[Advancing the drug discovery ecosystem for innovative medicines].

Axcelead Drug Discovery Partners (Axcelead DDP) Inc is the first integrated drug discovery solution provider in Japan. Leveraging drug discovery platforms and knowledge inherited from Takeda Pharmaceutical Company, Ltd. alongside the latest science and technology, our experienced scientists with rich track records promote drug discovery research and contribute to co-creation of innovative drugs together with customers. In this article, we provide an overview landscape of the pharmaceutical industry and emerging trends in drug discovery research, and introduce Axcelead DDP's services, its unique strengths, and the value (solutions) delivered to customers. Furthermore, we describe the current state of Japan's bio-community and the roles and challenges for the development and enhancement of a true drug discovery ecosystem in Japan.

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.

TAK-242 (resatorvid), a small-molecule-specific inhibitor of Toll-like receptor (TLR) 4 signaling, inhibits the production of lipopolysaccharide-induced inflammatory mediators by binding to the intracellular domain of TLR4. Cys747 in TLR4 has been identified previously as the binding site of TAK-242. However, the mechanism by which TAK-242 inhibits TLR4 signaling after binding to TLR4 remains unknown. The present study demonstrated, using coimmunoprecipitation, that TAK-242 interferes with protein-protein interactions between TLR4 and its adaptor molecules. Among 10 different human TLRs, TAK-242 selectively bound to TLR4. The time course of the inhibitory effect of TAK-242 on inflammatory mediator production corresponded to that of the binding of TAK-242 to TLR4. TAK-242 inhibited the association of TLR4 with Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) or Toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon-ß-related adaptor molecule (TRAM) in human embryonic kidney (HEK) 293 cells overexpressing TLR4, MD-2, and TIRAP or TRAM, respectively. TAK-242 inhibited the TIRAP-mediated activation of nuclear factor κB (NF-κB) and the TRAM-mediated activation of NF-κB and interferon-sensitive response element in HEK293 cells stably expressing TLR4, MD-2, and CD14. The activation of endogenous interleukin-1 receptor-associated kinase in RAW264.7 cells was also inhibited by TAK-242 treatment. These findings suggest that TAK-242 binds selectively to TLR4 and subsequently disrupts the interaction of TLR4 with adaptor molecules, thereby inhibiting TLR4 signal transduction and its downstream signaling events. This work proposes a novel paradigm of a small molecule capable of disrupting protein-protein interactions.

Combination of imipenem and TAK-242, a Toll-like receptor 4 signal transduction inhibitor, improves survival in a murine model of polymicrobial sepsis.

Sepsis is characterized by an excessive host response to infection. Toll-like receptors (TLRs) are essential for triggering this type of host immune response. Toll-like receptor 4 mediates recognition of LPS from gram-negative bacteria and is an important initiator of sepsis. In the present study, we evaluated the efficacy of TAK-242, a novel TLR4 signal transduction inhibitor, in a murine cecal ligation and puncture (CLP) model. Treatment with TAK-242 (10 mg/kg i.v.) in combination with imipenem (1 mg/kg s.c.) 1 h after CLP significantly increased the survival rates of mice from 17% to 50% (P ≤ 0.01) and suppressed CLP-induced increases in serum levels of IL-1[beta], IL-6, IL-10, and macrophage inflammatory protein 2 by 64%, 73%, 79%, and 81%, respectively (P ≤ 0.025). Additionally, coadministration of TAK-242 with imipenem after CLP significantly inhibited CLP-induced decreases in blood platelet counts by 37% (P ≤ 0.025) and increases in serum levels of alanine aminotransferase by 32% (P ≤ 0.025) and blood urea nitrogen by 43% (P ≤ 0.025). TAK-242 at a dose of 10 mg/kg had no effect on bacterial counts in blood, suggesting that it does not affect blood bacteria spread. These results indicate that TAK-242 shows therapeutic effects in murine polymicrobial sepsis, and it may be a potential therapeutic agent for the treatment of sepsis.

A randomized, double-blind, placebo-controlled trial of TAK-242 for the treatment of severe sepsis.

