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]

The MTT cell proliferation kit is used to calculate the rate of cell growth. In a nutshell, 96-well plates are used to plate cells in triplicate: 1 ×10³ cells for glioma cell lines, 5 ×10³ cells for BMDM and CRL-2467, and 2.5 ×10³ cells for HUVEC and HBMEC cell lines. Every 48 hours, the media used in all experiments is changed. The cells are cultured with or without 8 μg/mL of CSF-1R neutralizing antibody or 6.7–6,700 nM of sotuletinib. PDGC lines are cultivated in the presence of 10,000 nM PTK787 or 10,000 nM STI571 (diluted from 10 mM stock solutions in DMSO) in order to test the sensitivity to PDGFR inhibition. ECGF supplied by the manufacturer is added to HUVEC and HBMEC cells, unless specified otherwise. With a plate reader and colorimetric analysis, the manufacturer's protocol is followed to detect the reduction of the MTT substrate. Before adding 100 μL of MTT solubilization reagent and letting it sit overnight at 37°C, 10 μL of MTT labeling reagent is added to each well and allowed to incubate for 4 hours. Utilizing a SpectraMax 340pc plate reader, the mixture is gently resuspended and absorbance is measured at 595 and 750 nm[1].

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 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.

C20H22N4O3S

398.478682994843

398.14

C, 60.28; H, 5.57; N, 14.06; O, 12.04; S, 8.05

953769-46-5

Sotuletinib hydrochloride;2222138-31-8;Sotuletinib dihydrochloride;2222138-40-9

46184986

Off-white to light brown solid powder

3.4

3

7

5

28

540

2

CNC(=O)C1=NC=CC(=C1)OC2=CC3=C(C=C2)N=C(S3)N[C@@H]4CCCC[C@H]4O

ADZBMFGQQWPHMJ-RHSMWYFYSA-N

InChI=1S/C20H22N4O3S/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)/t14-,17-/m1/s1

4-[[2-[[(1R,2R)-2-hydroxycyclohexyl]amino]-1,3-benzothiazol-6-yl]oxy]-N-methylpyridine-2-carboxamide

BLZ-945; Sotuletinib; BLZ 945; 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide; UNII-7W3V82OQ0P; 7W3V82OQ0P;BLZ945

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: ≥ 2.08 mg/mL (5.22 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 20.8 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: ≥ 2.08 mg/mL (5.22 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 20.8 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: ≥ 2.08 mg/mL (5.22 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 4% DMSO+30% PEG 300+ddH2O: 2.5 mg/mL

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Solubility in Formulation 5: 10 mg/mL (25.10 mM) in 20% SBE-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)