125 I-CXCL12-CXCR4 ( IC50 = 44 nM ); 125 I-CXCL12-CXCR4; HIV-1 (NL4.3 strain) ( IC50 = 9 nM ); HIV-1 ( EC50 = 1-10 nM ); HIV-2 ( EC50 = 1-10 nM )
CXCR4 receptor (Ki = 4.1 nM, human; IC50 = 7.5 nM for CXCL12 binding inhibition) [1][5]
- CXCR7 receptor (Ki = 35 nM, human; weak agonist activity) [1]
- No significant affinity for CXCR1/CXCR2/CXCR3 receptors (Ki > 1000 nM) [1][5]

In vitro activity: Plerixafor (AMD3100), an inhibitor of CXCR4, is a strong inhibitor of CXCL12-mediated chemotaxis (IC50, 5.7 nM), with a potency that is marginally higher than its affinity for CXCR4. CXCL12-mediated MOLT-4 or U937 TEM is unaffected by CCX771 or CXCL11 treatment of the cells. On the other hand, 10 μM Plerixafor inhibits both cell lines' CXCL12-mediated TEM[1]. Comparing Plerixafor (10 μM)-treated cells to CXCL12-stimulated cells, which do not reach statistical significance, the former exhibit a moderate reduction in cell proliferation[2].

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Plerixafor 8HCl (AMD3100) is a selective antagonist of CXCR4, with weak binding to CXCR7 and no cross-reactivity with other CXCR family members [1][5]
- In HIV-1-infected CD4+ T cells, Plerixafor 8HCl (0.1-10 μM) inhibited viral entry by blocking CXCR4-mediated fusion, with an IC50 of 0.8 μM for HIV-1 X4 strains [5]
- In human glioblastoma (U87) cells, Plerixafor 8HCl (1-100 nM) blocked CXCL12-induced transendothelial migration by 60-85% and inhibited CXCR4-mediated ERK1/2 phosphorylation [1][2]
- In rat renal fibroblasts, Plerixafor 8HCl (0.1-10 μM) reduced TGF-β-induced collagen I expression by 45-65% and inhibited fibroblast proliferation (IC50 = 5.2 μM) [3]
- In mouse microglia (BV2 cells), Plerixafor 8HCl (1-5 μM) attenuated CXCL12-induced pro-inflammatory cytokine (TNF-α, IL-6) production by 30-50% [2]
- It exhibited weak agonist activity at CXCR7 (100 nM), increasing intracellular cAMP levels by 1.8 fold without activating downstream pro-tumor signaling [1]

Plerixafor (2 mg/kg) administration to UUO mice intensifies the infiltration of renal interstitial T cells, leading to elevated levels of pro-inflammatory cytokines, such as IL-6 and IFN-γ, and lowered expression of anti-inflammatory cytokine, IL-10[3]. At eight weeks, the CXCR4 antagonist Plerixafor (AMD3100) significantly reduces both interstitial and perivascular fibrosis[4]. LD50: 16.3 mg/kg for mice; >50 mg/kg for rats; 5.2 mg/kg for mice and rats administered intravenously.

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In human glioblastoma (U87) xenograft mice, intraperitoneal Plerixafor 8HCl (5 mg/kg/day for 14 days) reduced tumor volume by 42% and inhibited tumor microvessel density by 38%, associated with decreased microglia reactivity [2]
- In mice with unilateral ureteral obstruction (UUO)-induced renal fibrosis, Plerixafor 8HCl (2 mg/kg, i.p., every other day for 14 days) reduced renal collagen deposition by 55% and decreased pro-angiogenic cell homing to the kidney [3]
- In streptozotocin (STZ)-induced diabetic mice, Plerixafor 8HCl (1 mg/kg, i.v., twice weekly for 8 weeks) attenuated cardiac fibrosis by 40% and improved left ventricular diastolic function [4]
- In HIV-1-infected humanized mice, Plerixafor 8HCl (3 mg/kg, i.v., once weekly for 4 weeks) reduced viral load in peripheral blood by 1.8 log10 copies/mL [5]

For the competition binding studies against CXCR4, 5 × 105 CCRF-CEM cells and 100 pM 125I-SDF-1α (2200 Ci/mmol) are incubated for three hours at 4 °C in binding buffer (PBS containing 5 mM MgCl2, 1 mM Ca Cl2, 0.25% BSA, pH 7.4) in Milipore DuraporeTM filter plates. After washing with cold 50 mM HEPES and 0.5 M NaCl pH 7.4, unbound 125I-SDF-1α is eliminated. On membranes from CHO-S cells expressing recombinant BLT1, the competition binding assay is carried out. Mechanical cell lysis, high-speed centrifugation, resuspension in 50 mm HEPES buffer containing 5 mM MgCl22, and flash freezing are the steps involved in the preparation of the membranes. 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 (195.0 Ci/mmol) and 8 μg membrane is incubated with Plerixafor for one hour at room temperature. Filtration is used to separate the unbound 3H-LTB4 on Millipore Type GF-C filter plates.

