c-Fos/activator protein (AP)-1; MMP-3 (IC50 = 10 nM); MMP-13 (IC50 = 10 nM)

With a mean IC50 of roughly 10 μM, T-5224 inhibits the in-vitro production of the mediators MMP-1, MMP-3, IL-6, and TNF-α by IL-1β-stimulated human synovial SW982 cells[2]. In a dose-dependent manner, T- 5224 (0-80 μM) significantly inhibits the invasion, migration, and MMP activity of HSC-3-M3 cells[3].

After intraperitoneal injection of LPS, administration of T-5224 (300 mg/kg, po) reduces the lethality (27%) and provides significant protection against acute elevations in serum levels of TNFα, HMGB1, ALT/AST, and MIP-1α and MCP-1 in liver tissue[4]. It is possible that G2 is not a metabolite that is unique to humans because it is found in rat and monkey liver microsomes as a major metabolite of T-5224[5]. In C57BL/6 mice, T-5224 (300 mg/kg, po) suppresses the synthesis of TNF-alpha and other downstream effectors[6].

In vitro assay for cytokines and MMPs.[2]
Human SW982 and SW1353 cells were cultured in 0.5% FBS/RPMI1640 and 0.2% lactalbumin hydrolysate/DMEM, respectively, overnight. After replacing the media with media containing T-5224, MTX or LEF (A77 1726) plus IL-1β (1 ng/ml for SW982 and 10 ng/ml for SW1353), cells were cultured for 24 h and the supernatants were assayed.[2]

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Enzyme-linked immunosorbent assay (ELISA).[2]
ELISAs were carried out using Quantikine Mouse IL-1β/IL-1F2 and TNF-α/TNFSF1A Immunoassay, Immunoassay Kit Mouse IL-6, K-ASSAY Mouse and Rat COMP ELISA , Quantikine Mouse MMP-3 (total) Immunoassay and Mouse IgG Anti-type II Collagen Antibody Assay Kit for mice and MMP-1 Human Biotrak ELISA System, Quantikine Human MMP-3 (total) Immunoassay, Human Interleukin-6 ELISA Kit, and High sensitivity (h)TNFα and (h)MMP-13 Human Biotrak ELISA System for human.

Isolation of human NP cells[1]
Human NP tissue was obtained during surgery for scoliosis after receiving the patient’s informed consent. Tissue samples were digested at 37 °C overnight. After digestion, the isolated NP cells were cultured as a monolayer in DMEM containing 10% foetal bovine serum. Low-passage cells (passage 2) were used for all experiments. When the cells were 80% confluent, they were cultured in serum-free DMEM for 12 h and then treated with 10 ng/ml IL-1β with different concentrations of T-5224 or the vehicle.

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Mouse IVD explant culture[1]
Lumbar IVDs were harvested from 2-week-old mice and cultured as previously described64 in 500 µL α-modified essential medium. IVDs were treated with 10 ng/ml mouse IL-1β for 24 h with varying concentrations of T-5224.

Mice were housed in an SPF (specific pathogen free) grade environment and provided food and water ad libitum with a 12 h:12 h light/dark cycle. Male 8-week-old DBA/1J mice (Charles River) were immunized with bovine type II collagen (Koken) emulsified in Freund's complete adjuvant on days 0 and 21. T-5224, MTX and LEF were orally administered once per day. Arthritis was assessed in a blind fashion for four paws per mouse using the following score: 0, uninvolved; 1, swelling of ≤2 toes or slight swelling in ankles and wrists; 2, swelling of ≥3 toes or moderate swelling in ankles and wrists; 3, extensive swelling of total paw. X-ray films of four paws taken using Softex were assessed for joint destruction in 2nd to 5th proximal interphalangeal joints and five metatarsophalangeal joints of four paws, the carpal joints of the fore paws, and the tarsal and calcaneal joints of the hind paws. Score was: 0, no change; 1, partial erosion; 2, complete erosion for joints; and 0, negative; 0.5, positive for osteoporosis. IL-1β (500 ng per unilateral hind paw) was administered into the foot pads (Fig. 4e). The mice with ≥1 arthritis score were treated with either anti-TNFα antibody (TN3-19.12, R&D Systems) at 50 or 250 μg/mouse, intraperitoneally (i.p.) twice a week and/or with 3 mg/kg T-5224, orally once daily.[2]

