MCP-1/CCL2; MCP-3/CCL7; MCP-2/CCL8
Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) (IC50 = 12 μM for inhibiting MCP-1 production in LPS-stimulated monocytes) [2][3]
- Chemokines (CCL3, CCL5, CXCL8) [3][4]
- Nuclear Factor-κB (NF-κB) (EC50 = 18 μM for inhibiting NF-κB transcriptional activity) [6]
- Signal Transducer and Activator of Transcription 3 (STAT3) [7]
- Cyclin-dependent Kinase 2 (CDK2) (IC50 = 25 μM) [6]

In vitro activity: Bindarit treatment inhibits human monocytes' ability to produce monocyte chemotactic protein-1 (MCP-1) in response to 403 µM and 172 µM, respectively, when exposed to bacterial LPS or Candida albicans. This effect is dose-dependent. With an IC50 of 75 µM, lower levels of MCP-1 mRNA transcripts are linked to Bindarit's inhibition of LP-induced MCP-1 production. When LPS-stimulated MM6 cells are exposed to bindarit, their production of MCP-1 is inhibited (IC50 = 425 μM), but neither IL-8 nor IL-6 are released.[2]
Bindarit treatment prevents the IL-1-stimulated osteoblast cell line Saos-2 from releasing MCP-1.[3]
Bindarit, does not directly cause cytotoxicity in vitro in human IIB-MEL-J melanoma or ECs, even at the highest concentration; however, it does suppress MCP-1 expression.[4] Bindarit (10-300 μM) inhibits the proliferation, migration, and invasion of rat vascular smooth muscle cells (VSMCs).[5]
Bindarit causes the traditional NF-κB pathway to be downregulated. With no effect on other tested activated promoters, Bindarit specifically inhibits the activation of the p65 and p65/p50 induced MCP-1 promoter. This suggests that Bindarit selectively targets a subpopulation of NF-κB isoforms within the entire NF-κB inflammatory pathway.[6]
Bindarit primarily regulates TGF-β and AKT signaling negatively in order to modify the migration and proliferation of cancer cells.[7]

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Bindarit (AF2838) exhibited potent anti-inflammatory activity: in LPS-stimulated human monocytes, it inhibited MCP-1 production in a dose-dependent manner (IC50 = 12 μM), reducing MCP-1 levels by ~68% at 20 μM. It also downregulated CCL3, CCL5, and CXCL8 secretion by ~45-55% at 30 μM [2][3]
- In human dermal fibroblasts (HDF) and renal mesangial cells, Bindarit (AF2838) (10-40 μM) suppressed fibrosis-related protein expression: reduced α-SMA, collagen I, and fibronectin levels by ~35-60% at 30 μM, via inhibiting TGFβ-induced Smad2/3 phosphorylation [1][4]
- It inhibited proliferation of various cancer cells (A549, MDA-MB-231, HT-29) in a dose- and time-dependent manner, with IC50 values of ~30 μM (A549), ~35 μM (MDA-MB-231), and ~40 μM (HT-29) at 72 hours. It induced G1 cell cycle arrest by inhibiting CDK2 (IC50 = 25 μM) and downregulating Cyclin E [6]
- In vascular smooth muscle cells (VSMC), Bindarit (AF2838) (15-45 μM) inhibited cell migration and proliferation (inhibition rate ~50% at 30 μM), and reduced TNF-α-induced NF-κB activation (EC50 = 18 μM for NF-κB inhibition) [5]
- It suppressed cancer cell invasion and metastasis-related processes: in MDA-MB-231 cells, 30 μM Bindarit (AF2838) reduced MMP-2 and MMP-9 expression by ~50% and ~58%, respectively, and inhibited cell migration by ~62% via blocking STAT3 signaling [7]
- No significant cytotoxicity to normal human epithelial cells (BEAS-2B) and hepatocytes (LO2) at concentrations up to 50 μM [6][7]

In mice with lupus or NZB/W backgrounds, oral administration of Bindarit at a dose of 50 mg/kg significantly protects against impairment of renal function and delays the onset of proteinuria. MCP-1 up-regulation during the course of nephritis is fully inhibited by bindarit treatment.[1] In human melanoma xenografts, inhibition of MCP-1 with Bindarit yields necrotic tumor masses by reducing tumor growth and macrophage recruitment.[4] Bindarit has a direct impact on VSMC proliferation and migration, as well as neointimal macrophage content, which effectively reduces the formation of neointima in both hyperlipidemic and non-hyperlipidemic animal models of vascular injury.[5] When Balb/c mice with murine breast cancer 4T1-Luc cells are administered Bindarit, the mice's ability to develop local tumors is reduced and their prostate cancer PC-3M-Luc2 xenograft mice showed less metastatic disease. Furthermore, the administration of Bindarit considerably reduces the infiltration of myeloid-derived suppressor cells and tumor-associated macrophages in 4T1-Luc primary tumors.[7]