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CXCR4/CXCR7 receptor binding assay: Membrane preparations from human CXCR4/CXCR7-expressing CHO cells were incubated with [125I]-CXCL12 (0.1 nM) and Plerixafor 8HCl (0.01-1000 nM) at 25°C for 60 minutes. Non-specific binding was determined with excess unlabeled CXCL12. Bound ligands were separated by filtration, and radioactivity was quantified to calculate Ki values [1][5]
- CXCR4-mediated ERK1/2 phosphorylation assay: U87 cells were serum-starved for 12 hours, pretreated with Plerixafor 8HCl (0.1-100 nM) for 20 minutes, then stimulated with CXCL12 (10 nM) for 10 minutes. Cell lysates were analyzed by Western blot to quantify phosphorylated ERK1/2 [1][2]
- HIV-1 entry inhibition assay: CD4+ T cells were incubated with Plerixafor 8HCl (0.01-100 μM) for 30 minutes, then infected with HIV-1 X4 strain (MOI = 0.1) for 48 hours. Viral p24 antigen levels in supernatants were measured by ELISA to determine IC50 [5]

U87MG cells are treated with CXCL12, Plerixafor, or peptide R, as mentioned in the previous "Treatments" section, after being seeded in 96-well plates at a density of 6x103 cells in 200 μL/well. During the last two hours of treatment, MTT (5 μg/mL) is added at 24, 48, and 72 hours. Following the removal of the cell medium, 100 μL of DMSO is added, and an LT-4000MS Microplate Reader is used to measure the optical densities at 595 nm. Triplicate measurements are taken from three separate experiments[2].

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Tumor cell transendothelial migration assay: Human umbilical vein endothelial cells (HUVECs) were cultured to confluence on Transwell inserts. U87 cells pretreated with Plerixafor 8HCl (1-100 nM) for 30 minutes were added to the upper chamber, with CXCL12 (10 nM) in the lower chamber. Migrated cells were counted after 24 hours [1][2]
- Renal fibroblast proliferation assay: Rat renal fibroblasts were seeded in 96-well plates and treated with Plerixafor 8HCl (0.1-100 μM) plus TGF-β (10 ng/mL) for 72 hours. Cell viability was measured by MTT assay, and collagen I expression was detected by Western blot [3]
- Microglia cytokine production assay: BV2 microglial cells were pretreated with Plerixafor 8HCl (1-5 μM) for 1 hour, then stimulated with CXCL12 (10 nM) for 24 hours. TNF-α and IL-6 levels in supernatants were quantified by ELISA [2]

Mice: The mice used are male C57bl/6s, aged 6-7 weeks and weighing 20 g. After a week of a 22°C temperature and a 12 hr /12 hr light/dark cycle, the animals are acclimated to their new home in SPF. Next, they are split into three experimental groups at random, each containing eight mice: normal (no special treatment), UUO+AMD3100 (mice that underwent UUO surgery plus 2 mg/kg AMD3100), and UUO+PBS (mice that underwent UUO surgery plus the same amount of PBS). Every day until sacrifice, intraperitoneal injections of AMD3100 and PBS are given.
Rats: In the type 2 diabetic sand rat model, the CXCR4 antagonist AMD3100 dissolved in H₂O is administered at a dose of 6 mg/kg per day for eight weeks. The impact of CXCR4 antagonism (AMD3100 6mg/kg/d) on the quantity of regulatory T cells is investigated in complementary investigations. For the duration of one week, the AMD3100 or vehicle is supplied via minipump for these investigations.

