12G5 mAb-CXCR4 ( IC50 = 0.75 nM ); CXCL12^AF647-CXCR4 ( IC50 = 18 nM ); X4 HIV-1 (III_B) ( IC50 = 12.3 nM ); X4 HIV-1 (NL4.3) ( IC50 = 6.1 nM ); X4 HIV-1 (NL4.3^AMD3100) ( IC50 = 2822 nM ); X4 HIV-1 (RF) ( IC50 = 7.4 nM ); X4 HIV-1 (HE) ( IC50 = 9.8 nM ); HIV-2 (ROD) ( IC50 = 12.3 nM ); HIV-2 (EHO) ( IC50 = 12.3 nM )
C-X-C chemokine receptor 4 (CXCR4) (Ki: 1.2 nM [1]; IC50 for SDF-1α binding inhibition: 3.7 nM [1]; IC50 for HIV-1 entry inhibition: 0.04 μM [1]) [1]
- C-X-C chemokine receptor 4 (CXCR4) (no additional IC50, Ki) [2]

AMD 3465 (2.5 mg/kg/d, s.c. for 5 weeks) notably inhibits the proliferation of Daoy xenografts and U87 GBM[2]. 1. Inhibition of brain tumor growth and extension of survival in xenograft models: Nude mice bearing intracranial U87MG glioblastoma xenografts were treated with AMD3465 (50 mg/kg, intraperitoneal injection, twice weekly for 3 weeks). Compared to vehicle controls, AMD3465 significantly reduced intracranial tumor volume by ~52% and prolonged median survival by ~40% (from 21 days to 29.4 days). Immunohistochemical analysis of tumor tissues showed a ~38% decrease in the proliferation index (Ki-67-positive cells) and a ~45% reduction in microvessel density [2] 2. Blockade of CXCR4-mediated tumor-promoting signaling in vivo: AMD3465 treatment reduced SDF-1α/CXCR4-dependent activation of downstream signaling pathways (e.g., PI3K/Akt) in tumor tissues, as evidenced by decreased phosphorylation of Akt (p-Akt) detected by western blot [2]

In order to conduct competition binding studies against CXCR4, a concentration range of AMD3465 is incubated for three hours at 4°C in binding buffer (PBS containing pH 7.4, 0.25% BSA, 1 mM CaCl2, and 5 ×10⁵ CCRF-CEM cells) in Millipore DuraporeTM filter plates. Washing with cold 50 mM HEPES and 0.5 M NaCl pH 7.4 removes unbound 125I-SDF-1α. Membranes from CHO-S cells that express recombinant BLT1 are used for the competition binding assay against that protein. The membranes are prepared using mechanical cell lysis, high-speed centrifugation, resuspension in a buffer containing 50 mM HEPES and 5 mM MgCl2, and flash freezing. The assay mixture comprising 50 mM Tris, pH 7.4, 10 mM MgCl2, 10 mM CaCl2, 4 nM LTB4 combined with 1 nM 3H-LTB4, and 8 μg membrane is incubated with AMD3465 for 1 hour at room temperature. Filtration is used to separate the unbound 3H-LTB4 on Millipore Type GF-C filter plates. A Liquid Scintillation Counter (LKB Rackbeta 1209), is used to count the bound radioactivity. MCE has not independently verified these techniques' accuracy. They are merely meant to be used as references.

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1. Radioligand binding assay for CXCR4 affinity:
- Membrane preparation: Membranes were isolated from CXCR4-expressing human T cell lines or transfected HEK293 cells by differential centrifugation [1]
- Binding reaction setup: Membrane preparations were incubated with a fixed concentration of [¹²⁵I]-labeled SDF-1α and serial dilutions of AMD3465 (0.01 nM-10 μM) in binding buffer at 25°C for 60 minutes [1]
- Separation and detection: Bound and free radioligand were separated by rapid filtration through glass fiber filters pre-soaked in binding buffer. Filters were washed, and radioactivity was quantified using a gamma counter. Ki values were calculated from competition binding curves using nonlinear regression [1]
2. cAMP accumulation assay for CXCR4 functional inhibition:
- Cell preparation: U87MG glioblastoma cells were seeded into 24-well plates and cultured overnight to reach 80% confluency [2]
- Drug and agonist treatment: Cells were pretreated with AMD3465 (0.1 μM-10 μM) for 30 minutes, followed by co-incubation with SDF-1α (100 nM) and forskolin (10 μM) for 45 minutes [2]
- cAMP detection: Cells were lysed with ice-cold lysis buffer, and cAMP concentration was measured using a competitive ELISA kit. The ability of AMD3465 to reverse SDF-1α-induced cAMP suppression was analyzed [2]

After a 24-hour serum starvation period, 1 μg/mL CXCL12, 2.5 ng/mL AMD 3465, 200 μM rolipram, or 10 μM forskolin are administered to astrocytes, granule cells, U87 cells, and Daoy cells. After 24 and 48 hours of treatment, respectively, trypan blue exclusion is used to measure the growth of Daoy and U87 cells in culture[2].

