CXCR4 ( IC50 < 10 nM )
C-X-C chemokine receptor type 4 (CXCR4) - antagonist [1]

Benaxafortide significantly reduces pERK/pAKT signaling in the lymphoma lines Jurkat (IC50 < 400 nM) and Namalwa (IC50 < 200 nM). In MDA MB 231 breast cancer cells (IC50 < 20 nM), Namalwa and Jurkat cells (IC50 < 10 nM), benaxafortide effectively inhibits SDF-1 dependent chemotaxis (Table 1)[1].

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Balixafortide has been optimized with balanced plasma protein binding, increased plasma and microsomal stability, and favorable mouse absorption, distribution, metabolism, and excretion (ADME) properties in mind[1].

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Mobilization of Hematopoietic Stem and Progenitor Cells (HSPCs): In healthy male volunteers, a single intravenous infusion of balixafortide (500-2500 μg/kg) rapidly mobilized CD34+ HSPCs into peripheral blood in a dose-dependent manner. At doses ≥1500 μg/kg, peak mobilization reached 38.2 ± 2.8 CD34+ cells/μL (mean ± SEM). The mobilization response correlated well with the individual's response to G-CSF. [1]
Kinetics of HSPC Mobilization: At the 500 μg/kg dose, peak mobilization occurred 1 hour post-infusion. At higher doses (≥1000 μg/kg), the peak was observed approximately 4 hours after the end of the infusion. Circulating CD34+ cells remained elevated above baseline at 24 hours for doses ≥1000 μg/kg. [1]
Functional Activity of Mobilized HSPCs: The clonogenicity of balixafortide-mobilized CD34+ cells was lower than that of G-CSF-mobilized cells, with 1 CFU-C generated per 5.9 ± 0.5 CD34+ cells for balixafortide compared to 1 CFU-C per 3.2 ± 0.2 CD34+ cells for G-CSF. [1]
Mobilization of Mature Leukocytes: Balixafortide induced a dose-dependent, transient mixed leukocytosis. At doses of 1500-2500 μg/kg, white blood cell counts reached 25.3 ± 1.4 × 10³/μL. The lineage distribution differed from G-CSF, with higher relative and absolute numbers of B-cells, and lower numbers of neutrophils and monocytes. [1]
Mobilization of Plasmacytoid Dendritic Cell Progenitors (pro-pDCs): Balixafortide treatment mobilized a distinct population of pro-pDCs (CD34dimCD45dimCD45RA+CD123high), which comprised 22.4 ± 2.3% of the CD34+CD45dim cell fraction, significantly higher than after G-CSF mobilization. [1]
Comparison with G-CSF: Intra-individual comparison showed that the G-CSF regimen was approximately three times more effective in mobilizing CD34+ cells than balixafortide at the doses tested. However, volunteers subjectively rated balixafortide more favorably than G-CSF. [1]

Balixafortide was tested in a HTRF-based CXCR4 ligand binding assay, in functional assays (Calcium flux and beta arrestin), and further profiled in a large panel of other receptors including CXCR7. Effects on tumor cell sensitization were followed with an intracellular pERK / pAKT signaling assay. Tumor cell migration was assessed by chemotaxis assays, and inhibition of angiogenesis was determined by HUVEC sprouting. Evidence for immune cell activation came from evaluation of corresponding marker such as interferon gamma. Balixafortide was in detail profiled in an extensive in vitro ADME panel[1].

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Balixafortide binds CXCR4 with high affinity (IC50 < 10nM). It blocks beta arrestin recruitment and Calcium flux with IC50s < 10nM. A high 1000-fold selectivity window was demonstrated in a large panel of receptors including CXCR7. Balixafortide potently inhibits pERK / pAKT signaling in the lymphoma lines Namalwa (IC50 < 200 nM) and Jurkat (IC50 < 400 nM). Balixafortide efficiently blocks SDF-1 dependent chemotaxis of MDA MB 231 breast cancer cells (IC50 < 20 nM), Namalwa and Jurkat cells (IC50 < 10 nM). Receptor occupancy wash-out studies with competitive antibody 12G5 revealed prolonged binding of Balixafortide to CXCR4. In addition, Balixafortide was optimized for favorable mouse and human ADME properties with balanced plasma protein binding, greater plasma and microsomal stability[1].

