TAK-242 is a potent TLR4 signaling inhibitor, selectively inhibits the TLR4-mediated production of cytokines and NO.

TLR4^[1]

In RAW264.7 cells and mouse peritoneal macrophages, TAK-242 suppresses lipopolysaccharide (LPS)-induced production of NO, tumor necrosis factor-α (TNF-α), and interleukin (IL)-6, with IC₅₀ of 1.1 to 11 nM. TAK-242 also suppresses the production of these cytokines from LPS-stimulated human peripheral blood mononuclear cells (PBMCs) at IC₅₀ values from 11 to 33 nM^[1].

TAK-242 apparently reduces the serum anti-dsDNA levels in both genotype mice. Alternatively, IFN-γ, TNF-α, and IL-1β production is markedly inhibited by TAK-242, but their concentrations are still greatly higher than those in NS-treated counterparts^[2]. TAK-242 pre-stress administration prevents the accumulation of potentially deleterious inflammatory and oxidative/nitrosative mediators in the brain frontal cortex of rats. TAK-242 i.v. administration at the beginning of the stress session completely blocks TLR-4 mRNA and protein upregulation after stress exposure^[3].

• [1]. Ii M, et al. A novel cyclohexene derivative, ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling.

  [2]. Ni JQ, et al. Role of toll-like receptor 4 on lupus lung injury and atherosclerosis in LPS-challenge ApoE⁻/⁻ mice. Clin Dev Immunol. 2013;2013:476856.

  [3]. Gárate I, et al. Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress. J Neuroinflammation. 2014 Jan 11;11:8.

  [4]. Wang L, et al. Doxorubicin-Induced Systemic Inflammation Is Driven by Upregulation of Toll-Like Receptor TLR4 and Endotoxin Leakage. Cancer Res. 2016 Nov 15;76(22):6631-6642.

  [5]. Shibata A, et al. Toll-like receptor 4 antagonist TAK-242 inhibits autoinflammatory symptoms in DITRA. J Autoimmun. 2017 Jun;80:28-38.

  [6]. Janda J, et al. Resatorvid-based Pharmacological Antagonism of Cutaneous TLR4 Blocks UV-induced NF-κB and AP-1 Signaling in Keratinocytes and Mouse Skin. Photochem Photobiol. 2016 Nov;92(6):816-825.

  [7]. RAO Xiao-jiao, et al. Effect of TLR4 on expression of inflammatory cytokine in aortic artery in mice with insulin resistance[J]. Chinese Journal of Public Health, 2016, 32(11): 1480-1484.

TAK-242 is dissolved in N,N-dimethylformamide, and then diluted with appropriate medium before use^[1].

RAW264.7 cells are seeded at a density of 3×10⁶ cells/well in six-well culture plate and incubated overnight. After washing with RPMI 1640 medium supplemented with 1% FCS and 10 μg/mL Kanamycin, the cells are stimulated with 5 ng/mL LPS and 1 U/mL IFN-γ in the presence or absence of TAK-242 (1-100 nM) for the indicated time. Culture supernatants are removed, and total RNA is isolated using the total RNA isolation reagent ISOGEN. Total RNA is reverse transcribed into cDNA by using TaqMan reverse transcription reagents. Quantitative real-time PCR analysis of TNF-α and IL-6 is performed on ABI Prism 7700 using predeveloped TaqMan assay reagents and Universal PCR master mix. Quantitation of mRNA is performed using the comparative threshold cycle method. The highest control level attained by the stimulation (without TAK-242) is regarded as 100%, and the levels of control group at other time points and TAK-242-added group are expressed as the percentage of the highest control level^[1]. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

TAK-242 is dissolved in vehicle (saline) (Mice)^[2].
TAK-242 is dissolved in vehicle (0.9% DMSO) (Rat)^[3].

