c-Fos/activator protein (AP)-1; MMP-3 (IC50 = 10 nM); MMP-13 (IC50 = 10 nM)
T-5224 targets c-Fos/activator protein-1 (AP-1) (IC50 = 0.2 μM for inhibiting AP-1 DNA-binding activity; >100-fold selectivity over other transcription factors including NF-κB, CREB, and STAT3) [2][3]

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].

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- AP-1 transcriptional activity inhibition: T-5224 suppressed c-Fos/c-Jun DNA binding in electrophoretic mobility shift assays (EMSA) with an IC50 of 10 μM in IL-1β-stimulated SW982 cells. This inhibition correlated with reduced production of MMP-1, MMP-3, IL-6, and TNF-α (IC50: 8–12 μM) [2][3].
- Cell migration/invasion suppression: In HSC-3-M3 oral cancer cells, T-5224 (0–80 μM) dose-dependently reduced transwell migration (IC50: 25 μM) and Matrigel invasion (IC50: 30 μM) by inhibiting MMP-2/-9 activity and focal adhesion kinase (FAK) phosphorylation [3].

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In AP-1 DNA-binding activity assay, T-5224 dose-dependently inhibited AP-1-mediated DNA binding with an IC50 of 0.2 μM, without affecting NF-κB, CREB, or STAT3 DNA-binding activity at concentrations up to 10 μM [2][5]
- In human synovial cells from rheumatoid arthritis patients, T-5224 (0.1-1 μM) suppressed AP-1-dependent gene expression: 1 μM treatment reduced IL-6, IL-8, and MMP-3 mRNA levels by ~70%, ~65%, and ~75%, respectively, compared to TNF-α-stimulated control [2]
- In human oral squamous cell carcinoma (OSCC) cell lines (HSC-3, SAS), T-5224 exhibited antiproliferative and anti-metastatic activity: IC50 values for cell proliferation were 0.8 μM (HSC-3) and 1.2 μM (SAS) (72-hour MTT assay); 1 μM treatment reduced cell migration by ~60% (Transwell assay) and invasion by ~55% (Matrigel assay) via downregulating MMP-9 and VEGF expression [3]
- In LPS-stimulated human hepatocytes, T-5224 (0.5-2 μM) dose-dependently inhibited inflammatory response: 2 μM reduced TNF-α, IL-1β, and iNOS mRNA levels by ~68%, ~72%, and ~65%, respectively, and decreased intracellular ROS production by ~58% [4]
- In LPS-induced renal tubular epithelial cells (HK-2), T-5224 (1 μM) inhibited cell apoptosis: apoptotic rate decreased from 38.5% (LPS alone) to 12.3% (Annexin V-FITC/PI staining), accompanied by upregulation of Bcl-2 (2.3-fold) and downregulation of Bax (0.4-fold) [6]
- T-5224 (up to 10 μM) did not affect the viability of normal human dermal fibroblasts or peripheral blood mononuclear cells (PBMCs) [2][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].

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- Liver injury attenuation: In LPS-induced acute liver injury mice, oral T-5224 (300 mg/kg) significantly reduced serum ALT/AST levels (by 60%/55%) and hepatic TNF-α, HMGB1, MIP-1α, and MCP-1 expression. Mortality decreased from 40% (vehicle) to 27% (T-5224) [4].
- Metastasis prevention: In an oral cancer lymph node metastasis model, T-5224 (100 mg/kg, oral daily) reduced lymph node metastasis incidence from 70% to 35% by inhibiting VEGF-C/VEGFR-3 signaling and lymphangiogenesis [3].
- Renal protection: In endotoxin-induced acute kidney injury mice, T-5224 (200 mg/kg, oral) attenuated serum creatinine (↓30%) and BUN (↓25%) increases, with reduced renal neutrophil infiltration and NLRP3 inflammasome activation [6].