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C57BL/6NCrl wild-type (WT) mice, domestic (DanBred hybrid) female pigs
8 mg/kg, 3 mg/kg
IV

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Study Design and Volunteers:** This was a prospective, open-label, Phase I dose-escalation trial. Twenty-seven healthy male volunteers who had previously demonstrated an average mobilization response to G-CSF (filgrastim) were enrolled. After a minimum 6-week washout period, they received a single intravenous infusion of balixafortide. The study was conducted at the phase I clinical trial unit of Goethe University Medical Center. Volunteers were discharged 24 hours after treatment and returned for a follow-up 8-14 days later. [1]
* **Dosing Regimens:** Balixafortide was administered in normal saline at doses of 500, 1000, 1500, 2000, and 2500 μg/kg based on actual body weight. Infusion durations were 1 or 2 hours at a constant rate, or a 2-hour ramped (increasing) infusion rate. Twelve donors received a second dose after a minimum 4-week washout for intra-individual comparisons. Prophylactic or therapeutic anti-histamine medication was administered to some volunteers in higher dose groups (≥2000 μg/kg) to manage histamine release symptoms. [1]
* **Pharmacodynamic Assessments:** Serial blood samples were collected at defined time points. Complete blood counts were measured using a hematology analyzer. CD34+ cell counts were quantified by flow cytometry using a single-platform assay (SCE Kit) following ISHAGE guidelines. Multi-parametric flow cytometry was used to quantify T-cells (CD3+), B-cells (CD19+), NK-cells (CD56+16+), monocytes (CD14+), and pro-pDCs (CD45dimCD34dimCD45RA+CD123high). Circulating colony-forming units (CFU-C) were assessed by plating lysed peripheral blood in cytokine-replete methylcellulose media. [1]
* **Pharmacokinetic Assessments:** Plasma samples were collected at serial time points. Balixafortide concentrations were measured, and pharmacokinetic parameters (Cmax, AUC, volume of distribution, half-life, clearance) were calculated using Phoenix WinNonlin 6.4 software. [1]
* **Safety Assessments:** Vital signs were monitored, and blood samples were drawn for biochemical safety profiling. Adverse events were documented and their relationship to the study drug was assessed. Volunteers completed a questionnaire comparing their subjective experience with G-CSF versus balixafortide. [1]

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Study Design and Volunteers: This was a prospective, open-label, Phase I dose-escalation trial. Twenty-seven healthy male volunteers who had previously demonstrated an average mobilization response to G-CSF (filgrastim) were enrolled. After a minimum 6-week washout period, they received a single intravenous infusion of balixafortide. The study was conducted at the phase I clinical trial unit of Goethe University Medical Center. Volunteers were discharged 24 hours after treatment and returned for a follow-up 8-14 days later. [1]
Dosing Regimens: Balixafortide was administered in normal saline at doses of 500, 1000, 1500, 2000, and 2500 μg/kg based on actual body weight. Infusion durations were 1 or 2 hours at a constant rate, or a 2-hour ramped (increasing) infusion rate. Twelve donors received a second dose after a minimum 4-week washout for intra-individual comparisons. Prophylactic or therapeutic anti-histamine medication was administered to some volunteers in higher dose groups (≥2000 μg/kg) to manage histamine release symptoms. [1]
Pharmacodynamic Assessments: Serial blood samples were collected at defined time points. Complete blood counts were measured using a hematology analyzer. CD34+ cell counts were quantified by flow cytometry using a single-platform assay (SCE Kit) following ISHAGE guidelines. Multi-parametric flow cytometry was used to quantify T-cells (CD3+), B-cells (CD19+), NK-cells (CD56+16+), monocytes (CD14+), and pro-pDCs (CD45dimCD34dimCD45RA+CD123high). Circulating colony-forming units (CFU-C) were assessed by plating lysed peripheral blood in cytokine-replete methylcellulose media. [1]
Pharmacokinetic Assessments: Plasma samples were collected at serial time points. Balixafortide concentrations were measured, and pharmacokinetic parameters (Cmax, AUC, volume of distribution, half-life, clearance) were calculated using Phoenix WinNonlin 6.4 software. [1]
Safety Assessments: Vital signs were monitored, and blood samples were drawn for biochemical safety profiling. Adverse events were documented and their relationship to the study drug was assessed. Volunteers completed a questionnaire comparing their subjective experience with G-CSF versus balixafortide. [1]

