1. GPCR/G Protein
    Immunology/Inflammation
  2. CXCR

AMD 3465 hexahydrobromide (Synonyms: GENZ-644494 hexahydrobromide)

Cat. No.: HY-15971 Purity: 98.79%
Data Sheet SDS Handling Instructions

AMD 3465 (hexahydrobromide) is a potent, selective CXCR4 antagonist, and inhibits SDF-1α-ligand binding with Ki of 41.7 nM.

For research use only. We do not sell to patients.
AMD 3465 hexahydrobromide Chemical Structure

AMD 3465 hexahydrobromide Chemical Structure

CAS No. : 185991-07-5

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Other Forms of AMD 3465 hexahydrobromide:

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Description

AMD 3465 (hexahydrobromide) is a potent, selective CXCR4 antagonist, and inhibits SDF-1α-ligand binding with Ki of 41.7 nM.

IC50 & Target

Ki: 41.7 nM

In Vitro

The affinity of AMD3465 is 8-fold higher compared with AMD3100, in competition against the radiolabeled monoclonal antibody raised against CXCR4, 12G5. The affinity of AMD3465 is decreased >5000-fold in (D262N)-CXCR4 and 1913-fold in (A175F)-CXCR4. AMD3465 appears to interact with HisVII:-02 at the extracellular end of TM-VII (at the interface to extracellular loop 3) and HisIII:05. Both of these His residues are facing right into the main binding pocket of CXCR4[1]. AMD3465 inhibits 125I-SDF-1α ligand binding to CCRF-CEM cells. AMD3465 inhibits CXCR4 activation as measured by GTP binding with an IC50 of 10.38±1.99 nM, and inhibits SDF-1α mediated calcium flux with an IC50 of 12.07±2.42 nM[2].

In Vivo

AMD3465 (5 mg/kg, s.c.) significantly elevates total white blood cells in DBA/2, C57Bl/6 and BALB/c mice between 0.5 and 2 h. AMD3465 significantly increases the specific cell populations in all three strains of mice included neutrophils, lymphocytes, and monocytes[2].

References
Kinase Assay
[2]

For the competition binding studies against CXCR4, a concentration range of AMD3465 is incubated for 3 h at 4°C in binding buffer (PBS containing 5 mM MgCl2, 1 mM CaCl2, 0.25% BSA pH 7.4) with 5×105 CCRF-CEM cells and 100 pM 125I-SDF-1α in Millipore DuraporeTM filter plates. Unbound 125I-SDF-1α is removed by washing with cold 50 mM HEPES, 0.5 M NaCl pH 7.4. The competition binding assay against BLT1 is performed on membranes from CHO-S cells expressing recombinant BLT1. The membranes is prepisd by mechanical cell lysis followed by high speed centrifugation, resuspended in 50 mM HEPES, 5 mM MgCl2 buffer and flash frozen. The membrane preparation is incubated with AMD3465 for 1 h at room temperature in an assay mixture containing 50 mM Tris, pH 7.4, 10 mM MgCl2, 10 mM CaCl2, 4 nM LTB4 mixed with 1 nM 3H-LTB4 and 8 μg membrane. The unbound 3H-LTB4 is separated by filtration on Millipore Type GF-C filter plates. The bound radioactivity is counted using a LKB Rackbeta 1209 Liquid Scintillation Counter. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cell Assay
[1]

On the day prior to the experiment, the U87.CD4.CXCR4 transfectants is seeded in 0.1% gelatin-coated 96-well black wall microplates (Costar, Cambridge, MA) at 2×104 cells per well. On the day of the experiment, the cells is loaded with the fluorescent calcium indicator Fluo-3 acetoxymethyl at 4 μM for 45 min at 37°C. After thorough washing with calcium flux assay buffer (Hanks' balanced salt solution with 20 mM Hepes buffer and 0.2% bovine serum albumin, pH 7.4), the cells is preincubated for 15 min at 37°C with AMD3100 or AMD3465 (1 μg/mL) in the same buffer. Then, the intracellular calcium mobilization in response to 2-50 ng/mL CXCL12 is measured at 37°C by monitoring the fluorescence as a function of time simultaneously in all the wells using a Fluorometric Imaging Plate Reader. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Administration
[2]

The maximum tolerated dose (MTD) of AMD3465 is determined in mice. AMD3465 is given as a single subcutaneous injection to five male Swiss Webster mice per dose group (5, 10, 20, 50 and 100 mg/kg). Mice is observed for 48 h following administration; evaluations is made of mortality and morbidity, clinical observations, body weight and gross pathology. The pharmacokinetics of AMD3465 in male Swiss Webster mice is determined for a single 25 mg/kg subcutaneous dose. Blood is collected by cardiocentisis from three mice per time point at 0.25, 0.5, 1, 1.5, 2, 4, 8, 12, and 24 h post-administration. Plasma is obtained following centrifugation (3000 rpm, 10 min) and concentrations of AMD3465 in plasma is determined by LC-MS. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

References
M.Wt

410.6

Formula

C₂₄H₃₈N₆

CAS No.

185991-07-5

Storage
Powder -20°C 3 years
  4°C 2 years
In solvent -80°C 6 months
  -20°C 1 month
Shipping

Room temperature in continental US; may vary elsewhere

Solvent & Solubility

H2O: ≥ 38 mg/mL

* "<1 mg/mL" means slightly soluble or insoluble. "≥" means soluble, but saturation unknown.

Purity: 98.79%

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AMD 3465 hexahydrobromide
Cat. No.:
HY-15971
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