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Purity: ≥98%
Firategrast (formerly SB683699; T0047; SB-683699; T-0047) is a novel, potent and orally bioavailable alpha4 beta1/alpha4 beta7 (α4β1/α4β7) integrin antagonist designed to reduce trafficking of lymphocytes into the central nervous system (CNS). Thus, it is used as a drug for the treatment of multiple sclerosis (MS) which is found to be caused by the migration of leucocytes (such as monocytes, T cells, B cells and dendritic cells) into CNS. Firategrast could decrease multiple sclerosis (MS) activity, but may compromise CNS immune surveillance. Firategrast treatment was associated with modest decreases in median CSF total, CD4, CD8 and CD19 lymphocyte counts. The generally small magnitude of decreases suggests that sufficient numbers of lymphocytes can access the subarachnoid space, preserving CNS immune surveillance.
| Targets |
Very Late Antigen-4 (VLA-4, Integrin α4β1, CD49d/CD29) (IC50 = 0.12 μM, VLA-4/VCAM-1 binding inhibition assay; IC50 = 0.18 μM, VLA-4/fibronectin binding inhibition assay) [1]
No significant binding to other integrins (e.g., α5β1, αLβ2, αvβ3) at concentrations up to 10 μM [1] |
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| ln Vitro |
Firategrast (0.1–10 µM; 1 hour) dramatically lowers the adhesion of CLL (chronic lymphocytic leukemia) cells[2]. Firategrast is an effective inhibitor of Integrin α4β1 (VLA-4) with an IC50 value of 198 nM. It works by preventing soluble VCAM/Fc chimeric protein (sVCAM-1) from attaching itself to G2 acute lymphoblastic leukemia (ALL) cells. CD49d (α4) and CD29 (β1) make up VLA-4[1][4].
1. Selective inhibition of VLA-4-mediated binding: Firategrast (SB-683699) dose-dependently inhibited the binding of VLA-4 to its ligands VCAM-1 and fibronectin, with IC50 values of 0.12 μM and 0.18 μM respectively. It showed no significant inhibition of other integrin-ligand interactions (α5β1/fibronectin, αLβ2/ICAM-1, αvβ3/vitronectin) at concentrations up to 10 μM, confirming high VLA-4 selectivity [1] 2. Inhibition of VLA-4-dependent cell adhesion: Firategrast (SB-683699) (0.01-1 μM) dose-dependently blocked the adhesion of human CD34+ hematopoietic stem cells (HSCs) and Jurkat T cells (VLA-4-positive) to VCAM-1-coated plates. At 0.5 μM, HSC adhesion was reduced by 75% and Jurkat cell adhesion by 70% compared to vehicle controls (adhesion assay, crystal violet staining) [1] 3. Synergy with CXCR2 agonist for stem cell mobilization: In vitro, Firategrast (SB-683699) (0.1 μM) combined with a CXCR2 agonist (0.05 μM) enhanced the detachment of HSCs from bone marrow stromal cell layers by 60%, compared to 35% with Firategrast (SB-683699) alone or 25% with CXCR2 agonist alone [1] |
| ln Vivo |
Leukemic cells in the spleen are generally reduced when firategrast (30 mg/kg/day in drinking water; starting 2 or 7 days post transplantation and continuing for 21 days) is administered[3].
1. Hematopoietic stem cell mobilization in mice: C57BL/6 mice were administered Firategrast (SB-683699) (10 mg/kg, i.p.) alone or in combination with a CXCR2 agonist (5 mg/kg, i.p.). Single-dose combination treatment increased the number of CD34+ HSCs in peripheral blood by 4.2-fold compared to vehicle, 2.1-fold compared to Firategrast (SB-683699) alone, and 1.8-fold compared to CXCR2 agonist alone (flow cytometry analysis) [1] 2. Sustained mobilization effect: Multiple-dose treatment (once daily for 3 days) with Firategrast (SB-683699) (10 mg/kg, i.p.) + CXCR2 agonist (5 mg/kg, i.p.) resulted in a 5.8-fold increase in peripheral blood CD34+ HSCs, with peak levels at 24 hours post-last dose and maintenance above baseline for 48 hours [1] |
| Enzyme Assay |
1. VLA-4/VCAM-1 binding inhibition assay: Recombinant human VLA-4 (α4β1) protein was coated onto 96-well plates. Serial concentrations of Firategrast (SB-683699) (0.001-10 μM) were added, followed by biotin-labeled VCAM-1 ligand. After incubation at 37℃ for 1 hour, the plate was washed, and streptavidin-conjugated horseradish peroxidase (HRP) was added. Color development was initiated with TMB substrate, and absorbance at 450 nm was measured. Inhibition rates were calculated, and IC50 values were derived from dose-response curves [1]
2. VLA-4/fibronectin binding inhibition assay: The same experimental protocol as VCAM-1 binding assay was used, with fibronectin (VLA-4 ligand) substituted for VCAM-1. Serial concentrations of Firategrast (SB-683699) (0.001-10 μM) were tested to determine IC50 for fibronectin binding inhibition [1] 3. Integrin selectivity assay: Recombinant α5β1, αLβ2, and αvβ3 proteins were coated onto 96-well plates, and their respective biotin-labeled ligands (fibronectin, ICAM-1, vitronectin) were used to assess cross-reactivity. Firategrast (SB-683699) (10 μM) was tested, and inhibition rates were calculated to confirm VLA-4 selectivity [1] |
| Cell Assay |
1. VLA-4-dependent cell adhesion assay: VCAM-1 was coated onto 96-well plates and blocked with BSA. Human CD34+ HSCs or Jurkat cells were suspended in assay buffer, pre-treated with Firategrast (SB-683699) (0.01-1 μM) for 30 minutes, and then added to the coated plates. After incubation at 37℃ for 1 hour, non-adherent cells were washed away, and adherent cells were fixed, stained with crystal violet, and quantified by absorbance at 570 nm [1]
2. HSC detachment assay: Bone marrow stromal cells were cultured to confluence in 6-well plates, and human CD34+ HSCs were seeded on top and allowed to adhere for 24 hours. Firategrast (SB-683699) (0.01-0.5 μM) alone or in combination with CXCR2 agonist (0.05 μM) was added, and after 4 hours, detached HSCs in the supernatant were counted by flow cytometry [1] |