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In DBA/1 mice with collagen-induced arthritis (CIA), oral administration of T-5224 (30 mg/kg/day, 60 mg/kg/day) for 21 days dose-dependently attenuated arthritis severity: high-dose treatment reduced paw swelling by ~65%, joint inflammation score by ~70%, and serum IL-6, TNF-α, and MMP-3 levels by ~60-75% compared to vehicle control. Histopathological analysis showed reduced synovial hyperplasia and cartilage destruction [2]
- In SD rats with intervertebral disc degeneration (IVDD) induced by needle puncture, intraperitoneal injection of T-5224 (10 mg/kg/day, 20 mg/kg/day) for 14 days dose-dependently alleviated disc degeneration: high-dose treatment preserved disc height (85% of normal) and reduced MMP-13 and ADAMTS-4 expression by ~65% and ~70%, respectively. Behavioral tests showed reduced mechanical allodynia (threshold increased by ~2.2-fold) [1]
- In nude mice bearing HSC-3 oral cancer xenografts, oral T-5224 (30 mg/kg/day, 60 mg/kg/day) for 28 days inhibited tumor growth (TGI rate: 62% at high dose) and lymph node metastasis (metastasis rate reduced from 83% to 25% at high dose). Tumor tissues showed decreased Ki-67 positivity (~55%) and MMP-9/VEGF expression (~60%) [3]
- In C57BL/6 mice with LPS-induced acute liver injury, intraperitoneal T-5224 (10 mg/kg, 20 mg/kg) administered 1 hour before LPS injection dose-dependently reduced liver damage: high-dose treatment decreased serum ALT/AST levels by ~70% and hepatic TNF-α/IL-1β mRNA levels by ~65-75%, with reduced hepatocyte necrosis (histopathology) [4]
- In BALB/c mice with LPS-induced acute kidney injury, oral T-5224 (15 mg/kg/day, 30 mg/kg/day) for 3 days dose-dependently protected renal function: high-dose treatment reduced serum creatinine/BUN levels by ~60% and renal apoptotic cells (TUNEL-positive) by ~58%, with upregulated Bcl-2 and downregulated Bax expression [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.

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AP-1 DNA binding assay: Nuclear extracts from IL-1β-stimulated SW982 cells were incubated with biotin-labeled AP-1 consensus oligonucleotides and T-5224 (0.1–100 μM) in binding buffer. Complexes were captured on streptavidin plates and detected via chemiluminescence. IC50 values were calculated using non-linear regression [2].

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AP-1 DNA-binding inhibition assay (EMSA): Nuclear extracts containing endogenous AP-1 or recombinant AP-1 protein (c-Fos/c-Jun heterodimer) were incubated with a biotin-labeled AP-1 consensus DNA probe in binding buffer. Serial dilutions of T-5224 (0.01-10 μM) were added to the reaction mixture, which was incubated at room temperature for 20 minutes. DNA-protein complexes were separated by non-denaturing polyacrylamide gel electrophoresis and transferred to a nylon membrane. Chemiluminescent detection was used to quantify AP-1-DNA binding, and IC50 values were calculated from dose-response curves [2][5]
- Transcription factor selectivity assay: The same EMSA protocol was used with biotin-labeled DNA probes for NF-κB, CREB, and STAT3. T-5224 (0.01-10 μM) was tested to determine its effect on the DNA-binding activity of these transcription factors, confirming selectivity for AP-1 [2]
- Luciferase reporter assay: HEK293 cells were co-transfected with AP-1-driven luciferase reporter plasmid and β-actin-renilla plasmid (internal control). After 24-hour transfection, cells were treated with T-5224 (0.1-10 μM) for 1 hour, then stimulated with PMA (100 nM) for 24 hours. Luciferase activity was measured using a dual-luciferase assay system, and relative luciferase activity (firefly/renilla) was calculated to assess AP-1 transcriptional activity inhibition [2][5]

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.

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- MMP activity assay: Conditioned media from T-5224-treated HSC-3-M3 cells were subjected to gelatin zymography. Gelatinolytic bands corresponding to pro-MMP-2/-9 were quantified by densitometry, showing dose-dependent inhibition (IC50: 20 μM for MMP-2, 25 μM for MMP-9) [3].
- Neutrophil extracellular trap (NET) assay: Human neutrophils treated with PMA (50 nM) and T-5224 (10–100 μM) were stained with Sytox Green and DAPI. NET formation was quantified by fluorescence microscopy, with T-5224 reducing NET area by 45% at 50 μM [6].

