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

In bone marrow-derived macrophages (BMDM), sotuletinib hydrochloride suppresses CSF-1 suspension proliferation (EC50=67 nM) and decreases CSF-1R phosphorylation, which is likewise inhibited by CSF-1R inhibitors. Crucially, sotuletinib hydrochloride in culture did not affect ischemia in any PDG-derived tumor cell lines (all Csf-1r negative), U-87 MG human neuroastroma cells, or the formation of PDG spheroids by tumor cells. It also decreased the viability of CRL-2467 astrocytes, Ink4a/Arf/BMDM (PDG genetic background), and NOD/SCID BMDM. ..As a result, solutetinib hydrochloride directly affects neuroastroma cell ischemia and prevents CSF-1R-disrupted macrophage ischemia [1].

In the cohort treated with vehicles, the median survival was 5.7 weeks. Long-term survival was markedly increased by sotuletinib hydrochloride; 64.3% of patients receiving vehicle treatment reached the 26-week trial endpoint. This endpoint was selected because Ink4a/Arf/EGFR starts to spontaneously generate tumors at about 30 weeks, including sarcomas and severe tumors. In line with histological investigations, solutetinib hydrochloride was well tolerated over the long run with no discernible side effects. Tumors from all mice treated with vehicles had high-grade neuroastromas, according to histological grading. On the other hand, solutetinib hydrochloride-treated animals showed noticeably smaller tumors overall, and 55.6% of asymptomatic mice had no visible lesions at the end of the experiment [1]. Sotutinib hydrochloride-treated tumors and the stroma around them displayed decreased CSF1R staining, and CSF1R+ stromal macrophages were considerably lower than those of vehicle-treated tumors (P<0.05) [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 hydrochloride is an orally bioavailable colony-stimulating factor 1 receptor (CSF-1R) inhibitor with potential antitumor activity. The CSF1R inhibitor BLZ945 selectively binds to CSF1R expressed on the surface of tumor-associated macrophages (TAMs), blocking CSF1R activity and inhibiting CSF1R-mediated signal transduction pathways. This inhibits TAM activity and proliferation, and reshapes 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.

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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: We validated the prognostic value of myeloid cells by analyzing genomic datasets from neuroblastoma patients. This study used freshly isolated human CD34+ hematopoietic stem cells, primary human monocytes, and mouse bone marrow cells in an in vitro culture model to deeply analyze the effects of tumor-derived factors on myeloid hematopoiesis and the local induction of suppressive myeloid cells. To examine 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 human monocyte suppressive function 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) modulates the induction of human and mouse suppressive myeloid cells and effectively limits tumor progression. Although immune checkpoint inhibitors are insufficient to control tumor growth, the combination of BLZ945 with PD-1/PD-L1 blocking antibodies can achieve better tumor control. Conclusion: Our results indicate that CSF-1R signaling plays a key role in the induction of suppressive myeloid cells and highlight its clinical potential as a human cancer immunotherapy. [3] Increased number of tumor-infiltrating macrophages is 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 a pro-tumor phenotype. Currently, 12 clinical trials are evaluating 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 the number of macrophages in a tissue-specific manner. However, further research is needed to understand the mechanisms of macrophage reduction 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. Furthermore, we found that BLZ945 inhibits tumor progression in a transgenic human papillomavirus type 16 (K14-HPV-16) cervical cancer model expressing keratin 14. Our results suggest that tumor-associated macrophages (TAMs) are continuously renewed in a CSF1R-dependent manner, and that sustained inhibition of the CSF1R pathway may be key to 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 TAMs in a mouse model of preneural GBM, which significantly prolonged survival and caused established tumor regression. In addition, CSF-1R blockade also slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, the number of tumor-associated macrophages (TAMs) did not decrease in the treated mice. Conversely, glioma-secreted factors, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-γ (IFN-γ), promote TAM survival under CSF-1R inhibition. The expression of alternatively activated M2 markers is reduced in surviving TAMs, consistent with impaired tumor-promoting function. These genetic signatures are associated with prolonged survival in patients with neuronal progenitor glioblastoma (GBM). Our results suggest that TAMs are a promising therapeutic target for neuronal progenitor gliomas and demonstrate the transformative potential of CSF-1R inhibition in GBM treatment. [1]

C20H23CLN4O3S

434.94

434.117

C, 55.23; H, 5.33; Cl, 8.15; N, 12.88; O, 11.04; S, 7.37

2222138-31-8

Sotuletinib;953769-46-5;Sotuletinib dihydrochloride;2222138-40-9

141759984

Light yellow to brown solid powder

4

7

5

29

540

2

N([C@@H]1CCCC[C@H]1O)C1=NC2=CC=C(C=C2S1)OC1=CC=NC(=C1)C(=O)NC.Cl

IHWOVMRZEIHNGY-SATBOSKTSA-N

InChI=1S/C20H22N4O3S.ClH/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);1H/t14-,17-;/m1./s1

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

Sotuletinib HCl; Sotuletinib hydrochloride, Sotuletinib monohydrochloride; QJE0UTG9TS; BLZ-945 dihydrochloride; SOTULETINIB DIHYDROCHLORIDE; UNII-QJE0UTG9TS; 2222138-40-9; Sotuletinib (dihydrochloride); 2-Pyridinecarboxamide, 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)-6-benzothiazolyl)oxy)-N-methyl-, hydrochloride (1:2); Sotuletinib dihydrochloride?; BLZ945; BLZ 945; BLZ-945;

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)

H2O : ~100 mg/mL (~229.92 mM)
DMSO : ~100 mg/mL (~229.92 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMSO + 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 25.0 mg/mL clear DMSO 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: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMSO + 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 25.0 mg/mL clear DMSO 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: ≥ 2.5 mg/mL (5.75 mM) (saturation unknown) in 10% DMSO + 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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