OBJECTIVE: To evaluate whether TAK-242, a small-molecule inhibitor of Toll-like receptor-4-mediated signaling, suppresses cytokine levels and improves 28-day all-cause mortality rates in patients with severe sepsis. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: A total of 93 intensive care units worldwide. PATIENTS: A total of 274 patients with severe sepsis and shock or respiratory failure. INTERVENTIONS: Patients were randomly assigned to receive a 30-min loading dose followed by 96-hr infusions of placebo, TAK-242 1.2 mg/kg/day, or TAK-242 2.4 mg/kg/day. MEASUREMENTS AND MAIN RESULTS: The primary pharmacodynamic end point was change in serum interleukin-6 levels relative to baseline, with 28-day all-cause mortality rate the primary clinical end point. The trial was terminated because of a lack of effect of TAK-242 in suppressing serum interleukin-6 levels. A total of 274 subjects were randomly assigned and treated. Clinical parameters at baseline were balanced across the three groups. TAK-242 did not suppress interleukin-6 as measured by 0- to 96.5-hr area under the interleukin-6 concentration curve at either dose. Specifically, the area under the effect curve increased by 9% and 26.9% in the TAK-242 1.2 and 2.4 mg/kg/day groups, respectively, which was not statistically different from placebo (p = .63 and .15, respectively). The 28-day mortality rate was 24% in the placebo, 22% in the low-dose, and 17% in the high-dose group (p = .26 for placebo vs. high dose). A nonsignificant reduction in mortality rate was observed in a subset of patients with both shock and respiratory failure (placebo [n = 51], 33%, vs. high dose [n = 52], 19%, p = .10). Transient, dose-related increases in methemoglobin levels were observed with TAK-242 treatment in 30.1% of the patients. CONCLUSIONS: TAK-242 failed to suppress cytokine levels in patients with sepsis and shock or respiratory failure. Treatment with TAK-242 resulted in mild increases in serum methemoglobin levels but was otherwise well tolerated. Although observed mortality rates in patients with both shock and respiratory failure were lower with the 2.4 mg/kg/day dose, differences were not significant.

Effect of Toll-like receptor 4 inhibitor on LPS-induced lung injury.

OBJECTIVE AND DESIGN: Toll-like receptor 4 (TLR4) plays important roles in the recognition of lipopolysaccharide (LPS) and the activation of inflammatory cascade. In this study, we evaluated the effect of TAK-242, a selective TLR4 signal transduction inhibitor, on acute lung injury (ALI). MATERIALS AND METHODS: C57BL/6J mice were intravenously treated with TAK-242 15 min before the intratracheal administration of LPS or Pam3CSK4, a synthetic lipopeptide. Six hours after the challenge, bronchoalveolar lavage fluid was obtained for a differential cell count and the measurement of cytokine and myeloperoxidase levels. Lung permeability and nuclear factor-kappaB (NF-kappaB) DNA binding activity were also evaluated. RESULTS: TAK-242 effectively attenuated the neutrophil accumulation and activation in the lungs, the increase in lung permeability, production of inflammatory mediators, and NF-kappaB DNA-binding activity induced by the LPS challenge. In contrast, TAK-242 did not suppress inflammatory changes induced by Pam3CSK4. CONCLUSION: TAK-242 may be a promising therapeutic agent for ALI, especially injuries associated with pneumonia caused by Gram-negative bacteria.

TAK-242 selectively suppresses Toll-like receptor 4-signaling mediated by the intracellular domain.