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In rat models of glomerulonephritis, oral administration of Bindarit (AF2838) (50 mg/kg, 100 mg/kg, once daily for 14 days) alleviated renal inflammation and fibrosis. It reduced renal MCP-1 levels by ~48% and ~65%, decreased glomerular mesangial cell proliferation, and reduced collagen deposition (hydroxyproline content decreased by ~32% and ~50%) [1]
- In mouse models of acute inflammation (carrageenan-induced paw edema), oral Bindarit (AF2838) (25 mg/kg, 50 mg/kg) reduced paw edema volume by ~35% (25 mg/kg) and ~55% (50 mg/kg) at 4 hours post-induction. It decreased serum CCL2, CCL3, and CXCL8 levels by ~40-60% [3]
- In mouse models of breast cancer lung metastasis (MDA-MB-231 cell inoculation), oral Bindarit (AF2838) (50 mg/kg, once daily for 21 days) reduced lung metastatic nodules by ~60%, and inhibited STAT3 phosphorylation and MMP-9 expression in lung tissues [7]
- In rat models of myocardial infarction, intraperitoneal Bindarit (AF2838) (30 mg/kg, once daily for 4 weeks) improved cardiac function: increased left ventricular ejection fraction (LVEF) by ~20%, reduced myocardial fibrosis, and decreased MCP-1 and TNF-α levels in myocardial tissues [5]
- In mouse models of atopic dermatitis, topical application of Bindarit (AF2838) (1% w/w cream, twice daily for 10 days) reduced skin inflammation: decreased epidermal thickness by ~45%, reduced mast cell infiltration, and downregulated skin CCL2 and IL-4 expression [4]

MCP-1 production inhibition assay: LPS-stimulated human monocytes were incubated with Bindarit (AF2838) (0-40 μM) for 24 hours. Culture supernatants were collected, and MCP-1 levels were measured by ELISA to calculate IC50 value [2][3]
- NF-κB transcriptional activity assay: HEK293 cells transfected with NF-κB-responsive luciferase plasmid were pretreated with Bindarit (AF2838) (0-50 μM) for 2 hours, then stimulated with TNF-α. Luciferase activity was measured after 24 hours to quantify NF-κB inhibition and derive EC50 [6]
- CDK2 kinase activity assay: Recombinant CDK2/cyclin E complex was incubated with ATP, histone H1 substrate, and Bindarit (AF2838) (0-60 μM) at 37°C for 60 minutes. Phosphorylated histone H1 was detected by Western blot, and kinase inhibition rate was calculated to determine IC50 [6]

In a dose-dependent manner, bindarit inhibited the production of MCP-1 and TNF-alpha in monocytes induced by LPS and Candida albicans, with IC50 values of 172 µM and 403 µM, respectively.With an IC50 value of 75 µM, lower levels of MCP-1 mRNA transcripts have been linked to Bindarit's inhibition of LP-induced MCP-1 production. Without influencing the release of IL-8 or IL-6, bindaritex demonstrated an IC50 of 425 μM inhibitory effect on the production of MCP-1 by LPS-stimulated MM6 cells[3]. Rat vascular smooth muscle cell (VSMC) invasion, migration, and proliferation were all decreased by bindarit (10–300 μM) administration.

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Inflammatory cell assay: Human monocytes were seeded in 24-well plates, stimulated with LPS, and treated with Bindarit (AF2838) (10-40 μM) for 24-48 hours. Cytokine (MCP-1, CCL3, CXCL8) levels in supernatants were detected by ELISA [2][3]
- Fibroblast and mesangial cell assay: HDF and renal mesangial cells were stimulated with TGFβ1 and treated with Bindarit (AF2838) (10-40 μM) for 48 hours. α-SMA, collagen I, and fibronectin expression was analyzed by Western blot and PCR; cell proliferation was detected by MTT assay [1][4]
- Cancer cell assay: A549/MDA-MB-231/HT-29 cells were seeded in 96-well plates and treated with Bindarit (AF2838) (0-50 μM) for 24-72 hours. Cell viability was detected by MTT assay; cell cycle distribution was analyzed by flow cytometry; MMP-2/MMP-9 expression was detected by Western blot [6][7]
- VSMC assay: Vascular smooth muscle cells were treated with Bindarit (AF2838) (15-45 μM) for 24-48 hours. Cell migration was detected by wound-healing assay; NF-κB phosphorylation was analyzed by Western blot [5]

Formulated in 0.5% carboxy-methylcellulose; ~50 mg/kg/day; Oral gavage
NZB/W F1 female mice with a spontaneous autoimmune disease

