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 Dihydrochloride is an orally bioavailable inhibitor of colony stimulating factor 1 receptor (CSF-1R; CSF1R), with potential antineoplastic activity. CSF1R inhibitor BLZ945 selectively binds to CSF1R expressed on tumor-associated macrophages (TAMs), blocks the activity of CSF1R, and inhibits CSF1R-mediated signal transduction pathways. This inhibits the activity and proliferation of TAMs, and reprograms the immunosuppressive nature of existing TAMs. Altogether, this reduces TAM-mediated immune suppression in the tumor microenvironment, re-activates the immune system, and improves anti-tumor cell responses mediated by T-cells. CSF1R, also known as macrophage colony-stimulating factor receptor (M-CSFR) and CD115 (cluster of differentiation 115), is a cell-surface receptor for its ligand, colony stimulating factor 1 (CSF1); this receptor is overexpressed by TAMs in the tumor microenvironment, and plays a major role in both immune suppression and the induction of tumor cell proliferation.

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Purpose: Neuroblastoma is the most common extracranial solid cancer type in childhood, and high-risk patients have poor prognosis despite aggressive multimodal treatment. Neuroblastoma-driven inflammation contributes to the induction of suppressive myeloid cells that hamper efficient antitumor immune responses. Therefore, we sought to enhance antitumor immunity by removing immunosuppression mediated by myeloid cells.
Experimental Design: The prognostic values of myeloid cells are demonstrated by analyzing genomic datasets of neuroblastoma patients. The impact of tumor-derived factors on myelopoiesis and local induction of suppressive myeloid cells is dissected by in vitro culture models using freshly isolated human CD34+ hematopoietic stem cells, primary human monocytes, and murine bone marrow cells. To test the therapeutic efficacy of BLZ945 as a monotherapy or in combination with checkpoint inhibitors, we used a transgenic murine model (TH-MYCN) that develops aggressive spontaneous neuroblastoma.
Results: We report that infiltrating CSF-1R+ myeloid cells predict poor clinical outcome in patients with neuroblastoma. In vitro, neuroblastoma-derived factors interfere with early development of myeloid cells and enable suppressive functions on human monocytes through M-CSF/CSF-1R interaction. In a transgenic mouse model (TH-MYCN) resembling high-risk human neuroblastoma, antagonizing CSF-1R with a selective inhibitor (BLZ945) modulates the induction of human and murine suppressive myeloid cells and efficiently limit tumor progression. While checkpoint inhibitors are insufficient in controlling tumor growth, combining BLZ945 with PD-1/PD-L1 blocking antibodies results in superior tumor control.
Conclusions: Our results demonstrate the essential role of CSF-1R signaling during the induction of suppressive myeloid cells and emphasize its clinical potential as an immunotherapy for human cancers. [3]

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Increased numbers of tumor-infiltrating macrophages correlate with poor disease outcome in patients affected by several types of cancer, including breast and prostate carcinomas. The colony stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment of tumor-associated macrophages (TAMs) to the neoplastic microenvironment and promotes the differentiation of TAMs toward a pro-tumorigenic phenotype. Twelve clinical trials are currently evaluating agents that target the CSF1/CSF1R signaling pathway as a treatment against multiple malignancies, including breast carcinoma, leukemia, and glioblastoma. The blockade of CSF1R signaling has been shown to greatly decrease the number of macrophages in a tissue-specific manner. However, additional mechanistic insights are needed in order to understand how macrophages are depleted and the global effects of CSF1R inhibition on other tumor-infiltrating immune cells. Using BLZ945, a highly selective small molecule inhibitor of CSF1R, we show that CSF1R inhibition attenuates the turnover rate of TAMs while increasing the number of CD8+ T cells that infiltrate cervical and breast carcinomas. Specifically, we find that BLZ945 decreased the growth of malignant cells in the mouse mammary tumor virus-driven polyomavirus middle T antigen (MMTV-PyMT) model of mammary carcinogenesis. Furthermore, we show that BLZ945 prevents tumor progression in the keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) transgenic model of cervical carcinogenesis. Our results demonstrate that TAMs undergo a constant turnover in a CSF1R-dependent manner, and suggest that continuous inhibition of the CSF1R pathway may be essential to maintain efficacious macrophage depletion as an anticancer therapy.[2]

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Glioblastoma multiforme (GBM) comprises several molecular subtypes, including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumor-associated macrophages and microglia (TAMs). Macrophages depend on colony stimulating factor-1 (CSF-1) for differentiation and survival. We used an inhibitor of the CSF-1 receptor (CSF-1R) to target TAMs in a mouse proneural GBM model, which significantly increased survival and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, TAMs were not depleted in treated mice. Instead, glioma-secreted factors, including granulocyte-macrophage CSF (GM-CSF) and interferon-γ (IFN-γ), facilitated TAM survival in the context of CSF-1R inhibition. Expression of alternatively activated M2 markers decreased in surviving TAMs, which is consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in patients with proneural GBM. Our results identify TAMs as a promising therapeutic target for proneural gliomas and establish the translational potential of CSF-1R inhibition for GBM.[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.)