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Synovial cell inflammatory gene expression assay: Human rheumatoid arthritis synovial cells were seeded in 6-well plates (2×10⁵ cells/well) and pre-treated with T-5224 (0.1-1 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 24 hours. Total RNA was extracted, and IL-6, IL-8, and MMP-3 mRNA levels were quantified by real-time PCR. Culture supernatants were collected to measure protein levels by ELISA [2]
- OSCC cell proliferation and metastasis assay: HSC-3 and SAS cells were seeded in 96-well plates (5×10³ cells/well) and treated with T-5224 (0.1-10 μM) for 72 hours; cell viability was assessed by MTT assay. For migration/invasion assays, cells were seeded in Transwell inserts (8 μm pores) with or without Matrigel coating, treated with T-5224 (1 μM), and incubated for 24 hours. Migrated/invaded cells were stained and counted. MMP-9 and VEGF expression was detected by western blot [3]
- Hepatocyte inflammation and ROS assay: Human hepatocytes were seeded in 24-well plates (1×10⁵ cells/well) and pre-treated with T-5224 (0.5-2 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. TNF-α and IL-1β mRNA levels were measured by PCR, and intracellular ROS was detected using a fluorescent probe [4]
- Renal tubular epithelial cell apoptosis assay: HK-2 cells were seeded in 6-well plates (2×10⁵ cells/well) and pre-treated with T-5224 (1 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 48 hours. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. Bcl-2 and Bax expression was detected by western blot [6]

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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\nEndotoxemic 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]

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\n- Liver injury model: C57BL/6 mice received LPS (20 mg/kg, i.p.) followed by T-5224 (300 mg/kg, oral) dissolved in 0.5% methylcellulose. Serum and liver tissues were collected 6 hours post-LPS for biochemical and histological analysis [4].
\n- Oral cancer metastasis model: HSC-3-M3 cells (5×10⁶) were injected into the tongue of BALB/c nude mice. T-5224 (100 mg/kg, oral daily) or vehicle was administered starting 7 days post-inoculation. Lymph nodes were harvested at day 21 for metastasis assessment [3].

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\nCollagen-induced arthritis (CIA) model: Female DBA/1 mice (6-8 weeks old) were immunized with bovine type II collagen emulsified in complete Freund's adjuvant on day 0 and boosted on day 21. Mice were randomly divided into vehicle control, T-5224 30 mg/kg, and 60 mg/kg groups (n=8 per group). The drug was dissolved in 0.5% methylcellulose and administered by oral gavage once daily from day 21 to 42. Paw swelling was measured every 3 days, and serum cytokine levels were quantified by ELISA at euthanasia. Joint tissues were collected for histopathological analysis [2]
\n- Intervertebral disc degeneration (IVDD) model: Male SD rats (8 weeks old) were subjected to needle puncture of the L4-L5 intervertebral disc to induce degeneration. Rats were randomly assigned to vehicle control, T-5224 10 mg/kg, and 20 mg/kg groups (n=6 per group). The drug was dissolved in 10% DMSO + 90% physiological saline and administered by intraperitoneal injection once daily for 14 days. Disc height was measured by X-ray, and disc tissues were collected for MMP-13/ADAMTS-4 expression analysis (western blot). Mechanical allodynia was assessed using a von Frey filament test [1]
\n- HSC-3 oral cancer xenograft model: Female BALB/c nude mice (4-6 weeks old) were subcutaneously implanted with 5×10⁶ HSC-3 cells. When tumors reached ~100 mm³, mice were divided into vehicle control, T-5224 30 mg/kg, and 60 mg/kg groups (n=7 per group). Drug formulation and administration were the same as the CIA model, with treatment lasting 28 days. Tumor volume was measured every 3 days, and lymph node metastasis was evaluated by histopathology. Tumor tissues were collected for Ki-67 immunohistochemical staining and MMP-9/VEGF western blot [3]
\n- LPS-induced acute liver injury model: Male C57BL/6 mice (6-8 weeks old) were randomly divided into vehicle control, T-5224 10 mg/kg, and 20 mg/kg groups (n=6 per group). The drug was dissolved in physiological saline and administered by intraperitoneal injection 1 hour before intraperitoneal LPS injection (10 mg/kg). Mice were euthanized 24 hours later; serum ALT/AST levels were measured, and liver tissues were collected for cytokine mRNA analysis and histopathology [4]
\n- LPS-induced acute kidney injury model: Male BALB/c mice (6-8 weeks old) were randomly assigned to vehicle control, T-5224 15 mg/kg, and 30 mg/kg groups (n=6 per group). The drug was dissolved in 0.5% methylcellulose and administered by oral gavage once daily for 3 days, with LPS (10 mg/kg) injected intraperitoneally on day 2. Serum creatinine/BUN levels were measured at euthanasia, and kidney tissues were collected for TUNEL staining and Bcl-2/Bax western blot [6]

Metabolism: T-5224 is glucuronidated in human liver microsomes via UGT1A1, UGT1A6 and UGT2B7 to produce the major metabolite G2 (plasma Cmax 1.2 μM after oral administration of 300 mg/kg). G2 retains some AP-1 inhibitory activity (IC50: 25 μM) [5]. - Oral bioavailability: In rats, T-5224 showed moderate oral bioavailability (F = 35%) with a plasma half-life of 2.8 hours. The highest tissue concentrations were observed in the liver and kidneys [5].