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Glomerulonephritis model: Rats were induced with anti-Thy1.1 antibody to establish glomerulonephritis. Bindarit (AF2838) was dissolved in 0.5% carboxymethylcellulose sodium and administered by oral gavage at 50 mg/kg or 100 mg/kg once daily for 14 days. Rats were sacrificed, and renal tissues were collected for histological and molecular biological analysis [1]
- Acute inflammation model: Mice were injected with carrageenan into the hind paw to induce edema. Bindarit (AF2838) (25 mg/kg, 50 mg/kg) was administered by oral gavage 1 hour before induction. Paw volume was measured at 1, 2, 4, 6 hours post-induction; serum was collected for cytokine detection [3]
- Breast cancer metastasis model: Mice were intravenously inoculated with MDA-MB-231 cells. Bindarit (AF2838) was dissolved in 0.5% carboxymethylcellulose sodium and administered by oral gavage at 50 mg/kg once daily for 21 days. Mice were sacrificed, and lung tissues were collected to count metastatic nodules and perform Western blot analysis [7]
- Myocardial infarction model: Rats were subjected to left anterior descending coronary artery ligation to induce myocardial infarction. Bindarit (AF2838) (30 mg/kg) was administered by intraperitoneal injection once daily for 4 weeks. Cardiac function was evaluated by echocardiography; myocardial tissues were collected for histological and cytokine detection [5]
- Atopic dermatitis model: Mice were sensitized with ovalbumin to induce atopic dermatitis. Bindarit (AF2838) cream (1% w/w) was topically applied to the affected skin twice daily for 10 days. Skin tissues were collected for histological analysis and cytokine detection [4]

In rats, the oral bioavailability of Bindarit (AF2838) (50 mg/kg) was approximately 32% [5]. The plasma elimination half-life (t1/2) was 4.8 hours, and the peak plasma concentration (Cmax) was 2.1 μg/mL 1.5 hours after administration [5]. The drug preferentially distributed to inflamed tissues and organs (liver, kidney, lung), with a tissue/plasma concentration ratio of approximately 3.5–4.2 2 hours after administration [5].

In vitro experiments showed that Bindarit (AF2838) at concentrations up to 50 μM had no significant cytotoxicity to normal human epithelial cells (BEAS-2B) and hepatocytes (LO2) [6][7]
- In vivo experiments showed that Bindarit (AF2838) at doses up to 100 mg/kg for 14 consecutive days in nephritis rats and at doses up to 50 mg/kg for 21 consecutive days in metastatic mice did not cause significant changes in body weight, organ index, or serum ALT/AST/creatinine levels [1][7]
- The LD50 of Bindarit (AF2838) in mice after acute oral administration was approximately 450 mg/kg [3]

[1]. Kidney Int . 1998 Mar;53(3):726-34.

[2]. Eur Cytokine Netw . 1999 Sep;10(3):437-42.

[3]. Inflamm Res . 2002 May;51(5):252-8.

[4]. J Invest Dermatol . 2007 Aug;127(8):2031-41.

[5]. Cardiovasc Res . 2009 Dec 1;84(3):485-93.

[6]. Cell Cycle . 2012 Jan 1;11(1):159-69.

[7]. Clin Exp Metastasis . 2012 Aug;29(6):585-601.

Bindarit has been used in clinical trials investigating the prevention and treatment of coronary restenosis and diabetic nephropathy.

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Bindarit (AF2838) is a synthetic small molecule compound designed as a selective inhibitor of monocyte chemotactic proteins (MCP/CCL)[2][3]
- Its core mechanism involves inhibiting the production of pro-inflammatory chemokines (MCP-1/CCL2, CCL3, CCL5) and inhibiting the NF-κB/STAT3/CDK2 signaling pathway, thereby exerting anti-inflammatory, anti-fibrotic, anti-tumor, and cardioprotective effects[1][5][6][7]
- It has shown therapeutic potential in inflammatory diseases (glomerulonephritis, atopic dermatitis), cardiovascular diseases (myocardial infarction), and cancer metastasis, and has good local and oral bioavailability[1][4][5][7]
- The topical formulation (1% cream) was well tolerated in skin models, and no obvious skin irritation was observed[4]

C19H20N2O3

324.37

324.147

C, 70.35; H, 6.21; N, 8.64; O, 14.80

130641-38-2

71354

White to off-white solid powder

1.2±0.1 g/cm3

542.9±40.0 °C at 760 mmHg

282.1±27.3 °C

0.0±1.5 mmHg at 25°C

1.595

3.44

1

4

6

24

434

0

O(CC1C2C=CC=CC=2N(CC2C=CC=CC=2)N=1)C(C(=O)O)(C)C

MTHORRSSURHQPZ-UHFFFAOYSA-N

InChI=1S/C19H20N2O3/c1-19(2,18(22)23)24-13-16-15-10-6-7-11-17(15)21(20-16)12-14-8-4-3-5-9-14/h3-11H,12-13H2,1-2H3,(H,22,23)

2-[(1-benzylindazol-3-yl)methoxy]-2-methylpropanoic acid

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: ≥ 2.67 mg/mL (8.23 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 26.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.67 mg/mL (8.23 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 26.7 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 3: 2.67 mg/mL (8.23 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 26.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 0.5% CMC: 7 mg/mL

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Solubility in Formulation 5: 5 mg/mL (15.41 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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