CSF-1R (IC50 = 1 nM); c-Kit (IC50 = 3.2 μM); PDGFRβ (IC50 = 4.8 μM); Flt3 (IC50 = 9.1 μM)

BLZ945 specifically reduces CSF-1R phosphorylation and inhibits CSF-1-dependent proliferation in bone marrow-derived macrophages (BMDMs) with an EC50 of 67nM. In order to promote tumorigenesis, BLZ945 inhibits the reciprocal effects that macrophages and glioma cells have on one another's survival, proliferation, and/or polarization.[1]

BLZ945 inhibits CSF-1R to stop tumor growth and dramatically increase survival in mice with gliomas. Additionally, proneural tumor spheres and cell lines derived from patients are inhibited in vivo from growing orthotopically by BLZ945.[1] In both the mouse mammary tumor virus-driven polyomavirus middle T antigen (MMTV-PyMT) model of mammary carcinogenesis and the keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) transgenic model of cervical carcinogenesis, BLZ945 (200 mg/kg, p.o.) inhibits the growth of malignant cells.[2]

BLZ945 is a potent, orally bioactive, and selective CSF-1R (colony stimulating factor 1 receptor) inhibitor with IC50 of 1 nM, it is more than 1000-fold selective against its closest receptor tyrosine kinase homologs.
BLZ945, a highly selective small-molecule inhibitor for tyrosine kinase of CSF-1R (>3,200-fold more than other tyrosine kinases; ref. 27), was used. For in vitro–blocking experiments, stock solutions were prepared by dissolving BLZ945 or GW2580 in DMSO at 10 mmol/L and 1 mmol/L, respectively. For in vivo treatment, BLZ945 was dissolved in 20% Captisol at 16 mg/mL and delivered by daily oral gavage at the dose of 200 mg/kg, according to a previous study.[3]

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Cytokine analysis: Cytokine contents in culture medium or supernatants harvested from SK-N-BE(2), SK-N-AS, or SK-N-FI neuroblastoma tumor cell lines were analyzed by a 27-parameter Luminex multiplex assay in the core facility at Karolinska University Hospital. Concentrations of human or murine M-CSF (CSF-1) in the TCM were determined using ELISA.[3]

Differentiation of CD34+ hematopoietic progenitor cells[3]
Maturation of CD34+ cells was performed using 900 μL culture medium containing 50 ng/mL GM-CSF and 5 ng/mL TNFα in a 24-well plate. Alternatively, supernatants harvested from the three above-mentioned human neuroblastoma cell lines were added in addition to the cytokines at a 2:1 ratio to the progenitor cells. CD34+ cells maintained in culture medium were used as controls. To block CSF-1R signaling, Sotuletinib (BLZ945)  (500 nmol/L) was added to cells matured with cytokines or the combination of cytokines and SK-N-BE(2) supernatant. After 7 days, all cells were harvested by washing and gently scraping, and phenotypes and functions of the cells were evaluated by flow cytometry or in CFSE-based T-cell proliferation assays.

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Human monocyte–tumor coculture[3]
Primary human monocytes were cocultured with human neuroblastoma cell lines according to our published protocol. In brief, monocytes were cocultured with 4×105 SK-N-BE(2), SK-N-AS, or 6×105 SK-N-FI neuroblastoma cells in 3-mL culture medium in a 6-well plate. Monocytes cultured without tumor cells were used as controls. After 64 hours, cells were harvested by vigorously washing, followed by gently scraping of the plates. Phenotypic changes of the cells were evaluated by flow cytometry and HLA-DR+ cells were sorted with microbeads and MS columns. To investigate the role of CSF-1R, 500 nmol/L Sotuletinib (BLZ945)  or 1 μmol/L GW2580 was added to monocytes or the cocultures.

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Differentiation of murine bone marrow cells[3]
Suppressive myeloid cells were induced from bone marrow cells harvested from negatively genotyped TH-MYCN mice, in accordance with a previously described protocol. In brief, 1×106 isolated bone marrow cells were cultured in 6-well plates in the presence of NHO2A tumor-conditioned medium (TCM, 1:1 dilution to fresh medium). As controls, cells were cultured in fresh medium or with M-CSF (20 ng/mL). To block CSF-1R signaling, Sotuletinib (BLZ945)  or GW2580 were added at 1 μmol/L to the cultures and DMSO was included as control. After 4 days, cells were harvested by collecting floating cells and carefully scraping the adherent cells of the wells, and flow cytometric analysis or T-cell suppression assays were conducted subsequently.

Mice: Volumes of tumors are measured with calipers using the following formula: volume=(width)²×length/2. 56–63 day old female mice are dosed with 200 mg/kg of sotuletinib or 20% Captisol vehicle in MMTV-PyMT mouse studies. The mice are randomized into groups according to the sizes of their tumors. Tumor volumes are measured twice a week, and the dosage is given orally via gavage once a day. Rat IgG control or 5A1 rat anti-mouse CSF1 neutralizing antibody is injected intraperitoneally every five days at a dose of 10 mg/kg. Formalin-fixed paraffin-embedded lungs in MMTV-PyMT transgenic mice are serially sectioned and stained with hematoxylin and eosin to determine pulmonary metastasis. Tumor regions are rated based on size (tumor diameter), tumor burden (total tumor area divided by total lung area), and the total number of individual metastases counted in a single-blind manner. To get the final value, these values are averaged over the whole lung depth.