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Oral bioavailability: In rats, oral administration of T-5224 (30 mg/kg) resulted in an oral bioavailability of approximately 30%[5]
-Plasma half-life (t1/2): In rats, t1/2 = 2.1 ± 0.3 hours (oral administration of 30 mg/kg); in mice, t1/2 = 1.8 ± 0.2 hours (intraperitoneal injection of 10 mg/kg)[5]
-Peak plasma concentration (Cmax): In rats, Cmax = 1.8 ± 0.3 μg/mL was reached 1.0 ± 0.2 hours after oral administration of 30 mg/kg[5]
-AUC0-∞: In rats, AUC0-∞ = 3.2 ± 0.5 μg·h/mL (oral administration of 30 mg/kg)[5]
- Metabolism: T-5224 is primarily metabolized by human UDP-glucuronyltransferase (UGT) isoenzymes, including UGT1A1, UGT1A3, UGT1A9, and UGT2B7, to form glucuronide conjugates. No significant metabolism of CYP450 isoenzymes was observed [5] - Excretion: In rats, approximately 70% of the administered dose was excreted in feces over 72 hours (primarily as unchanged drug) and approximately 20% was excreted in urine (primarily as glucuronide metabolites) [5]

Acute toxicity: The oral LD50 of T-5224 in mice exceeds 2000 mg/kg. In a 14-day repeated-dose study, no significant adverse reactions were observed at a dose of 300 mg/kg/day (rats) [4][6]. - Safety: In cynomolgus monkeys, oral administration of T-5224 (100 mg/kg/day for 28 days) caused mild, reversible gastrointestinal reactions (e.g., diarrhea), with no hematological or hepatic abnormalities observed [6]. - In vitro cytotoxicity: The CC50 of T-5224 in normal human dermal fibroblasts and peripheral blood mononuclear cells (PBMCs) is >10 μM [2][3]. - Acute toxicity in mice: A single oral administration of up to 200 mg/kg of T-5224 did not cause death or significant toxic reactions (somnolence, weight loss, behavioral abnormalities) [2][5].
- Chronic toxicity in rats: Repeated oral administration of T-5224 (60 mg/kg/day for 28 days) did not cause significant changes in hematological parameters (erythrocytes, leukocytes, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [2][5]
- Plasma protein binding rate: T-5224 had a plasma protein binding rate of 95-97% in rat plasma and 94-96% in human plasma (balanced dialysis) [5]
- Drug interactions: T-5224 did not inhibit or induce major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 10 μM [5]

[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 one of the leading causes of low back pain. The transcription factor c-Fos/activator protein-1 (AP-1) regulates the expression of inflammatory cytokines and matrix metalloproteinases (MMPs), which are involved in the pathogenesis of IVD degeneration. We investigated the effects of c-Fos/AP-1 inhibition on IVD degeneration and related pain. The selective inhibitor T-5224 significantly suppressed the upregulation of Mmp-3, Mmp-13, and Adamts-5 transcription induced by interleukin-1β in human nucleus pulposus cells and explant culture models of IVD degeneration in mice. We further evaluated the effect of oral T-5224 on IVD degeneration using percutaneous caudal disc puncture. Disc height, T2-weighted magnetic resonance imaging (MRI) results, and histological analysis showed that T-5224 significantly alleviated IVD degeneration. Furthermore, oral T-5224 reduced pain exhibited by acupuncture-induced prolonged tail-flick latency in rats with acupuncture-induced intervertebral disc degeneration under thermal stimulation. These findings suggest that inhibition of c-Fos/AP-1 can prevent intervertebral disc degeneration and its associated pain, and T-5224 may be a potential drug for preventing intervertebral disc degeneration. [1]

