Table 2.

Summary of the effect of synthetic TLR4 antagonists in pre-clinical in vitro and in vivo research studies

Study	Study model	TLR4 antagonist	Outcome
Mullarkey et al. [83]	Sepsis In vivo: C57Bl/6 mice, Hartley guinea pigs, Fischer rats challenged with i.v. LPS	Eritoran (E5564)	Mice: E5564 (100, 300, 1000 μg/kg) co-treatment was associated with 37%, 81% and 93% inhibition of LPS-induced (100 μg/kg) response as measured by serum TNF-α levels, respectively (p < 0.05 vs. control). Guinea pigs: E5564 (30, 100, 300 μg/kg) co-treatment was associated with 29%, 57% and 94% inhibition of LPS-induced (1000 μg/kg) response as measured by serum TNF-α levels, respectively (p < 0.05 vs. control). Rats: E5564 (10, 100, 1000 μg/kg) co-treatment was associated with 84%, 97% and 100% inhibition of LPS-induced (3 μg/kg) response as measured by serum TNF-α levels, respectively (p < 0.05 vs. control).
Kitazawa et al. [84]	Acute liver failure (ALF) In vivo: Wistar rats challenged with d-galactosamine (GalN) and LPS		Rats treated with E5564 after ALF (500 mg/kg GalN + 50 μg/kg LPS) displayed a decrease in serum TNF-α levels and had an improved survival rate of 42.9% compared to untreated rats (p < 0.05).
Liu et al. [85]	Inflammatory effects of ischemia-reperfusion in kidneys In vivo: Fisher rats with kidney nephrectomy and ischemia performed.		Rats treated with E5564 displayed a significant improvement in renal function as measured by serum creatinine levels (p < 0.05) and higher survival rates (p < 0.05) vs. vehicle controls.
Sha et al. [86]	Endotoxin shock In vivo: BALB/c mice treated with LPS i.p.	TAK-242 (Resatorvid)	Pre-treatment of TAK-242 (0.1, 0.3, 1 and 3 mg/kg) was associated with a decrease in LPS-induced (10 mg/kg) responses as measured by IL-6, IL-10, MIP-2, IL-1β and NO serum levels vs. vehicle control (p < 0.025). A 40% increase in survival rate of mice was also observed vs vehicle control (p ≤ 0.05). Post-treatment of TAK-242 (1 mg/kg) was associated with a decrease in LPS-induced (5 mg/kg) response as measured by IL-6 and MIP-2 serum levels vs. vehicle control (p ≤ 0.01). A survival rate of 45% was also observed vs. vehicle control (p ≤ 0.01).
Kuno et al. [87]	Endotoxemia In vivo: Hartley guinea pigs treated with LPS i.v.		TAK-242 (3 and 10 mg/kg) pre-treatment was associated with a dose-dependent decline in colonic muscle tension (p=0.001 and p<0.001, respectively) and mean arterial pressure (p = 0.036 and p = 0.004, respectively) caused by LPS (10 mg/kg, i.v.). A 50% survival rate was observed when pre-treated with TAK-242 at 10 mg/kg vs. the 10% observed in the control group.
Garate et al. [88]	Neuroinflammation In vivo: Wistar Hannover rats restrained to induce stress.		Pre-treatment of TAK-242 (0.5 mg/kg, i.v.) decreased expression of the pro-inflammatory enzymes: IL-1β, COX-2 and iNOS expression levels, p < 0.05 vs control, p < 0.05 vs stress only group.
Hua et al. [89]	Cerebral ischemia In vivo: C57Bl/6 mice induced with focal cerebral ischemia/reperfusion		Treatment with TAK-242 (3 mg/kg) was associated with reduce levels of serum TNF receptor II, monocyte chemotactic protein-1, macrophage inflammatory protein-1γ and tissue inhibitor of metalloproteinases-1 (p < 0.05 vs. untreated mice). An 8.8% reduction in brain infarct size and improved neurologic function score (6.73) were also observed (p < 0.05 vs. untreated mice).
