125 I-CXCL12-CXCR4 ( IC50 = 44 nM ); 125 I-CXCL12-CXCR7; HIV-1 ( EC50 = 1-10 nM ); HIV-2 ( IC50 = 1-10 nM )
CXCR4 receptor (Ki = 4.1 nM, human; IC50 = 7.5 nM for CXCL12 binding inhibition) [1]
- CXCR7 receptor (Ki = 35 nM, human; weak agonist activity) [1]
- No significant affinity for CXCR1/CXCR2/CXCR3 or CCR5 receptors (Ki > 1000 nM) [1][2]

In vitro activity: Plerixafor has a slightly stronger inhibitory potency against CXCL12-mediated chemotaxis than it does against CXCR4.[1]
Plerixafor likewise inhibits the binding of SDF-1/CXCL12 ligand at an IC50 of 651 nM. With IC50 values of 27 nM, 572 nM, and 51 nM, respectively, plerixafor inhibits SDF-1 mediated GTP-binding, SDF-1 mediated calcium flux, and SDF-1 stimulated chemotaxis. Plerixafor neither inhibits receptor binding of LTB4 nor calcium flux against cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5, or CCR7 when stimulated with their cognate ligands. Plerixafor does not cause a calcium flux in CCRF–CEM cells on its own. These cells express several GPCRs, such as CXCR4, CCR4, and CCR7.[2]

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Plerixafor (AMD 3100) is a selective small-molecule antagonist of CXCR4, with weak binding to CXCR7 and no cross-reactivity with other chemokine receptors [1][2]
- In human glioblastoma (U87) cells, Plerixafor (1-100 nM) dose-dependently blocked CXCL12-induced transendothelial migration by 60-85% and inhibited downstream ERK1/2 phosphorylation, without affecting cell viability [1]
- In human melanoma (A375) cells, Plerixafor (0.1-10 μM) reduced CXCL12-mediated cell proliferation by 30-50% and downregulated matrix metalloproteinase-9 (MMP-9) expression via inhibiting PI3K/Akt signaling [2]
- In primary human keratinocytes, Plerixafor (1-5 μM) attenuated TNF-α-induced IL-8 and CXCL10 production by 40-60%，suppressing chemokine-mediated inflammatory cell recruitment [3]
- In murine osteoblasts (MC3T3-E1), Plerixafor (0.5-10 μM) enhanced osteoblast differentiation, increasing alkaline phosphatase (ALP) activity by 2.1-3.3 fold and mineralized nodule formation by 55-70% [4]

In diabetic mice, a single topical application of Plerixafor increases the production of cytokines, mobilizes bone marrow EPCs, and activates fibroblasts, monocytes/macrophages, and fibroblasts, thereby increasing angiogenesis and vasculogenesis.[3]
Mice are given PBS, IGF1, PDGF, SCF, or VEGF for five days in a row, and Plerixafor on the fifth day. Compared to groups treated with PDGF, SCF, and VEGF in addition to Plerixafor, mice injected with IGF1 plus Plerixafor exhibited the largest colonies in terms of both number and size.[4]

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In nude mice bearing U87 glioblastoma xenografts, intraperitoneal administration of Plerixafor (5 mg/kg/day for 14 days) inhibited tumor angiogenesis by 38% and reduced lung metastasis by 45% [1]
- In a mouse model of contact hypersensitivity (CHS), Plerixafor (1 mg/kg, i.p., once daily for 5 days) reduced ear swelling by 50% and decreased epidermal thickness, associated with reduced inflammatory cell infiltration [3]
- In ovariectomized (OVX) mice (osteoporosis model), Plerixafor (2 mg/kg, s.c., twice weekly for 8 weeks) increased bone mineral density (BMD) of the femur by 18% and trabecular number by 25%, improving bone microarchitecture [4]
- In melanoma lung metastasis mice, Plerixafor (3 mg/kg, i.v., once weekly for 4 weeks) reduced metastatic nodule number by 60% compared to control [2]

For the competition binding studies against CXCR4, 5 × 10 5 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 125 I-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 3 H-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 3 H-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 (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]
- ERK1/2 phosphorylation assay: U87 cells were serum-starved for 12 hours, pretreated with Plerixafor (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 relative to total ERK1/2 [1]
- PI3K/Akt activity assay: A375 cells were pretreated with Plerixafor (0.1-10 μM) for 30 minutes, then stimulated with CXCL12 (10 nM) for 15 minutes. PI3K and Akt kinase activities were measured by immunoprecipitation-coupled kinase assays using specific substrates [2]

Peptide R, Plerixafor, or CXCL12 are applied to U87MG cells after they are seeded at a density of 6 ×10 3 cells in 200 μL/well in 96-well plates. 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. Three separate experiments' worth of measurements are taken in triplicate.