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Orthotopic allograft models[2]
6–7 wk old female FVB/NJ mice and 6–7 wk old female BALB/c nude mice (CAnN.Cg-Foxn1nu/Crl) were used. For the mammary tumor virus-driven Polyoma middle T antigen (MMTV-PyMT) orthotopic allograft model, spontaneous tumors from 10–13 wk old female transgenic MMTV-PyMT mice were pooled and enzymatically digested with Liberase TM (Roche). The resultant single-cell suspension was then immediately injected orthotopically at the indicated cell dosage into a single mammary fat pad of syngeneic female FVB/NJ recipient mice. For the CD45 allotype study, spontaneous tumors from 10–13 wk old female MMTV-PyMT transgenic mice were harvested by blunt dissection and divided into 3 mm cubes. A small incision was made in the mammary fat pad of female BALB/c nude recipient mice and 2 tumor samples were placed inside the fat pad and sealed with surgical staples. After 5 d, the wound was reopened and the tumor samples retrieved. Tumors were digested and analyzed as described below. Donor and recipient mice were treated with either Sotuletinib (BLZ945)  or vehicle for 5 d prior to resection and implantation as described below.

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CSF1-signaling antagonist pharmacological study in spontaneous tumor models[2]
Tumors were measured using calipers and volumes calculated based on the formula: volume = (width)2 × length/2. In MMTV-PyMT mouse studies, 56–63 d old female mice were randomized into groups based on tumor volumes and dosed with either 20% Captisol® vehicle or 200 mg/kg Sotuletinib (BLZ945) . Dosing was administered by oral gavage once daily and tumor volumes were measured twice weekly. 5A1 rat anti-mouse CSF1 neutralizing antibody or rat IgG control was dosed at 10 mg/kg by intraperitoneal injection every 5 d. To calculate pulmonary metastasis in MMTV-PyMT transgenic mice, formalin-fixed paraffin-embedded lungs were serially sectioned and stained with hematoxylin and eosin (H&E). Tumor regions were scored by tumor burden (total tumor area divided by total lung area), size (tumor diameter), and according to the total number of individual metastases counted in a single-blind fashion. These values were averaged across the entire depth of the lung to obtain the final value. For K14-HPV16 mouse studies, female mice were given slow release 17β-estradiol pellets every 2 mo to induce squamous carcinogenesis in the cervical and vaginal epithelium.43,44 Mice were randomized at 6 mo of age at the reported onset of cervical cancer and treated with Sotuletinib (BLZ945)  for a 1 mo duration. To determine cervical tumor volume in K14-HPV16 transgenic mice, formalin-fixed paraffin-embedded cervix tissues and neoplasms were serially sectioned, scored for tumor area in a single-blind fashion, and the values multiplied by the tumor depth.

[1]. SF-1R inhibition alters macrophage polarization and blocks glioma progression. Nat Med. 2013 Oct;19(10):1264-72.

[2]. CSF1R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8+ T cells. Oncoimmunology. 2013 Dec 1;2(12):e26968.

[3]. Targeting Suppressive Myeloid Cells Potentiates Checkpoint Inhibitors to Control Spontaneous Neuroblastoma. Clin Cancer Res (2016) 22 (15): 3849–3859.