Perrin-Cocon et al. [90]	Lethal influenza infection In vitro: monocyte-derived dendritic cells (DCs) challenged with influenza virus, strain A/PR/8/34 In vivo: C57Bl/6 infected with mouse-adapted influenza virus, strain A/PR/8/34	FP7	In vitro: FP7 (1 and 10 μM) treatment was associated with decreased levels of LPS-induced (10 ng/mL) responses as measured by supernatant levels of IL-8, IL-6, MIP-1β, TNF-α, IL-12 and IL-10 in both monocytes and DCs (p < 0.05 vs. LPS). In vivo: Mice treated with FP7 (200 μg/mouse, i.v.) after influenza infection displayed reduced gene production of TNF-α, IL-1β, IFN-β, murine IL-8 (p < 0.01) and IL-6 (p < 0.05) in the lungs. FP7-treated mice had decreased viral load (log FP7-treated titre = 4.1 ± 0.39) vs. vehicle-treated mice (log vehicle-treated titre = 5.27 ± 0.15) as measured by a virus titration assay (p = 0.0225).
Palmer et al. [91]	Cardiovascular inflammatory-based diseases In vitro: Human umbilical vein endothelial cells (HUVEC), THP-1 and mouse RAW-264.7 macrophages challenged with LPS In vivo: Angiotensin II-infused apolipoprotein E-deficient mice		In vitro: FP7 (0–10 μM) negatively regulated LPS-induced production (100 ng/mL) of pro-inflammatory cytokines in a dose-dependent manner: THP-1: IL-8 (p < 0.001), IL-6 (p < 0.01), MIP-1α (p < 0.001) at 5 μM and IL-1β (p < 0.001) at 0.1, 1, 5 μM vs. LPS. RAW-264.7: p65 NF-κB at 1, 5, 10 μM (p < 0.001), IL-6 at 5 μM (p < 0.05), 10 μM (p < 0.001) and p38 MAPK at 0.1 (p < 0.05), 1 (p < 0.01), 5 and 10 μM (p < 0.001) vs. LPS. HUVEC: p38 MAPK and p65 NF-κB at 0.1, 0.5 and 1 μM (p < 0.01, p < 0.05, p < 0.01, respectively), MCP-1 at 1 μM (p < 0.05) vs. LPS. In vivo: FP7 (3 mg/kg/day) inhibited angiotensin II-driven production of pro-inflammatory proteins, and MIP-1γ and JNK phosphorylation (p < 0.05 vs. angiotensin II group).
Facchini et al. [92]	Inflammatory bowel disease In vitro: Peripheral blood mononuclear cells and lamina propria mononuclear cells collected from patients with IBD In vivo: BALB/c mice with DSS administered in their water.		In vitro: FP7 at 10 μM negatively regulated LPS-induced production (100 ng/mL) of pro-inflammatory cytokines: mRNA relative expression: TNF-α (p < 0.001); IL-1β (p < 0.05); IL-6 (p < 0.05). ELISA: TNF-α, IL-1β and IL-6 (p < 0.05). In vivo: FP7 (250 μg/kg) treatment was associated with a lower histological score (p < 0.01 vs. DSS) and significantly reduced the release of inflammatory cytokines TNF-α (p < 0.05), IL-1β (p < 0.001) and IL-6 (p < 0.05).
Huggins et al. [93]	Abdominal aortic aneurysm (AAA) In vitro: HUVEC challenged with LPS In vivo: C57Bl/6 mice induced with AAA	IAXO-102	In vitro: IAXO-102 (10 μM) blocked LPS-stimulated (100 ng/mL) production of JNK, ERK, p65 NF-κB (p < 0.05) and p38, MCP-1, IL-8 (p < 0.01) vs. LPS. In vivo: IAXO-102 (3 mg/kg/day) blocked angiotensin II-induced response as measured by protein expression of JNK, ERK, p65, NF-κB (p < 0.05) vs. angiotensin II only group. IAXO-102 also downregulated expression of MIP-1γ and TLR4 (p < 0.05 vs. angiotensin II group) and reduced incidence of AAA (30% IAXO-102-treated vs. 86% angiotensin II group).
Zhang et al. [94]	Acute lung injury (ALI) In vitro: Mouse RAW 264.7 macrophages challenged with LPS In vivo: Sprague-Dawley rats with ALI induced by intratracheal LPS instillation	Chalcone derivatives - Compound 20	In vitro: Fluorescent probe determined compound 20 is a specific inhibitor of MD2 (KD = 189 μM). Addition of compound 20 (10 μM) inhibited LPS-induced (0.5 μg/mL) secretion of TNF-α, IL-1β, COX-2 (p < 0.01) and IL-6 (p < 0.05) vs. LPS. In vivo: Compound 20 (20 mg/kg) reduced LPS-induced (5 mg/kg) pulmonary edema as measure by the decrease in lung wet/dry weight ratio (p < 0.01) vs. LPS. Compound 20 also inhibited IL-1β secretion (p < 0.01) and MPO activity (p < 0.05) vs. LPS.