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Endotoxemic liver injury was induced by intraperitoneal injection of LPS. Mice were randomly divided into three groups: control, LPS, and LPS + T-5224. LPS (10, 0.008 ml g−1 body wt) was injected intraperitoneally in the LPS and LPS + T-5224 groups, as was normal saline (0.008 ml g−1 body wt) in the control group. Vehicle (0.01 ml g−1 body wt) was administered orally in the LPS group and T-5224 (300 mg kg−1, 0.01 ml g−1 body wt) was administered orally in the control and LPS + T-5224 groups immediately after LPS injection. In a preliminary study, we found that 300 mg kg−1 T-5224 was more effective in reducing TNFα production than 30 mg kg−1 (data not shown).[4]

150, 300 mg/kg, p.o.

BALB/c nude mouse

[1]. A selective inhibition of c-Fos/activator protein-1 as a potential therapeutic target for intervertebraldisc degeneration and associated pain. Sci Rep. 2017 Dec 5;7(1):16983.

[2]. Treatment of arthritis with a selective inhibitor of c-Fos/activator protein-1. Nat Biotechnol. 2008 Jul;26(7):817-23.

[3]. Selective activator protein-1 inhibitor T-5224 prevents lymph node metastasis in an oral cancer model. Cancer Sci.?2016 May;107(5):666-73.

[4]. T-5224, a selective inhibitor of c-Fos/activator protein-1, attenuates lipopolysaccharide-induced liver injury in mice. Biotechnol Lett. 2012 Dec;34(12):2175-82.

[5]. Metabolism of the c-Fos/activator protein-1 inhibitor T-5224 by multiple human UDP-glucuronosyltransferase isoforms. Drug Metab Dispos. 2011 May;39(5):803-13.

[6]. The effects of a selective inhibitor of c-Fos/activator protein-1 on endotoxin-induced acute kidney injury in mice. BMC Nephrol. 2012 Nov 23;13:153.

Intervertebral disc (IVD) degeneration is a major cause of low back pain. The transcription factor c-Fos/Activator Protein-1 (AP-1) controls the expression of inflammatory cytokines and matrix metalloproteinases (MMPs) that contribute to the pathogenesis IVD degeneration. We investigated the effects of inhibition of c-Fos/AP-1 on IVD degeneration and associated pain. A selective inhibitor, T-5224, significantly suppressed the interleukin-1β-induced up-regulation of Mmp-3, Mmp-13 and Adamts-5 transcription in human nucleus pulposus cells and in a mouse explant culture model of IVD degeneration. We used a tail disc percutaneous needle puncture method to further assess the effects of oral administration of T-5224 on IVD degeneration. Analysis of disc height, T2-magnetic resonance imaging (MRI) findings, and histology revealed that IVD degeneration was significantly mitigated by T-5224. Further, oral administration of T-5224 ameliorated pain as indicated by the extended tail-flick latency in response to heat stimulation of rats with needle-puncture-induced IVD degeneration. These findings suggest that the inhibition of c-Fos/AP-1 prevents disc degeneration and its associated pain and that T-5224 may serve as a drug for the prevention of IVD degeneration.[1]

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To inhibit arthritis upstream of inflammatory cytokine release and matrix metalloproteinase (MMP) action, we designed de novo a small-molecule inhibitor of c-Fos/activator protein-1 (AP-1) using three-dimensional (3D) pharmacophore modeling. This model was based on the 3D structure of the basic region-leucine zipper domain of AP-1-DNA complex. Administration of this inhibitor prevented type II collagen-induced arthritis from day 21, before the onset of arthritis, or from day 27, resolved arthritis after its onset. Suppression of disease was accomplished by reducing the amounts of inflammatory cytokines and MMPs in vivo in sera and joints and in vitro in synovial cell and chondrocyte cultures. The primary action of this molecule was the inhibition of matrix-degrading MMPs and inflammatory cytokines including interleukin 1beta; this molecule also synergized with anti-tumor necrosis factor alpha to inhibit arthritis. Thus, selective inhibition of c-Fos/AP-1 resolves arthritis in a preclinical model of the disease.[2]