Pharmacokinetic Profile in Humans: Following intravenous infusion in healthy volunteers, balixafortide exhibited dose-linear pharmacokinetics for both Cmax and AUC across the 500-2500 μg/kg dose range. The volume of distribution was approximately 500-600 mL/kg. The terminal half-life was approximately 5 hours (mean 5:45 ± 0:35 h). Clearance was similar to the glomerular filtration rate, suggesting mainly renal elimination. [1]
Effect of Infusion Regimen: Different infusion durations (1 h vs. 2 h at 1000 μg/kg) did not notably influence the overall PK profile, except for an earlier Cmax with the shorter infusion. Constant-rate and ramped infusions at 2000 μg/kg resulted in virtually identical AUC, although Cmax was higher with the ramped infusion. [1]

Safety and Tolerability in Humans: Balixafortide was generally well tolerated. No severe adverse events (SAEs) were observed. The most common adverse events were mild skin reactions consistent with histamine release (flushing, urticaria, pruritus, erythema), which occurred with increasing frequency at higher doses. These symptoms were manageable with H1/H2 antihistamine treatment. [1]
Adverse Events: A summary of adverse events possibly related to the study drug included mild bone pain (1 subject), elevated serum creatinine kinase (2 subjects), and systolic blood pressure >150 mmHg (2 subjects). [1]
Subjective Preference: When questioned at follow-up, volunteers overwhelmingly preferred balixafortide over G-CSF as a mobilizing agent, rating it as easier to use and more convenient overall. [1]

[1]. Anti-tumor cell activity and in vitro profile of the next generation CXCR4 antagonist Balixafortide. Ann Oncol. 2018 Oct;29 Suppl 8:viii103.

[2]. Mobilization of hematopoietic stem cells with the novel CXCR4 antagonist POL6326 (balixafortide) in healthy volunteers-results of a dose escalation trial. J Transl Med. 2017 Jan 3;15(1):2.

Balixafortide is being investigated in the clinical trial NCT03786094 (a pivotal study of HER2-negative, locally recurrent, or metastatic breast cancer). Balixafortide is an orally bioavailable CXC chemokine receptor 4 (CXCR4) inhibitor with receptor-binding and hematopoietic stem cell mobilization activity. Balixafortide binds to the chemokine receptor CXCR4, thereby preventing stromal cell-derived factor-1 (SDF-1 or CXCL12) from binding to the CXCR4 receptor, thus inhibiting receptor activation. This may induce the mobilization of hematopoietic stem cells and progenitor cells from the bone marrow into the bloodstream. CXCR4 is a chemokine receptor belonging to the G protein-coupled receptor (GPCR) gene family, playing an important role in chemotaxis and angiogenesis, and is upregulated in various tumor cell types; the CXCL12/CXCR4 interaction induces hematopoietic cell retention in the bone marrow.

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Drug Indications
Therapy of breast cancer

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Background and Rationale: Balixafortide is a synthetic protein epitope mimetic (PEM) peptidic CXCR4 antagonist. It was developed as an alternative to G-CSF for mobilizing hematopoietic stem cells for transplantation, aiming to overcome disadvantages of G-CSF such as the need for multiple daily injections over several days, associated side effects (bone pain, flu-like symptoms), and potential contraindications. [1]
Mechanism of Action: By antagonizing the CXCR4 receptor, balixafortide disrupts the CXCR4/CXCL12 (SDF-1) axis, which is crucial for retaining hematopoietic stem cells in the bone marrow niche. This results in the rapid release or "mobilization" of stem and progenitor cells into the peripheral blood. [1]
Clinical Potential: Based on the mobilization levels achieved (≥1500 μg/kg dose), the study predicts that a single apheresis procedure would yield a standard dose (≥4 × 10⁶ CD34+ cells/kg recipient weight) for most individuals donating for a weight-matched recipient. The study suggests that balixafortide could be a safe, well-tolerated, and convenient single-dose alternative to G-CSF for stem cell mobilization in both donors and patients. Its mechanism and rapid elimination may make it suitable for individuals with contraindications to G-CSF. [1]