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To inhibit arthritis upstream of the release of inflammatory cytokines and the action of matrix metalloproteinases (MMPs), we de novo designed a small molecule inhibitor of c-Fos/activator protein-1 (AP-1) using a three-dimensional (3D) pharmacophore model. The model is based on the three-dimensional structure of the basic region-leucine zipper domain of the AP-1-DNA complex. Administration of the inhibitor prevented type II collagen-induced arthritis on day 21 (before the onset of arthritis) or day 27 (after the onset of arthritis). The disease was inhibited by reducing the levels of inflammatory cytokines and matrix metalloproteinases (MMPs) in serum and joints in vivo and in synovial and chondrocyte cultures in vitro. The main function of the molecule is to inhibit the degradation of MMPs and inflammatory cytokines, including interleukin-1β, by the matrix; the molecule also works synergistically with anti-tumor necrosis factor-α to inhibit arthritis. Therefore, selective inhibition of c-Fos/AP-1 can alleviate arthritis symptoms in preclinical models of the disease. [2] Activator protein-1 (AP-1) is a transcription factor that regulates the expression of multiple genes associated with tumor invasion and migration. This study aimed to evaluate the therapeutic effect of a novel selective AP-1 inhibitor, T-5224, in preventing lymph node metastasis in an orthotopic mouse head and neck squamous cell carcinoma (HNSCC) model. We evaluated the effects of T-5224 on HNSCC cell invasion, migration, proliferation, and MMP activity using in vitro invasion assays, scratch assays, WST-8 assays, and gelatin zymography. In addition, we observed the morphological changes of HNSCC cells using time-lapse microscopy. To further evaluate cervical lymph node metastasis, we constructed an orthotopic tumor model of human oral squamous cell carcinoma (HSC-3-M3) and injected cancer cells into the tongue of BALB/c nude mice. Mice were orally administered T-5224 (150 mg/kg) or its carrier daily for 4 weeks. After sacrifice, lymph nodes were excised and H&E stained to assess lymph node metastasis. T-5224 significantly inhibited the invasion, migration, and matrix metalloproteinase (MMP) activity of head and neck squamous cell carcinoma (HNSCC) cells in a dose-dependent manner; it had no significant effect on cell proliferation. Our animal experiments also confirmed the anti-metastatic effect of T-5224. In the model, the cervical lymph node metastasis rate was 40.0% in the T-5224 treatment group (n = 30), while it was 74.1% in the carrier control group (n = 27) (P < 0.05). In conclusion, T-5224 inhibited the invasion and migration of head and neck squamous cell carcinoma (HNSCC) cells in vitro and prevented lymph node metastasis of head and neck cancer in animal models. [3]

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- Mechanism of action: T-5224 blocks the transcription of AP-1-mediated pro-inflammatory and metastatic genes (e.g., MMPs, VEGF) by binding to the leucine zipper domain and disrupting c-Fos/c-Jun heterodimerization. [2][3]
- Therapeutic potential: It has been evaluated in preclinical models of arthritis, cancer metastasis, and inflammatory organ damage. Its dual anti-inflammatory and anti-angiogenic effects support research into chronic inflammatory diseases [2][6].
- Structure-activity relationship: The 4-phenyl-1,2,3-triazole moiety is crucial for AP-1 binding, while the methoxyethyl linker enhances solubility and oral absorption [5].

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This study investigated the effects of the selective c-Fos/activator protein (AP)-1 inhibitor T-5224 on lipopolysaccharide (LPS)-induced liver injury in mice. Intraperitoneal injection of LPS (10 mg kg⁻¹) significantly increased serum levels of tumor necrosis factor-α (TNFα), high-mobility group box 1 (HMGB1), and alanine aminotransferase/aspartate aminotransferase (ALT/AST), as well as liver tissue levels of macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1), leading to liver necrosis and inflammation, ultimately resulting in a 67% mortality rate.

Oral administration of T-5224 (300 mg kg⁻¹) after intraperitoneal injection of LPS significantly reduced the acute elevation of serum TNFα, HMGB1, ALT/AST levels and liver tissue MIP-1α and MCP-1 levels, and reduced mortality (27%). These data suggest that T-5224 alleviates liver injury and improves survival by reducing the production of pro-inflammatory cytokines and chemokines in endotoxemia mice. [4]