Wang et al. [95]	Septic shock and lung injury In vitro: Mouse primary peritoneal macrophages challenged with LPS In vivo: C57Bl/6 mice injected with LPS	Curcumin analogues - L48H37	In vitro: Fluorescent probe determined L48H37 is a specific inhibitor of MD2 (KD = 11.3 μM). L48H37 (1, 2.5, 5 or 10 μM) inhibited LPS-induced (0.5 μg/mL) phosphorylation in a dose-dependent manner: ERK at 1, 2.5, 5 and 10 μM (p < 0.01), p38 at 2.5 μM (p < 0.05), 5 and 10 μM (p < 0.01), and JNK at 5 and 10 μM (p < 0.01) vs. LPS. L48H37 (10 μM) inhibited secretion of TNF-α, IL-6, IL-1β and iNOS (p < 0.01 vs. LPS-treated group); IL-10 and COX-2 (p < 0.05 vs. LPS-treated group). In vivo: L48H37-treated (10 mg/kg) mice had higher survival rates vs. LPS (20 mg/kg, i.v.) (p < 0.01). Pulmonary damage and LPS-injured tissue structure of lungs was amended.
Hodgkinson and Ye. [96]	In vitro inflammation model In vitro: Human embryonic kidney (HEK) 293-CD14- MD2 cells challenged with LPS	Statins - Simvastatin - Pravastatin	Both simvastatin and pravastatin (2 μM) pre-treatment was associated with the inhibition of LPS-induced (5 ng/mL) response as measured by supernatant concentrations of NF-κB, IL-6 and TNF-α (p < 0.05 vs. LPS).
Katsargyris et al. [97]	Carotid atherosclerotic plaques Ex vivo: atherosclerotic plaques from patients		Patients who used statins had lower TLR4 expression in their endothelial cells and atherosclerotic plaques vs. non-statin patients (p = 0.02 and p = 0.03, respectively). Prevalence cerebrovascular accident was 18.6% in statin group vs. 61.4% of non-statin group (odds ratio [95% CI] 0.14 [0.07–0.31] p < 0.001).
Fort et al. [98]	Inflammatory bowel disease BALB/c mice with DSS administered in their water.	Lipid A-mimetic - CRX-526	CRX-526 (2, 10, 50 μg) treatment was associated with a lower DAI (p = 0.421, 0.056, 0.016, respectively) and histological score (p = 0.032, 0.008, 0.008, respectively) vs. DSS in a dose-dependent manner.

E5564 eritoran, LPS lipopolysaccharide, IV intravenous, TNF-α tumor necrosis factor alpha, ALF acute liver failure, GalN d-galactosamine, i.p. intraperitoneal, Resatorvid TAK-242, IL-6 interleukin 6, IL-10 interleukin 10, MIP-2 macrophage inflammatory protein 2, IL-1β interleukin 1 beta, NO nitric oxide, COX-2 cyclooxygenase 2, iNOS nitric oxide synthase, DCs dendritic cells, IL-8 interleukin 8, MIP-1β macrophage inflammatory protein 1 beta, IL-12 interleukin 12, IFN-β interferon beta, HUVEC human umbilical vein endothelial cells, THP-1 human acute monocytic leukemia cell, MAPK mitogen-activated protein kinase, MIP-1α macrophage inflammatory protein 1 alpha, MAPK mitogen-activated protein kinase, MIP-1γ macrophage inflammatory protein 1 gamma, JNK c-Jun N-terminal kinase, IBD inflammatory bowel disease, DSS dextran sulphate sodium, mRNA messenger RNA, AAA abdominal aortic aneurysm, HUVEC human umbilical vein endothelial cell, ERK extracellular signal-regulated kinase, ALI acute lung injury, MD2 myeloid differentiation factor 2, KD equilibrium dissociation constant, HEK human embryonic kidney, CD14 cluster of differentiation 14, DAI disease activity index