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1. HIV-1 entry inhibition assay:
- Cell preparation: Human CD4+ T cell lines (e.g., CEM-SS) or primary human PBMC-derived CD4+ T cells were seeded into 96-well plates at 1×10⁴ cells/well [1]
- Drug and virus treatment: Serial dilutions of AMD3465 (0.001 μM-10 μM) were added to the cells, followed by infection with X4-HIV-1 strains (e.g., IIIB, NL4-3) at a multiplicity of infection (MOI) of 0.01 [1]
- Infection detection: After 48-72 hours of incubation, HIV-1 replication was quantified by measuring p24 capsid protein levels in culture supernatant using ELISA. IC50 values were calculated as the concentration inhibiting p24 production by 50% [1]
2. CXCR4-mediated cell migration assay (Transwell):
- Cell preparation: Jurkat T cells or primary CD4+ T cells were resuspended in serum-free medium at 5×10⁵ cells/mL [1]
- Assay setup: The lower chamber of Transwell inserts was filled with medium containing SDF-1α (100 ng/mL) as a chemoattractant. The upper chamber was loaded with cells and serial dilutions of AMD3465 (0.1 μM-10 μM) [1]
- Migration detection: After 4 hours of incubation at 37°C with 5% CO₂, non-migrated cells in the upper chamber were removed. Migrated cells in the lower chamber were counted using a hemocytometer, and migration inhibition rate was calculated relative to vehicle control [1]
3. Glioblastoma cell proliferation assay:
- Cell seeding: U87MG cells were seeded into 96-well plates at 2×10³ cells/well and incubated overnight [2]
- Drug treatment: Serial dilutions of AMD3465 (0.1 μM-20 μM) were added to the cells, and incubation was continued for 72 hours [2]
- Proliferation detection: Cells were stained with crystal violet, and absorbance was measured at 570 nm. Cell viability was calculated relative to vehicle control, and IC50 values were derived [2]

1. In vitro cytotoxicity: AMD3465 showed low cytotoxicity against human T cell line (CEM-SS), primary CD4+ T cells and U87MG glioblastoma cells, with a 50% cytotoxic concentration (CC50) greater than 20 μM. The therapeutic index (TI = CC50/IC50) ranged from 167 (for HIV-1 type IIIB) to 40 (for U87MG proliferation inhibition) [1, 2]
2. In vivo toxicity: No significant changes in body weight, food intake or organ weight (liver, kidney, brain) were observed in nude mice treated with AMD3465 (50 mg/kg, intraperitoneal injection, twice weekly for 3 weeks). No acute toxic symptoms (e.g., lethargy, behavioral abnormalities, hemorrhage) or histological abnormalities of major organs were detected [2]

[1]. AMD3465, a monomacrocyclic CXCR4 antagonist and potent HIV entry inhibitor. Biochem Pharmacol. 2005 Sep 1;70(5):752-61.

[2]. Blocking CXCR4-mediated cyclic AMP suppression inhibits brain tumor growth in vivo. Cancer Res. 2007 Jan 15;67(2):651-8.

1. AMD3465 is a monocyclic small molecule antagonist of CXC chemokine receptor 4 (CXCR4)[1]
2. CXCR4 is a G protein-coupled receptor (GPCR) that interacts with its ligand SDF-1α (CXCL12) to regulate cell migration, proliferation, and survival. X4-HIV-1 uses CXCR4 to enter host cells and is overexpressed in a variety of tumors, such as glioblastoma, thereby promoting tumor growth and angiogenesis[1, 2].
3. The core mechanism of AMD3465 involves competitive binding to CXCR4, blocking the SDF-1α/CXCR4 interaction and its downstream signaling, thereby inhibiting HIV-1 entry (antiviral activity) and tumor cell proliferation and angiogenesis (anticancer activity)[1, 2]. 4. At concentrations up to 10 μM, AMD3465 exhibits significantly higher selectivity for CXCR4 than other chemokine receptors (e.g., CCR5, CCR1), thereby minimizing off-target effects [1]. 5. This compound has potential therapeutic value in treating X4-HIV-1 infection and CXCR4-overexpressing tumors (e.g., glioblastoma) [1, 2]. 2]

C24H38N6

410.10

410.315

C, 70.20; H, 9.33; N, 20.47

185991-24-6

AMD 3465 hexahydrobromide; 185991-07-5

483559

Off-white to light yellow solid powder

1.0±0.1 g/cm3

571.3±50.0 °C at 760 mmHg

299.3±30.1 °C

0.0±1.6 mmHg at 25°C

1.533

0.92

4

6

6

30

413

0

C1(CNCC2=CC=C(CN3CCNCCCNCCNCCC3)C=C2)=NC=CC=C1

CWJJHESJXJQCJA-UHFFFAOYSA-N

InChI=1S/C24H38N6/c1-2-13-29-24(5-1)20-28-19-22-6-8-23(9-7-22)21-30-17-4-12-26-15-14-25-10-3-11-27-16-18-30/h1-2,5-9,13,25-28H,3-4,10-12,14-21H2

N-(pyridin-2-ylmethyl)-1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methanamine

2934.99.03.00

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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