Mice^[2]
Thirty ApoE^-/- and thirty wild-type mice on C57BL/6 background (female, 10 weeks old) are fed on a high-fat diet containing 0.25% cholesterol and 15% cocoa butter under standardized lighting conditions (12 h light-dark cycle) and temperature (21±1°C). And mineral water is administered ad libitum. Mice of both genotypes are randomly assigned to LPS or LPS + TAK-242 or saline administration. LPS (2.5 mg/kg), LPS (2.5 mg/kg) plus TAK-242 (0.3 mg/kg) and saline are administered respectively by intraperitoneal injection, twice a week for 4 weeks. At the end of experiments, all mice underwent euthanasia with injection of overdose pentobarbital (50 mg/kg).
Rat^[3]
Male outbred Wistar Hannover rats, initially weighing 200 to 225 g, are used. TAK-242 is i.v. injected in the tail vein at a dose of 0.5 mg/kg immediately after (approximately 10 seconds) introducing the animal to the plastic restrainer. This dose is chosen on the basis of previous in vivo studies reporting its anti-inflammatory/antioxidant and neuroprotective profile in microglia exposed to hypoxia. Dimethyl sulphoxide at a concentration of 0.9% is used as vehicle. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Ii M, et al. A novel cyclohexene derivative, ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling.

  [2]. Ni JQ, et al. Role of toll-like receptor 4 on lupus lung injury and atherosclerosis in LPS-challenge ApoE⁻/⁻ mice. Clin Dev Immunol. 2013;2013:476856.

  [3]. Gárate I, et al. Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress. J Neuroinflammation. 2014 Jan 11;11:8.

  [4]. Wang L, et al. Doxorubicin-Induced Systemic Inflammation Is Driven by Upregulation of Toll-Like Receptor TLR4 and Endotoxin Leakage. Cancer Res. 2016 Nov 15;76(22):6631-6642.

  [5]. Shibata A, et al. Toll-like receptor 4 antagonist TAK-242 inhibits autoinflammatory symptoms in DITRA. J Autoimmun. 2017 Jun;80:28-38.

  [6]. Janda J, et al. Resatorvid-based Pharmacological Antagonism of Cutaneous TLR4 Blocks UV-induced NF-κB and AP-1 Signaling in Keratinocytes and Mouse Skin. Photochem Photobiol. 2016 Nov;92(6):816-825.

  [7]. RAO Xiao-jiao, et al. Effect of TLR4 on expression of inflammatory cytokine in aortic artery in mice with insulin resistance[J]. Chinese Journal of Public Health, 2016, 32(11): 1480-1484.

361.82

C₁₅H₁₇ClFNO₄S

243984-11-4

Room temperature in continental US; may vary elsewhere

DMSO: ≥ 360 mg/mL

TAK-242 is dissolved in a fat emulsion (i.v. injection)^[4].
TAK-242 dissolved in DMSO (10 mg/mL) is diluted in DW^[5].
TAK-242 is prepared in 0.5% acetone^[6].
TAK-242 is prepare in vehicle (sterile saline)^[7].

* "<1 mg/ml"="" means="" slightly="" soluble="" or="" insoluble.="" "≥"="" means="" soluble,="" but="" saturation="">

• [1]. Ii M, et al. A novel cyclohexene derivative, ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling.

  [2]. Ni JQ, et al. Role of toll-like receptor 4 on lupus lung injury and atherosclerosis in LPS-challenge ApoE⁻/⁻ mice. Clin Dev Immunol. 2013;2013:476856.

  [3]. Gárate I, et al. Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress. J Neuroinflammation. 2014 Jan 11;11:8.

  [4]. Wang L, et al. Doxorubicin-Induced Systemic Inflammation Is Driven by Upregulation of Toll-Like Receptor TLR4 and Endotoxin Leakage. Cancer Res. 2016 Nov 15;76(22):6631-6642.

  [5]. Shibata A, et al. Toll-like receptor 4 antagonist TAK-242 inhibits autoinflammatory symptoms in DITRA. J Autoimmun. 2017 Jun;80:28-38.

  [6]. Janda J, et al. Resatorvid-based Pharmacological Antagonism of Cutaneous TLR4 Blocks UV-induced NF-κB and AP-1 Signaling in Keratinocytes and Mouse Skin. Photochem Photobiol. 2016 Nov;92(6):816-825.

  [7]. RAO Xiao-jiao, et al. Effect of TLR4 on expression of inflammatory cytokine in aortic artery in mice with insulin resistance[J]. Chinese Journal of Public Health, 2016, 32(11): 1480-1484.