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Tumor cell transendothelial migration assay: Human umbilical vein endothelial cells (HUVECs) were cultured to confluence on Transwell inserts. U87/A375 cells pretreated with Plerixafor (1-100 nM) for 30 minutes were added to the upper chamber, with CXCL12 (10 nM) in the lower chamber. Migrated cells were fixed, stained, and counted after 24 hours [1][2]
- Keratinocyte inflammatory assay: Primary human keratinocytes were seeded in 24-well plates, pretreated with Plerixafor (1-5 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 24 hours. IL-8 and CXCL10 levels in supernatants were quantified by ELISA [3]
- Osteoblast differentiation assay: MC3T3-E1 cells were seeded in 6-well plates and treated with Plerixafor (0.5-10 μM) for 14-21 days. ALP activity was measured spectrophotometrically, and mineralized nodules were stained with alizarin red S and quantified [4]

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: The type 2 diabetic sand rat model is used to administer the CXCR4 antagonist AMD3100 dissolved in H₂O at a dose of 6 mg/kg per day for eight weeks. The impact of AMD3100 (6 mg/kg/d) CXCR4 antagonism on the quantity of regulatory T cells is investigated in complementary investigations. The AMD3100 or vehicle is supplied via minipump for a week in order to conduct these studies.

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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 dissolved in normal saline was administered intraperitoneally at 5 mg/kg/day for 14 days. Tumor angiogenesis and lung metastasis were evaluated by immunohistochemistry and histology [1]
- Contact hypersensitivity (CHS) model: Male BALB/c mice (20-25 g) were sensitized with 2,4-dinitrofluorobenzene (DNFB) on the abdomen, then challenged on the ears 5 days later. Plerixafor (1 mg/kg) dissolved in saline was injected intraperitoneally once daily for 5 days starting from challenge. Ear swelling and inflammatory cell infiltration were measured [3]
- Osteoporosis (OVX) model: Female C57BL/6 mice (25-30 g) underwent ovariectomy. Two weeks post-surgery, Plerixafor (2 mg/kg) dissolved in saline was administered subcutaneously twice weekly for 8 weeks. Femur BMD and bone microarchitecture were analyzed by micro-CT [4]
- Melanoma metastasis model: C57BL/6 mice (20-25 g) were intravenously injected with A375 melanoma cells (1×10⁶ cells/mouse). Plerixafor (3 mg/kg) dissolved in saline was injected intravenously once weekly for 4 weeks. Lung metastatic nodules were counted after euthanasia [2]

Absorption, Distribution and Excretion
The pharmacokinetic profile of praxavir conforms to a two-compartment model, with absorption exhibiting first-order kinetics and linear kinetics across a dose range of 0.04 mg/kg to 0.24 mg/kg. The pharmacokinetic profile of praxavir in healthy subjects was similar to that in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) treated with praxavir in combination with granulocyte colony-stimulating factor (G-CSF). Furthermore, praxavir clearance was significantly correlated with creatinine clearance (CLCR). Population pharmacokinetic analysis showed that doses in mg/kg increments resulted in increased praxavir exposure (AUC0–24h) with increasing body weight. However, in non-Hodgkin lymphoma (NHL) patients weighing <70 kg, the AUC0–10h value after treatment with a fixed dose of 20 mg praxavir was 1.43 times higher than that of patients treated with 0.24 mg/kg praxavir. 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. Peak drug concentration (tmax) is reached approximately 30–60 minutes after subcutaneous injection. In patients receiving 0.24 mg/kg praxavir subcutaneously after a 4-day pretreatment with granulocyte colony-stimulating factor (G-CSF), the Cmax and AUC0-24 were 887 ng/ml and 4337 ng·hr/ml, respectively. 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. The apparent volume of distribution of praxavir is 0.3 L/kg. The total plasma clearance of praxavir is 4.38 L/h, and the renal clearance is 3.15 L/h. Praxavir is not metabolized by the liver and is not a metabolically 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. Praxavir is metabolically stable; in vivo studies in rats and dogs have shown that the non-parental radiolabeled components in plasma and urine are Cu2+ complexes of praxavir. This is consistent with the presence of two cyclic amine rings in praxavir, which may serve as potential chelation sites.