C84H118N24O21S2

1864.1

1862.83

C, 49.00; H, 5.53; F, 7.75; N, 15.24; O, 19.58; S, 2.91

1051366-32-5

Balixafortide TFA; 1051366-32-5

138752609

Cyclo(Ala-Cys-Ser-Ala-{D-Pro}-{Dab}-Arg-Tyr-Cys-Tyr-Gln-Lys-{D-Pro}-Pro-Tyr-His) (Disulfide bridge: Cys2-Cys9)

Cyclo(ACSA-{D-Pro}-{Dab}-RYCYQK-{D-Pro}-PYH) (Disulfide bridge: Cys2-Cys9)

Solid powder

-3.5

24

29

23

131

3970

16

C[C@H]1C(=O)N[C@H]2CSSC[C@@H](C(=O)N[C@H](C(=O)C(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]3CCCN3C(=O)[C@@H](NC(=O)[C@@H](NC2=O)CO)C)CCN)CCCNC(=N)N)CC4=CC=C(C=C4)O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]5CCCN5C(=O)[C@@H]6CCCN6C(=O)[C@@H](NC(=O)[C@@H](NN1)CC7=CN=CN7)CC8=CC=C(C=C8)O)CCCCN)CCC(=O)N)CC9=CC=C(C=C9)O

OYWQJZAVFWOOBF-WBMPNIIXSA-N

InChI=1S/C84H118N24O21S2/c1-44-70(116)102-62-41-130-131-42-63(77(123)98-57(35-46-14-20-50(110)21-15-46)69(115)68(114)53(10-5-31-91-84(88)89)94-73(119)56(28-30-86)97-79(125)64-11-6-32-106(64)81(127)45(2)93-76(122)61(40-109)101-78(62)124)103-74(120)58(36-47-16-22-51(111)23-17-47)99-72(118)55(26-27-67(87)113)95-71(117)54(9-3-4-29-85)96-80(126)65-12-7-33-107(65)83(129)66-13-8-34-108(66)82(128)60(37-48-18-24-52(112)25-19-48)100-75(121)59(105-104-44)38-49-39-90-43-92-49/h14-25,39,43-45,53-66,104-105,109-112H,3-13,26-38,40-42,85-86H2,1-2H3,(H2,87,113)(H,90,92)(H,93,122)(H,94,119)(H,95,117)(H,96,126)(H,97,125)(H,98,123)(H,99,118)(H,100,121)(H,101,124)(H,102,116)(H,103,120)(H4,88,89,91)/t44-,45-,53-,54-,55-,56-,57-,58-,59-,60-,61-,62-,63-,64+,65+,66-/m0/s1

3-[(1R,4S,7S,10S,16S,22R,25S,28S,31S,34R,37S,40S,43S,46R,52S,55S)-25-(4-aminobutyl)-43-(2-aminoethyl)-40-(3-carbamimidamidopropyl)-55-(hydroxymethyl)-10,31,37-tris[(4-hydroxyphenyl)methyl]-7-(1H-imidazol-5-ylmethyl)-4,52-dimethyl-3,8,11,17,23,26,29,32,35,38,39,42,45,51,54,57-hexadecaoxo-59,60-dithia-2,5,6,9,12,18,24,27,30,33,36,41,44,50,53,56-hexadecazapentacyclo[32.23.4.012,16.018,22.046,50]henhexacontan-28-yl]propanamide

Balixafortide; POL 6326; POL6326; POL6326; Cyclo(L-alanyl-L-cysteinyl-L-seryl-L-alanyl-D-prolyl-(2S)-2,4-diaminobutanoyl-L-arginyl-L-tyrosyl-L-cysteinyl-L-tyrosyl-L-glutaminyl-L-lysyl-D-prolyl-L-prolyl-L-tyrosyl-L-histidyl), cyclic (2->9)-disulfide; Balixafortide [INN]; Balixafortide [USAN]; 11-Dideuterolinoleic acid; Balixafortide(POL6326); POL6326

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)

Water: ~100 mg/mL

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.)