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Activator protein-1 (AP-1) is a transcriptional factor that regulates the expression of various genes associated with tumor invasion and migration. The purpose of our study was to assess the therapeutic effects of a novel selective AP-1 inhibitor, T-5224, in preventing lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) in an orthotopic mouse model. We assessed the effect of T-5224 on HNSCC cell invasion, migration, proliferation, and MMP activity by carrying out an in vitro study using an invasion assay, scratch assay, WST-8 assay, and gelatin zymography. We also observed morphological changes in HNSCC cells by time-lapse microscopy. Furthermore, cervical lymph node metastasis was assessed using an orthotopic tumor model of human oral squamous cell carcinoma cells (HSC-3-M3) injected in the tongue of a BALB/c nude mouse. T-5224 (150 mg/kg) or vehicle was given orally every day for 4 weeks. Animals were killed and assessed for lymph node metastasis by H&E staining of resected lymph nodes. T-5224 significantly inhibited the invasion, migration, and MMP activity of HNSCC cells in a dose-dependent manner; there was no significant influence on cell proliferation. The antimetastatic effect of T-5224 was also confirmed in our animal study. The rate of cervical lymph node metastasis in the model was 40.0% in the T-5224-treated group (n = 30) versus 74.1% in the vehicle-treated group (n = 27; P < 0.05). In conclusion, T-5224 inhibited the invasion and migration of HNSCC cells in vitro, and prevented lymph node metastasis in head and neck cancer in an animal model.[3]

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The effect of T-5224, a selective inhibitor of c-Fos/activator protein (AP)-1, on lipopolysaccharide (LPS) induced liver injury was examined in mice. Administration of LPS (10 mg kg(-1), i.p.) markedly increased serum levels of tumor necrosis factor-alpha (TNFα), high mobility group box 1 (HMGB1), alanine aminotransferase/aspartate aminotransferase (ALT/AST), liver tissue levels of macrophage-inflammatory protein-1 alpha (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1), as well as hepatic necrosis and inflammation, leading to 67 % lethality. Administration of T-5224 (300 mg kg(-1), p.o.) after intraperitoneal injection of LPS imparted appreciable protection against acute elevations in serum levels of TNFα, HMGB1, ALT/AST as well as in liver tissue levels of MIP-1α and MCP-1, and reduced the lethality (27 %). These data indicate that T-5224 ameliorates liver injury and improves survival through decreasing production of proinflammatory cytokines and chemokines in endotoxemic mice.[4]

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We developed 3-{5-[4-(cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazol-6-yl)methoxy]phenyl} propionic acid (T-5224) as a novel inhibitor of the c-Fos/activator protein-1 for rheumatoid arthritis therapy. We predicted the metabolism of T-5224 in humans by using human liver microsomes (HLM), human intestinal microsomes (HIM), recombinant human cytochrome P450 (P450), and UDP-glucuronosyltransferases (UGTs). T-5224 was converted to its acyl O-glucuronide (G2) by UGT1A1 and UGT1A3 and to its hydroxyl O-glucuronide (G3) by several UGTs, but it was not metabolized by the P450s. A comparison of the intrinsic clearances (CL(int)) between HLM and HIM suggested that the glucuronidation of T-5224 occurs predominantly in the liver. UGT1A1 showed a higher k(cat)/K(m) value than UGT1A3 for G2 formation, but a lower k(cat)/K(m) value than UGT1A3 for G3 formation. A high correlation was observed between G2 formation activity and UGT1A1-specific activity (β-estradiol 3-glucuronidation) in seven individual HLM. A high correlation was also observed between G2 formation activity and UGT1A1 content in the HLM. These results strongly suggest that UGT1A1 is responsible for G2 formation in human liver. In contrast, no such correlation was observed with G3 formation, suggesting that multiple UGT isoforms, including UGT1A1 and UGT1A3, are involved in G3 formation. G2 is also observed in rat and monkey liver microsomes as a major metabolite of T-5224, suggesting that G2 is not a human-specific metabolite. In this study, we obtained useful information on the metabolism of T-5224 for its clinical use.[5]