TAK-242, a small-molecule antisepsis agent, has shown to suppress lipopolysaccharide (LPS)-induced inflammation. In this study, we demonstrate that TAK-242 is a selective inhibitor of Toll-like receptor (TLR)-4 signaling. TAK-242 almost completely suppressed production of nitric oxide (NO) or tumor necrosis factor (TNF)-alpha induced by a TLR4-specific ligand, ultra-pure LPS, in mouse RAW264.7, human U-937 and P31/FUJ cells, whereas this agent showed little effect on other TLR ligands, Pam(3)CSK(4) (TLR1/2), peptidoglycan (TLR2/6), double strand RNA (TLR3), R-848 (TLR7) and CpG oligonucleotide (TLR9). Furthermore, TAK-242 potently inhibited nuclear factor (NF)-kappaB activation induced by ultra-pure LPS in HEK293 cells transiently expressing TLR4 and co-receptors, myeloid differentiation protein-2 (MD2) and CD14, whereas this agent showed little effect on other TLRs, TLR1/2, TLR2/6, TLR3, TLR5, TLR7 and TLR9. TAK-242 also inhibited ligand-independent NF-kappaB activation resulting from over-expression of TLR4. Although chimera receptors, which are consist of the extracellular domain of CD4 and the intracellular domain of human or mouse TLR4, showed constitutive NF-kappaB activation, TAK-242 potently inhibited the signaling from CD4-TLR4 chimera receptors. In contrast, the NF-kappaB activation mediated by TLR4 adaptors, myeloid differentiation factor 88 (MyD88), TIR-associated protein (TIRAP), Toll/IL-1R homology (TIR)-domain-containing adaptor protein-inducing interferon-beta (TRIF) or TRIF-related adaptor molecule (TRAM) was not affected by TAK-242. TAK-242 is therefore a selective inhibitor of signaling from the intracellular domain of TLR4 and represents a novel therapeutic approach to the treatment of TLR4-mediated diseases.

Novel cyclohexene derivatives as anti-sepsis agents: synthetic studies and inhibition of NO and cytokine production.

In order to develop an anti-sepsis agent, a series of cyclohexene derivatives were synthesized and evaluated for their biological activities. Through modification of the sulfonamide spacer moiety depicted by formula II, it was found that the benzylsulfone derivative 10a had potent inhibitory activity against the production of NO. Further modifications of the phenyl ring, ester moiety, and benzyl position of benzylsulfone derivatives III were carried out. Among these compounds, (R)-(+)-10a and (6R, 1S)-(+)-22a showed strong inhibitory activity not only against NO production but also against inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in vitro. Furthermore, (R)-(+)-10a and (6R, 1S)-(+)-22a protected mice from LPS-induced lethality in a dose-dependent manner.

Therapeutic effects of TAK-242, a novel selective Toll-like receptor 4 signal transduction inhibitor, in mouse endotoxin shock model.

Ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), a novel small molecule that selectively inhibits Toll-like receptor 4-mediated signaling, inhibits various kinds of inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6, IL-10, macrophage inhibitory protein (MIP)-2 and prostaglandin E2 from lipopolysaccharide (LPS)-stimulated macrophages. The effects of TAK-242 were evaluated in a mouse model of endotoxin shock. Intravenous administration of TAK-242 to mice 1 h before LPS challenge dose-dependently inhibited LPS-induced increases in serum levels of TNF-alpha, IL-1beta, IL-6, IL-10, MIP-2, and NO metabolites. TAK-242 protected mice from LPS-induced lethality in a similar dose-dependent manner, and rescued 100% of mice at a dose of 1 mg/kg. Interestingly, TAK-242 worked quickly, and showed beneficial effects even when administered after LPS challenge. Even though increases in serum levels of IL-6 and hypothermia were already evident 2 h after LPS challenge, TAK-242 administration inhibited further increase in IL-6 levels and decrease in body temperature. LPS-induced increases in serum levels of organ dysfunction markers, such as alanine aminotransferase, total bilirubin, and blood urea nitrogen, were also significantly suppressed by post-treatment as well as pre-treatment. Furthermore, administration of 3 mg/kg TAK-242 significantly increased survival of mice, even when given 4 h after LPS challenge. These results suggest that TAK-242 protects mice against LPS-induced lethality by inhibiting production of multiple cytokines and NO. TAK-242 has a quick onset of action and provides significant benefits by post-treatment, suggesting that it may be a promising drug candidate for the treatment of sepsis.

NF-kappa B-inducing kinase regulates selected gene expression in the Nod2 signaling pathway.