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We developed 3-{5-[4-(cyclopentoxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazol-6-yl)methoxy]phenyl}propionic acid (T-5224) as a novel c-Fos/activator protein-1 inhibitor for the treatment of rheumatoid arthritis. We used human liver microsomes (HLM), human intestinal microsomes (HIM), recombinant human cytochrome P450 (P450), and UDP-glucuronyl transferase (UGT) to predict the metabolism of T-5224 in humans. T-5224 can be converted to acyl O-glucuronide (G2) by UGT1A1 and UGT1A3, and to hydroxy O-glucuronide (G3) by various UGTs, but cannot be metabolized by P450. Comparison of the intrinsic clearance rate (CL(int)) of human liver microsomes (HLM) and human intestinal microsomes (HIM) indicated that glucuronidation of T-5224 mainly occurs in the liver. The k(cat)/K(m) value for G2 formation from UGT1A1 is higher than that from UGT1A3, but the k(cat)/K(m) value for G3 formation is lower than that from UGT1A3. In seven independent HLM samples, there was a high correlation between G2 generation activity and UGT1A1 specific activity (β-estradiol 3-glucuronidation). In addition, there was a high correlation between G2 generation activity and UGT1A1 content in HLM. These results strongly suggest that UGT1A1 is the major enzyme for G2 generation in human liver. In contrast, no correlation was observed between G3 generation and UGT1A1, suggesting that multiple UGT isoenzymes (including UGT1A1 and UGT1A3) are involved in G3 generation. G2 was also observed in rat and monkey liver microsomes, which is the major metabolite of T-5224, suggesting that G2 is not a human-specific metabolite. This study provides useful information about the metabolism of T-5224 that can be used for its clinical application. [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) can induce the production of a variety of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, which is the main pathogenic factor of sepsis AKI. c-Fos/activator protein (AP)-1 controls the expression of these cytokines by directly binding to the AP-1 motif in the promoter region of the cytokines. T-5224 is a novel drug developed by computer-aided drug design that can selectively inhibit the binding of c-Fos/AP-1 to DNA. This study aims to investigate whether T-5224 can inhibit LPS-induced acute kidney injury (AKI) by inhibiting the TNF-α inflammatory response and other downstream effectors. [6] Methods: To verify this hypothesis, 7-week-old male C57BL/6 mice were divided into three groups (control group, LPS group and T-5224 group). The control group mice were injected intraperitoneally with saline and orally administered polyvinylpyrrolidone solution. Mice in the LPS group were intraperitoneally injected with a dose of 6 mg/kg of LPS and immediately given polyvinylpyrrolidone solution after injection. In the T-5224 group, mice were orally administered a dose of 300 mg/kg of T-5224 immediately after LPS injection. The concentrations of TNF-α, interleukin (IL)-1β, IL-6 and IL-10 in serum were detected by ELISA. In addition, the expression of intercellular adhesion molecule (ICAM)-1 mRNA in the kidney was detected by quantitative real-time RT-PCR. Finally, we evaluated the histological changes in the kidney. [6] Results: LPS injection induced an increase in serum TNF-α, IL-1β and IL-6 levels. However, administration of T-5224 inhibited the increase in these cytokine levels induced by LPS. Serum IL-10 levels were significantly increased in both the LPS group and the T-5224 group compared with the control group. T-5224 also inhibited LPS-induced ICAM-1 mRNA expression. Furthermore, histological studies also support the anti-inflammatory effects of T-5224. [6] Conclusion: In endotoxin-induced acute kidney injury, T-5224 inhibited the production of TNF-α and other downstream effector factors. Conversely, T-5224 did not inhibit the anti-inflammatory cytokine IL-10. These data support the use of T-5224 for the treatment of septic kidney injury as a promising new therapy. [6] T-5224 is a potent, orally effective, and selective small molecule c-Fos/activator protein-1 (AP-1) inhibitor. [1][2][3][4][5][6]
-T-5224's therapeutic mechanism involves selectively inhibiting the DNA-binding activity of AP-1 and blocking the downstream transcription of pro-inflammatory genes (IL-6, TNF-α), degenerative genes (MMPs, ADAMTS), and oncogenes (VEGF, MMP-9), thereby exerting anti-inflammatory, anti-degenerative, anti-tumor, and organ-protective effects. [1][2][3][4][6]
-T-5224 was originally developed for the treatment of AP-1-mediated septic kidney injury. A range of diseases including rheumatoid arthritis, intervertebral disc degeneration, oral cancer, and LPS-induced organ damage (liver, kidney) [1][2][3][4][6] - Preclinical data show that T-5224 has demonstrated significant efficacy in various in vitro and in vivo models, and has good pharmacokinetic characteristics (high oral bioavailability, short half-life) and low toxicity, supporting its potential as a targeted therapy for AP-1-driven diseases [1][2][3][4][5][6] - T-5224 is metabolized by UGT isoenzymes and does not involve CYP450, thereby reducing the risk of drug interactions with CYP450 substrates [5]

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.)