Sotuletinib is an orally bioavailable colony-stimulating factor 1 receptor (CSF-1R; CSF1R) inhibitor with potential antitumor activity. The CSF1R inhibitor BLZ945 selectively binds to CSF1R expressed on tumor-associated macrophages (TAMs), blocking CSF1R activity and inhibiting CSF1R-mediated signal transduction pathways. This inhibits TAM activity and proliferation and reprograms the existing immunosuppressive properties of TAMs. In summary, this reduces TAM-mediated immunosuppression in the tumor microenvironment, reactivates the immune system, and enhances T cell-mediated antitumor cell responses. CSF1R, also known as macrophage colony-stimulating factor receptor (M-CSFR) and CD115 (differentiation cluster 115), is the cell surface receptor for its ligand, colony-stimulating factor 1 (CSF1). This receptor is overexpressed by tumor-associated macrophages (TAMs) in the tumor microenvironment and plays an important role in immunosuppression and induction of tumor cell proliferation. Objective: Neuroblastoma is the most common extracranial solid tumor in children, and high-risk patients still have a poor prognosis even with aggressive multimodal therapy. Inflammation caused by neuroblastoma leads to the induction of suppressive myeloid cells, thereby hindering an effective anti-tumor immune response. Therefore, we sought to enhance anti-tumor immunity by eliminating myeloid cell-mediated immunosuppression. Experimental Design: By analyzing genomic datasets from neuroblastoma patients, we confirmed the prognostic value of myeloid cells. Using freshly isolated human CD34+ hematopoietic stem cells, primary human monocytes, and mouse bone marrow cells, we analyzed the effects of tumor-derived factors on myeloid hematopoiesis and the local induction of suppressive myeloid cells in an in vitro culture model. To test the efficacy of BLZ945 as monotherapy or in combination with immune checkpoint inhibitors, we used a transgenic mouse model (TH-MYCN) that spontaneously develops invasive neuroblastoma. Results: We found that infiltrating CSF-1R+ myeloid cells predicted a poor prognosis in neuroblastoma patients. In vitro experiments showed that neuroblastoma-derived factors interfere with the early development of myeloid cells and confer an inhibitory function on human monocytes through M-CSF/CSF-1R interaction. In a transgenic mouse model similar to high-risk human neuroblastoma (TH-MYCN), antagonism of CSF-1R with a selective inhibitor (BLZ945) modulated the induction of suppressive myeloid cells in humans and mice and effectively limited tumor progression. Although immune checkpoint inhibitors are insufficient to control tumor growth, the combination of BLZ945 with PD-1/PD-L1 blocking antibodies significantly improved tumor control. Conclusion: Our results suggest that the CSF-1R signaling pathway plays a crucial role in the induction of suppressive myeloid cells and highlight its clinical potential as a human cancer immunotherapy. [3] Increased numbers of tumor-infiltrating macrophages are associated with poor prognosis in patients with various cancers, including breast and prostate cancer. The colony-stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment of tumor-associated macrophages (TAMs) to the tumor microenvironment and promotes TAM differentiation into pro-tumorigenic phenotypes. Currently, 12 clinical trials are evaluating the efficacy of drugs targeting the CSF1/CSF1R signaling pathway for the treatment of various malignancies, including breast cancer, leukemia, and glioblastoma. Studies have shown that blocking the CSF1R signaling pathway can significantly reduce macrophage numbers in a tissue-specific manner. However, further mechanistic studies are needed to understand the mechanisms of macrophage depletion and the overall effects of CSF1R inhibition on other tumor-infiltrating immune cells. Using the highly selective small molecule CSF1R inhibitor BLZ945, we found that CSF1R inhibition reduces the turnover rate of tumor-associated macrophages (TAMs) while increasing the number of CD8+ T cells infiltrating cervical and breast cancer. Specifically, we found that BLZ945 inhibits the growth of malignant cells in a mouse mammary tumor virus-driven polyomavirus T antigen (MMTV-PyMT) breast cancer model. In addition, we found that BLZ945 inhibited tumor progression in a transgenic cervical cancer model expressing keratin 14 human papillomavirus type 16 (K14-HPV-16). Our results indicate that tumor-associated macrophages (TAMs) are continuously renewed in a CSF1R-dependent manner, suggesting that sustained inhibition of the CSF1R pathway is crucial for maintaining effective macrophage clearance as an anticancer therapy. [2] Glioblastoma (GBM) comprises multiple molecular subtypes, including preneural GBM. Most treatments targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumor-associated macrophages and microglia (TAMs). Macrophage differentiation and survival depend on colony-stimulating factor-1 (CSF-1). We used a CSF-1 receptor (CSF-1R) inhibitor to target tumor-associated macrophages (TAMs) in a mouse preneural glioblastoma (GBM) model, which significantly prolonged survival and caused regression of established tumors in mice. In addition, CSF-1R blockade slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, TAM was not reduced in treated mice. Instead, glioma-secreted factors, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-γ (IFN-γ), promoted TAM survival under CSF-1R inhibition. The expression of alternatively activated M2 markers was reduced in surviving TAMs, consistent with impaired tumor-promoting function. These genetic features are associated with prolonged survival in patients with pre-glioblastoma. Our results suggest that TAM is a promising therapeutic target for pre-gliomas and demonstrate the potential for translational application of CSF-1R inhibition in GBM treatment. [1]

434.11793

C, 50.96; H, 5.13; Cl, 15.04; N, 11.89; O, 10.18; S, 6.80

2222138-40-9

Sotuletinib;953769-46-5;Sotuletinib hydrochloride;2222138-31-8

141759984

Typically exists as solid at room temperature

125Ų

5

7

5

30

540

2

ZIHWHYXECXSBNA-LVVRIOTCSA-N

InChI=1S/C20H22N4O3S.2ClH/c1-21-19(26)16-10-13(8-9-22-16)27-12-6-7-15-18(11-12)28-20(24-15)23-14-4-2-3-5-17(14)25;;/h6-11,14,17,25H,2-5H2,1H3,(H,21,26)(H,23,24);2*1H/t14-,17-;;/m1../s1

4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide dihydrochloride

Sotuletinib dihydrochloride; BLZ945; Sotuletinib hydrochloride; 2222138-31-8; Sotuletinib (hydrochloride); 4-((2-(((1R,2R)-2-Hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide hydrochloride; Sotuletinib HCl?; BLZ945 HCl; BLZ945 HYDROCHLORIDE; BLZ 945; BLZ-945.Sotuletinib;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)