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

体外活性：在骨髓源性巨噬细胞 (BMDM) 中，BLZ945 特异性抑制 CSF-1 依赖性增殖，EC50 为 67nM，并降低 CSF-1R 磷酸化。 BLZ945 阻断巨噬细胞和神经胶质瘤细胞之间对彼此存活、增殖和/或极化的相互作用，从而促进肿瘤发生。激酶测定：BLZ945 是一种有效的、口服生物活性的、选择性的 CSF-1R（集落刺激因子 1 受体）抑制剂，IC50 为 1 nM，对其最接近的受体酪氨酸激酶同源物的选择性超过 1000 倍。细胞测定：使用MTT细胞增殖试剂盒测定细胞生长速率。简而言之，将细胞一式三份接种在 96 孔板中：神经胶质瘤细胞系每孔 1,000 个细胞，BMDM 和 CRL-2467 每孔 5 x 1,000 个细胞，HUVEC 和 HBMEC 细胞系每孔 2.5 x 1,000 个细胞。对于所有实验，介质每 48 小时更换一次。细胞在存在或不存在 6.7–6,700 nM BLZ945 或 8 μg/mL CSF-1R 中和抗体的情况下生长。 BMDM 和 CRL-2467 细胞分别补充 10 ng/mL 和 30 ng/mL 重组小鼠 CSF-1。根据制造商的方案，使用读板器通过比色分析检测 MTT 底物的还原，并在 SpectraMax 340pc 读板器上在 595 nm 和 750 nm 处进行测量。

在患有神经胶质瘤的小鼠中，BLZ945 通过抑制 CSF-1R 来阻止肿瘤进展并显着提高生存率。 BLZ945 还在体内抑制患者源性原神经肿瘤球和细胞系的原位肿瘤生长。 BLZ945（200 mg/kg，口服）可降低小鼠乳腺肿瘤病毒驱动的多瘤病毒中 T 抗原 (MMTV-PyMT) 乳腺癌模型和表达角蛋白 14 的人乳头瘤病毒 16 型 (K14-HPV- 16）宫颈癌转基因模型。

BLZ945 是一种有效的、口服生物活性的、选择性的 CSF-1R（集落刺激因子 1 受体）抑制剂，IC50 为 1 nM，对其最接近的受体酪氨酸激酶同源物的选择性超过 1000 倍。
BLZ945， CSF-1R酪氨酸激酶的高选择性小分子抑制剂（比其他酪氨酸激酶高3200倍）；参考文献27)。在体外阻断实验中，将BLZ945和GW2580分别溶于10 mmol/L和1 mmol/L的DMSO中制备原液。在体内治疗方面，根据先前的研究，BLZ945溶解在20% Captisol中，剂量为16 mg/mL，每日灌胃剂量为200 mg/kg。[3]

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细胞因子分析：在卡罗林斯卡大学医院的核心设施中，通过27参数Luminex多路试验 分析从SK-N-BE(2)、SK-N-AS或SK-N-FI神经母细胞瘤细胞系中收集的培养基或上清液中的细胞因子含量。采用ELISA法测定中药中人或鼠M-CSF （CSF-1）的浓度。[3]

MTT细胞增殖试剂盒用于计算细胞生长速率。简而言之，96 孔板用于将细胞一式三份铺板：神经胶质瘤细胞系 1 ×10³ 细胞，BMDM 和 CRL-5 ×10³ 细胞2467 和 2.5 ×10³ 细胞（适用于 HUVEC 和 HBMEC 细胞系）。每 48 小时，所有实验中使用的培养基都会更换一次。细胞在有或没有 8 μg/mL CSF-1R 中和抗体或 6.7–6,700 nM sotuletinib 的情况下培养。 PDGC 系在 10,000 nM PTK787 或 10,000 nM STI571（用 10 mM DMSO 储备液稀释）存在下培养，以测试对 PDGFR 抑制的敏感性。除非另有说明，否则将制造商提供的 ECGF 添加到 HUVEC 和 HBMEC 细胞中。通过读板器和比色分析，遵循制造商的方案来检测 MTT 底物的还原。在加入 100 μL MTT 增溶试剂并在 37°C 下静置过夜之前，向每孔中加入 10 μL MTT 标记试剂并孵育 4 小时。使用 SpectraMax 340pc 酶标仪，轻轻地重悬混合物，并在 595 和 750 nm 处测量吸光度[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

125

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.22 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (5.22 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (5.22 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 4% DMSO+30% PEG 300+ddH2O: 2.5 mg/mL

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配方 5 中的溶解度: 10 mg/mL (25.10 mM) in 20% SBE-β-CD in Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。