box protein-1 promotes cerebral edema after traumatic brain injury via activation of toll-like receptor 4. Glia 2014;62:26-38.
Arslan F, Keogh B, McGuirk P, Parker AE. TLR2 and TLR4 in ischemia reperfusion injury. Mediators Inflamm 2010;2010:704202.
Mittermayer F, Caveney E, De Oliveira C, Fleming GA, Gourgiotis L, Puri M, et al.
Addressing unmet medical needs in type 1 diabetes: A Review of drugs under development. Curr Diabetes Rev 2017;13:300-14.
Stojanovic I, Cuzzocrea S, Mangano K, Mazzon E, Miljkovic D, Wang M, et al. In vitro, ex vivo
and in vivo
immunopharmacological activities of the isoxazoline compound VGX-1027: Modulation of cytokine synthesis and prevention of both organ-specific and systemic autoimmune diseases in murine models. Clin Immunol 2007;123:311-23.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]