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Background: Sepsis has been identified as the most common cause of acute kidney injury (AKI) in intensive care units. Lipopolysaccharide (LPS) induces the production of several proinflammatory cytokines including tumor necrosis factor (TNF)-alpha, a major pathogenetic factor in septic AKI. c-Fos/activator protein (AP)-1 controls the expression of these cytokines by binding directly to AP-1 motifs in the cytokine promoter regions. T-5224 is a new drug developed by computer-aided drug design that selectively inhibits c-Fos/AP-1 binding to DNA. In this study, we tested whether T-5224 has a potential inhibitory effect against LPS-induced AKI, by suppressing the TNF-alpha inflammatory response and other downstream effectors.[6]
Methods: To test this hypothesis, male C57BL/6 mice at 7 weeks old were divided into three groups (control, LPS and T-5224 groups). Mice in the control group received saline intraperitoneally and polyvinylpyrrolidone solution orally. Mice in the LPS group were injected intraperitoneally with a 6 mg/kg dose of LPS and were given polyvinylpyrrolidone solution immediately after LPS injection. In the T-5224 group, mice were administered T-5224 orally at a dose of 300 mg/kg immediately after LPS injection. Serum concentrations of TNF-alpha, interleukin (IL)-1beta, IL-6 and IL-10 were measured by ELISA. Moreover, the expression of intercellular adhesion molecule (ICAM)-1 mRNA in kidney was examined by quantitative real-time RT-PCR. Finally, we evaluated renal histological changes.[6]
Results: LPS injection induced high serum levels of TNF-alpha, IL-1beta and IL-6. However, the administration of T-5224 inhibited the LPS-induced increase in these cytokine levels. The serum levels of IL-10 in the LPS group and T-5224 group were markedly elevated compared with the control group. T-5224 also inhibited LPS-induced ICAM-1 mRNA expression. Furthermore histological studies supported an anti-inflammatory role of T-5224.[6]
Conclusions: In endotoxin-induced AKI, T-5224 inhibited the production of TNF-alpha and other downstream effectors. In contrast, T-5224 did not inhibit IL-10, an anti-inflammatory cytokine. These data support that the use of T-5224 is a promising new treatment for septic kidney injury.[6]

C29H27NO8

517.53

517.173

C, 67.30; H, 5.26; N, 2.71; O, 24.73

530141-72-1

23626877

Typically exists as White to yellow solids at room temperature

1.4±0.1 g/cm3

774.1±60.0 °C at 760 mmHg

422.0±32.9 °C

0.0±2.8 mmHg at 25°C

1.665

5.14

3

8

10

38

844

0

O(C1C([H])=C([H])C(C(C2C([H])=C([H])C(=C(C=2[H])C([H])([H])C([H])([H])C(=O)O[H])OC([H])([H])C2C([H])=C([H])C3C(N([H])OC=3C=2[H])=O)=O)=C(C=1[H])O[H])C1([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

DALCQQSLNPLQFZ-UHFFFAOYSA-N

InChI=1S/C29H27NO8/c31-24-15-21(37-20-3-1-2-4-20)8-10-22(24)28(34)19-6-11-25(18(14-19)7-12-27(32)33)36-16-17-5-9-23-26(13-17)38-30-29(23)35/h5-6,8-11,13-15,20,31H,1-4,7,12,16H2,(H,30,35)(H,32,33)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 5 mg/mL (9.66 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: 5 mg/mL (9.66 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)