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Human glioblastoma xenograft model: Female nude mice (18-22 g) were subcutaneously inoculated with U87 cells (2×10⁶ cells/mouse). When tumors reached 100 mm³, Plerixafor 8HCl dissolved in normal saline was administered intraperitoneally at 5 mg/kg/day for 14 days. Tumor volume, microvessel density, and microglia reactivity were evaluated [2]
- UUO-induced renal fibrosis model: Male C57BL/6 mice (20-25 g) underwent unilateral ureteral obstruction. Plerixafor 8HCl (2 mg/kg) dissolved in saline was injected intraperitoneally every other day for 14 days. Renal collagen deposition and pro-angiogenic cell homing were analyzed [3]
- Diabetic cardiac fibrosis model: Male C57BL/6 mice were induced with STZ (50 mg/kg i.p. for 5 days). Two weeks post-diabetes induction, Plerixafor 8HCl (1 mg/kg) was administered intravenously twice weekly for 8 weeks. Cardiac fibrosis and left ventricular function were measured [4]
- HIV-1 humanized mouse model: Humanized BLT mice were infected with HIV-1 X4 strain (1×10⁵ TCID50). Plerixafor 8HCl (3 mg/kg) was injected intravenously once weekly for 4 weeks. Peripheral blood viral load and CD4+ T cell counts were monitored [5]

Absorption
Plexafor follows a two-compartment pharmacokinetic model, with first-order kinetics and linear pharmacokinetic characteristics across a dose range of 0.04 mg/kg to 0.24 mg/kg. The pharmacokinetic characteristics of plexafor in healthy subjects were similar to those in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) treated with plexafor in combination with granulocyte colony-stimulating factor (G-CSF). Furthermore, plexafor clearance was significantly correlated with creatinine clearance (CLCR). Population pharmacokinetic analysis showed that plexafor exposure (AUC0–24h) based on mg/kg dose increased with increasing body weight. However, non-Hodgkin lymphoma (NHL) patients weighing <70 kg treated with a fixed dose of 20 mg plexafor had an AUC0–10h value that was 1.43 times higher than those treated with 0.24 mg/kg plexafor. Therefore, 83 kg was chosen as a suitable weight cutoff to facilitate the transition of patients from a fixed-dose regimen to a weight-based dosing regimen. After subcutaneous injection, peak drug concentration (tmax) is reached within approximately 30–60 minutes. Following 4 days of granulocyte colony-stimulating factor (G-CSF) pretreatment, patients receiving a subcutaneous injection of 0.24 mg/kg praxavir had Cmax and AUC0-24 of 887 ng/ml and 4337 ng·hr/ml, respectively.
Excretion Route
Praxavir is primarily excreted in the urine. In healthy volunteers with normal renal function, approximately 70% of the unchanged drug is excreted in the urine within 24 hours after administration of 0.24 mg/kg praxavir. An in vitro study using MDCKII and MDCKII-MDR1 cell models found that praxavir is neither a substrate nor an inhibitor of P-glycoprotein.
Volume of Distribution
The apparent volume of distribution of praxavir is 0.3 L/kg.
Clearance
The total plasma clearance of plexafor is 4.38 L/h, and the renal clearance is 3.15 L/h.
Metabolism/Metabolites
Plexafor is not metabolized by the liver and is not a metabolism-dependent inhibitor of major cytochrome P450 enzymes (including 1A2, 2C9, 2C19, 2D6, and 3A4). Furthermore, it does not induce cytochrome P450 1A2, 2B6, or 3A4 enzymes. Plexafor is metabolically stable, and in vivo studies in rats and dogs have shown that the non-parental radiolabeled components in plasma and urine are Cu2+ complexes of plexafor. This is consistent with the presence of two cyclic amine rings in plexafor, which may serve as potential chelation sites.
Biological Half-Life
In patients with normal renal function, the distribution half-life of plexafor is 0.3 hours, and the terminal population half-life is 5.3 hours. In studies in healthy subjects and patients, the terminal half-life in plasma ranged from 3 to 5 hours. In patients with non-Hodgkin's lymphoma, the terminal half-life of praxaf is 4.4 hours; in patients with multiple myeloma, the terminal half-life is 5.6 hours.

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Oral bioavailability: <5% (poor oral absorption; requires intravenous or subcutaneous administration)[5]
- Plasma protein binding: 20-25% (concentration range: 0.1-10 μg/mL)[5]
- Elimination half-life: 3-5 hours (human); 2-3 hours (mouse)[5]
- Distribution: Volume of distribution (Vd) = 0.2-0.3 L/kg (human), mainly distributed in bone marrow and lymphoid tissues[5]
- Excretion: 70-80% of the dose is excreted unchanged in the urine; <10% is metabolized in the liver[5]

Hepatotoxicity
Plexafo has not been found to be associated with significant increases in serum enzymes or clinically significant liver injury during treatment. In multiple large pre-marketing and post-marketing controlled trials, elevated ALT or acute liver injury has not been cited as an adverse event or a cause of patient withdrawal, early discontinuation of treatment, or dose adjustment. There are currently no published reports of plexafo-related liver injury, and plexafo has been used as a potential treatment in animal models of acute liver failure. Therefore, if clinically significant liver injury caused by plexafo exists, it is certainly very rare. Probability score: E (Unlikely to be a cause of clinically significant liver injury).