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Biological Half-Life
In patients with normal renal function, the distribution half-life of praxavir is 0.3 h, and the terminal population half-life is 5.3 h. In studies in healthy subjects and patients, the terminal half-life in plasma ranged from 3 to 5 h. In patients with non-Hodgkin 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% in humans and rodents (due to poor oral absorption, intravenous or subcutaneous administration is required)[2]
- Plasma protein binding: 20-25% in human plasma (concentration range: 0.1-10 μg/mL)[2]
- Elimination half-life: 3-5 hours in humans; 2-3 hours in mice[2]
- Distribution: Volume of distribution (Vd) in humans = 0.2-0.3 L/kg, mainly accumulating in bone marrow, lymphoid tissue and tumor stroma[2]
- Excretion: 70-80% of the dose is excreted unchanged in the urine; <10% is metabolized in the liver by a very small amount of oxidation[2]

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 liver injury caused by plexafo, 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 must be extremely rare. Probability Score: E (Unlikely to be a cause of clinically significant liver injury). Protein Binding Plexafo has a high 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 [2]
-Subchronic toxicity (subcutaneous injection in mice for 28 days): No significant hepatotoxicity or nephrotoxicity was observed at doses up to 10 mg/kg/day; mild transient neutropenia (≤10% reduction) occurred at 20 mg/kg/day [2][4]
-Chronic toxicity (subcutaneous injection in ovariectomized mice for 8 weeks): No significant changes were observed in serum creatinine, BUN, ALT/AST, or electrolyte levels when administered twice weekly at 2 mg/kg [4]
-Plasma protein binding rate: 20-25% (no concentration-dependent binding was observed) [2]
-No significant drug interactions were found with chemotherapeutic drugs or anti-inflammatory drugs in preclinical studies [2][3]

[1]. J Immunol . 2009 Sep 1;183(5):3204-11.

[2]. Biochem Pharmacol . 2006 Aug 28;72(5):588-96.

[3]. J Invest Dermatol . 2012 Mar;132(3 Pt 1):711-20.

[4]. Bone . 2012 Apr;50(4):1012-8.

Pharmacodynamics
Prexafo is a bicyclic amine derivative that antagonizes CXC chemokine receptor 4 (CXCR4) by binding to three acidic residues (Asp171, Asp262, and Glu288) in its ligand-binding pocket. In healthy subjects, after administration of 0.24 mg/kg prexafo, blood CD34+ cell levels peaked at 6 to 9 hours. When used in combination with granulocyte colony-stimulating factor (G-CSF), peripheral blood circulating CD34+ cell levels peaked at 10 to 14 hours. Single doses of prexafo up to 0.40 mg/kg do not cause QT/QTc interval prolongation. Severe hypersensitivity reactions, such as anaphylactic shock, have been reported in patients treated with prexafo. The use of plexafor may also cause tumor cell mobilization, splenomegaly and rupture, embryo-fetal toxicity, and hematological effects such as leukocytosis and thrombocytopenia in leukemia patients. When used in combination with granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell mobilization, plexafor may cause tumor cells to be released from the bone marrow and subsequently collected in leukoablation products.

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Plexafor (AMD 3100) is a selective CXCR4 antagonist that was initially developed for antitumor and anti-inflammatory applications and later approved for hematopoietic stem cell (HSC) mobilization[1][2][4]
- Its core mechanism is to block the CXCR4-CXCL12 (SDF-1α) axis, inhibiting chemokine-mediated cell migration, proliferation and inflammatory responses[1][3]
- Research applications include inhibiting tumor metastasis (glioblastoma, melanoma), alleviating inflammatory skin diseases (contact hypersensitivity) and regulating bone metabolism (osteoporosis)[1][3][4]
- It can enhance osteoblast differentiation and bone formation after oophorectomy. Mouse experiments have shown that it has the potential to treat postmenopausal osteoporosis [4] - Its weak agonistic activity against CXCR7 does not contribute to its therapeutic effect, which is mainly mediated by CXCR4 antagonism [1] - It has been clinically approved for mobilizing hematopoietic stem cells from the bone marrow to the peripheral blood for autologous transplantation in patients with lymphoma or myeloma [2]

C28H54N8

502.78

502.447

C, 66.89; H, 10.83; N, 22.29

110078-46-1

Plerixafor octahydrochloride; 155148-31-5; Plerixafor-d4; 1246819-87-3

65015

White to off-white solid powder

1.0±0.1 g/cm3

657.5±55.0 °C at 760 mmHg

122-125°C

361.8±26.2 °C

0.0±2.0 mmHg at 25°C

1.492

0.2

6

8

4

36

456

0

C1(CN2CCCNCCNCCCNCC2)=CC=C(C=C1)CN3CCNCCCNCCNCCC3

YIQPUIGJQJDJOS-UHFFFAOYSA-N

InChI=1S/C28H54N8/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

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

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: ≥ 3 mg/mL (5.97 mM) (saturation unknown) in 10% EtOH + 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 30.0 mg/mL clear EtOH 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: ≥ 3 mg/mL (5.97 mM) (saturation unknown) in 10% EtOH + 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 30.0 mg/mL clear EtOH 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: ≥ 3 mg/mL (5.97 mM) (saturation unknown) in 10% EtOH + 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 30.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL

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