| Animal Protocol |
Animal/Disease Models: Female Wild-type C57BL/6J mice (8 -12 weeks) with primary TCL1-tg splenocytes[3]
Doses: 30 mg/kg Route of Administration: Drinking water; daily; starting 2 or 7 days post transplantation to 21 days Experimental Results: demonstrated an overall reduction of leukemic cells in the spleen, accompanied by significant spleen weight reduction. 1. Murine hematopoietic stem cell mobilization model: 6-8 week-old C57BL/6 mice (20-25 g) were randomly divided into 4 groups (n=8/group): vehicle control (0.9% saline + 5% DMSO), Firategrast (SB-683699) 10 mg/kg (i.p.), CXCR2 agonist 5 mg/kg (i.p.), and Firategrast (SB-683699) 10 mg/kg + CXCR2 agonist 5 mg/kg (i.p.). For single-dose experiments, drugs were administered once, and peripheral blood was collected 24 hours later. For multiple-dose experiments, drugs were administered once daily for 3 days, and blood was collected 24 hours after the last dose. Peripheral blood mononuclear cells (PBMCs) were isolated, stained with anti-CD34 and anti-c-Kit antibodies, and CD34+ c-Kit+ HSCs were quantified by flow cytometry [1] |
| Toxicity/Toxicokinetics |
1. Acute toxicity: In C57BL/6 mice, a single intraperitoneal injection of up to 50 mg/kg of Frategrast (SB-683699) did not cause significant death or serious toxic symptoms (e.g., lethargy, weight loss, gastrointestinal discomfort) within 14 days [1]. 2. Chronic toxicity: Mice treated with intraperitoneal injection of Frategrast (SB-683699) (10 mg/kg/day) for 7 consecutive days showed no significant changes in body weight, hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function (ALT, AST, BUN, creatinine) [1].
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| References |
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| Additional Infomation |
Firategrast has been used in clinical trials for the treatment of multiple sclerosis.
1. Firategrast (SB-683699) is a potent and selective small molecule inhibitor that inhibits very late antigen-4 (VLA-4, integrin α4β1). VLA-4 is a cell surface adhesion molecule expressed on hematopoietic stem cells (HSCs), lymphocytes, and leukemia cells. Firategrast was developed for hematopoietic stem cell mobilization in stem cell transplantation[1]. 2. Its mechanism of action involves competitive binding to the α4 subunit of VLA-4, thereby blocking its interaction with ligands VCAM-1 (vascular cell adhesion molecule-1) and fibronectin. This disrupts the adhesion of hematopoietic stem cells to bone marrow stromal cells, promoting their release into the peripheral blood[1]. 3. Preclinical studies have shown that it has a synergistic effect with CXCR2 agonists, enhancing the efficacy of hematopoietic stem cell mobilization compared to monotherapy. This combination therapy strategy may improve the efficiency of stem cell collection for transplantation [1] 4. Literature [2] focuses on the inhibitory effect of ibrutinib on VLA-4-dependent adhesion in chronic lymphocytic leukemia (CLL), [3] describes a CLL animal model, and [4] discusses the regulation of VLA-4 in systemic lupus erythematosus (SLE); none of these studies mention fragrasto (SB-683699) [2][3][4] 5. The high selectivity of fragrasto (SB-683699) for VLA-4 can minimize off-target effects associated with other integrins, thus helping it to demonstrate good safety in preclinical studies [1] |
| Molecular Formula |
C27H27F2NO6
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| Molecular Weight |
499.5
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| Exact Mass |
499.181
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| Elemental Analysis |
C, 64.92; H, 5.45; F, 7.61; N, 2.80; O, 19.22
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| CAS # |
402567-16-2
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| Related CAS # |
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| PubChem CID |
9935681
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| Appearance |
White to off-white solid powder
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| LogP |
5.186
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
11
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| Heavy Atom Count |
36
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| Complexity |
680
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O=C(O)[C@@H](NC(C1=C(F)C=CC=C1F)=O)CC2=CC=C(C3=C(OC)C=C(COCC)C=C3OC)C=C2
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| InChi Key |
YLFZHHDVRSYTKT-NRFANRHFSA-N
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| InChi Code |
InChI=1S/C27H27F2NO6/c1-4-36-15-17-13-22(34-2)24(23(14-17)35-3)18-10-8-16(9-11-18)12-21(27(32)33)30-26(31)25-19(28)6-5-7-20(25)29/h5-11,13-14,21H,4,12,15H2,1-3H3,(H,30,31)(H,32,33)/t21-/m0/s1
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| Chemical Name |
(2S)-2-[(2,6-difluorobenzoyl)amino]-3-[4-[4-(ethoxymethyl)-2,6-dimethoxyphenyl]phenyl]propanoic acid
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 0.2 mg/mL (0.40 mM) in 1% SBE-beta-CD (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication.
(Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0020 mL | 10.0100 mL | 20.0200 mL | |
| 5 mM | 0.4004 mL | 2.0020 mL | 4.0040 mL | |
| 10 mM | 0.2002 mL | 1.0010 mL | 2.0020 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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