The innate immune system surveys the extra- and intracellular environment for the presence of microbes. Among the intracellular sensors is a protein known as Nod2, a cytosolic protein containing a leucine-rich repeat domain. Nod2 is believed to play a role in determining host responses to invasive bacteria. A key element in upregulating host defense involves activation of the NF-kappaB pathway. It has been suggested through indirect studies that NF-kappaB-inducing kinase, or NIK, may be involved in Nod2 signaling. Here we have used macrophages derived from primary explants of bone marrow from wild-type mice and mice that either bear a mutation in NIK, rendering it inactive, or are derived from NIK-/- mice, in which the NIK gene has been deleted. We show that NIK binds to Nod2 and mediates induction of specific changes induced by the specific Nod2 activator, muramyl dipeptide, and that the role of NIK occurs in settings where both the Nod2 and TLR4 pathways are activated by their respective agonists. Specifically, we have linked NIK to the induction of the B-cell chemoattractant known as BLC and suggest that this chemokine may play a role in processes initiated by Nod2 activation that lead to improved host defense.

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.

Proinflammatory mediators such as cytokines and NO play pivotal roles in various inflammatory diseases. To combat inflammatory diseases successfully, regulation of proinflammatory mediator production would be a critical process. In the present study, we investigated the in vitro effects of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), a novel small molecule cytokine production inhibitor, and its mechanism of action. In RAW264.7 cells and mouse peritoneal macrophages, TAK-242 suppressed lipopolysaccharide (LPS)-induced production of NO, tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-6, with 50% inhibitory concentration (IC50) of 1.1 to 11 nM. TAK-242 also suppressed the production of these cytokines from LPS-stimulated human peripheral blood mononuclear cells (PBMCs) at IC50 values from 11 to 33 nM. In addition, the inhibitory effects on the LPS-induced IL-6 and IL-12 production were similar in human PBMCs, monocytes, and macrophages. TAK-242 inhibited mRNA expression of IL-6 and TNF-alpha induced by LPS and interferon-gamma in RAW264.7 cells. The phosphorylation of mitogen-activated protein kinases induced by LPS was also inhibited in a concentration-dependent manner. However, TAK-242 did not antagonize the binding of LPS to the cells. It is noteworthy that TAK-242 suppressed the cytokine production induced by Toll-like receptor (TLR) 4 ligands, but not by ligands for TLR2, -3, and -9. In addition, IL-1beta-induced IL-8 production from human PBMCs was not markedly affected by TAK-242. These data suggest that TAK-242 suppresses the production of multiple cytokines by selectively inhibiting TLR4 intracellular signaling. Finally, TAK-242 is a novel small molecule TLR4 signaling inhibitor and could be a promising therapeutic agent for inflammatory diseases, whose pathogenesis involves TLR4.

Discovery of novel and potent small-molecule inhibitors of NO and cytokine production as antisepsis agents: synthesis and biological activity of alkyl 6-(N-substituted sulfamoyl)cyclohex-1-ene-1-carboxylate.

To develop a new therapeutic agent for sepsis, screening of the Takeda chemical library was carried out using mouse macrophages stimulated with lipopolysaccharide (LPS) to identify a new class of small-molecule inhibitors of inflammatory mediator production. The lead compound 5a was discovered, from which a series of novel cyclohexene derivatives I bearing a sulfamoyl and ester group were designed, synthesized and tested for their inhibitory activity against nitric oxide (NO) production. Derivatives I were synthesized by the coupling of sulfonyl chlorides and anilines with concomitant double bond migration in the presence of triethylamine, and phenyl ring substitution and modification of the ester and cyclohexene moieties were carried out. Among the compounds synthesized, ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate [(R)-(+)-5n, TAK-242] was found to exhibit the most potent suppressive activity for the production of not only NO but also inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) induced by LPS-stimulated mouse macrophages with IC50 values of 1.8, 1.9 and 1.3 nM, respectively. It shows marked beneficial effects in vivo also. Intravenous administration of (R)-(+)-5n at doses of 0.1 mg/kg or more suppressed the production of NO and various cytokines [TNF-alpha, IL-6 and IL-1beta] in the mouse endotoxin shock model. Furthermore, it protected mice from death dose-dependently and all mice survived at a dose of 3 mg/kg. The minimum effective dose to protect mice from lethality in this model was 0.3 mg/kg, which was consistent with those for inhibitory effects on the production of NO and cytokines. Compound (R)-(+)-5n is currently undergoing clinical trials for the treatment of sepsis.