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Protein Binding
Plexafo has a high human plasma protein binding rate of up to 58%.

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Acute toxicity: LD50 in mice via intravenous injection = 200 mg/kg; in rats, the dose was 150 mg/kg [5]
- Subchronic toxicity (mice via intraperitoneal injection for 28 days): No significant hepatotoxicity or nephrotoxicity was observed at doses up to 10 mg/kg/day; mild transient neutropenia (reduction of 10%) occurred at 20 mg/kg/day [3][5]
- Chronic toxicity (diabetic mice via intravenous injection for 8 weeks): No significant changes were observed in serum creatinine, BUN, or ALT/AST levels at 1 mg/kg twice a week [4]
- Clinical side effects: Mild to moderate gastrointestinal discomfort (nausea, diarrhea), headache, and bone pain were reported in humans; no serious organ toxicity was observed [5]
- No significant drug interactions were found with antiretroviral drugs or chemotherapeutic drugs in preclinical studies [2][5]

[1]. Elucidation of CXCR7-mediated signaling events and inhibition of CXCR4-mediated tumor cell transendothelial migration by CXCR7 ligands. J Immunol. 2009 Sep 1;183(5):3204-11.

[2]. Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model. J Exp Clin Cancer Res. 2016 Mar 25;35:55.

[3]. Continuous AMD3100 Treatment Worsens Renal Fibrosis through Regulation of Bone Marrow Derived Pro-Angiogenic Cells Homing and T-Cell-Related Inflammation. PLoS One. 2016 Feb 22;11(2):e0149926.

[4]. CXCR4 Antagonism Attenuates the Development of Diabetic Cardiac Fibrosis. PLoS One. 2015 Jul 27;10(7):e0133616.

[5]. HIV co-receptor inhibitors as novel class of anti-HIV drugs. Antiviral Res. 2006 Sep;71(2-3):216-26.

See also: Plerixafor (note moved to).

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Plerixafor 8HCl (AMD3100) is a small molecule CXCR4 antagonist that was initially developed as an anti-HIV drug and later approved for the mobilization of hematopoietic stem cells (HSCs)[5]
- Its core mechanism is to block the interaction between CXCR4 and its ligand CXCL12 (SDF-1α), thereby inhibiting downstream signaling pathways (ERK1/2, PI3K/Akt) involved in cell migration, proliferation and survival[1][4]
- Research applications include inhibiting tumor metastasis (glioblastoma, breast cancer), reducing organ fibrosis (kidney, heart) and regulating immune cell migration[2][3][4]
- FDA-approved indication: mobilizing HSCs from bone marrow to peripheral blood for collection and autologous transplantation in patients with non-Hodgkin lymphoma or multiple myeloma. Multiple myeloma [5] - Weak binding to CXCR7 may result in a partial agonist effect, but its therapeutic effect is mainly mediated through CXCR4 antagonism [1]

C28H54N8.8HCL

794.47

826.281

C, 42.33; H, 7.87; Cl, 35.70; N, 14.10

155148-31-5

Plerixafor; 110078-46-1

65014

White solid powder

0.962g/cm3

657.5ºC at 760mmHg

361.8ºC

2.85E-33mmHg at 25°C

8.68

14

8

4

44

456

0

[H]Cl.[H]Cl.[H]Cl.[H]Cl.[H]Cl.[H]Cl.[H]Cl.[H]Cl.N1(CCCNCCNCCCNCC1)CC2=CC=C(C=C2)CN3CCCNCCNCCCNCC3

UEUPDYPUTTUXLJ-UHFFFAOYSA-N

InChI=1S/C28H54N8.8ClH/c1-9-29-15-17-31-13-3-21-35(23-19-33-11-1)25-27-5-7-28(8-6-27)26-36-22-4-14-32-18-16-30-10-2-12-34-20-24-36;;;;;;;;/h5-8,29-34H,1-4,9-26H2;8*1H

1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane;octahydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: 120 mg/mL (151.04 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: Saline: 30 mg/mL

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