GDC-0575 dihydrochloride (ARRY-575 dihydrochloride) is an orally bioavailable CHK1 inhibitor, with an IC₅₀ of 1.2 nM, and has antitumor activity.

GDC-0575 dihydrochloride is a selective and orally bioavailable CHK1 inhibitor, with an IC₅₀ of 1.2 nM. GDC-0575 (100 nM) suppressses CHK1 activation induced by AraC by decreasing the level of Tyr15-phosphorylated CDK2. GDC-0575 (100 nM) has no effect on the viability of AML cells, but significantly reduces cell viability and induces apoptosis in combination with AraC. In addition, GDC-0575 plus AraC shows no effect on normal hematopoietic stem and progenitor cells (HSPCs)^[1]. GDC-0575 shows cytotoxic activity against most of 20 melanoma cell lines tested, but several cell lines grown as tumour sphere (TS) are relatively insensitive^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

GDC-0575 (7.5 mg/kg, p.o.) in combination with AraC alomost completely eradicates leukemic burden in mice transplanted with U937-Luc cells, and shows more efficient activity than AraC alone. Furthermore, GDC-0575 elevates the cytotoxicity of AraC in different primary AML models in vivo^[1]. GDC-0575 (25, 50 mg/kg, p.o.) dose-dependently inhibits the growth of tumor in D20 and C002 xenografts^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

451.19

C₁₆H₂₂BrCl₂N₅O

1657014-42-0

Room temperature in continental US; may vary elsewhere.

4°C, sealed storage, away from moisture

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

DMSO : 65 mg/mL (144.06 mM; Need ultrasonic)

H₂O : 25 mg/mL (55.41 mM; Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.17 mg/mL (4.81 mM); Clear solution

  此方案可获得 ≥ 2.17 mg/mL (4.81 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 21.7 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.17 mg/mL (4.81 mM); Clear solution

  此方案可获得 ≥ 2.17 mg/mL (4.81 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 21.7 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.17 mg/mL (4.81 mM); Clear solution

  此方案可获得 ≥ 2.17 mg/mL (4.81 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 21.7 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Di Tullio A, et al. The combination of CHK1 inhibitor with G-CSF overrides cytarabine resistance in human acute myeloid leukemia. Nat Commun. 2017 Nov 22;8(1):1679.

  [2]. Oo ZY, et al. Endogenous Replication Stress Marks Melanomas Sensitive to CHEK1 Inhibitors In Vivo. Clin Cancer Res. 2018 Jun 15;24(12):2901-2912.

For co-culture experiments, 2 days before initiating the co-culture, feeder cells are plated onto type-I collagen-coated 96-well or 6-well plates and allowed to reach confluence. One day before starting co-culture, they are irradiated at 6.8 Gy and the culture media exchanged. On day 0 of the co-culture, AmL cells are plated at 2 × 10⁵ cells/mL using the correspondent AmL medium. Cells are cultured at 37°C in 5% CO₂-humidified incubators at indicated oxygen concentrations. For short-term culture (STC), cells are kept for 1 week in hypoxia (5% O₂) with the indicated drugs: 500 nM AraC and/or 100 nM GDC-0575^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
NSG mice are injected intravenously with 1 × 10⁵-10⁶ cells of AmL and 1-3 × 10⁵ cells of hCB CD34⁺/hBM CD34⁺. After demonstrating AmL engraftment at 9-11 weeks through FACS analysis of tibia bone marrow aspiration, mice are treated accordingly to proper 7-day treatment regimen with daily 10 mg/kg AraC via subcutaneous injection, 7.5 mg/kg GDC-0575 suspension administered via oral gavage on every other day schedule, and/or 300 μg/kg G-CSF administered daily for 5 days via intraperitoneal injection. One week after the final dosing, mice are killed by cervical dislocation. The femurs, tibias, and pelvis are dissected and flushed with PBS. Red blood cells are lyzed via ammonium chloride. Cells are stained with human-specific FITC-conjugated anti-CD19, PE-conjugated anti-CD33, PE-Cy7-conjugated anti-CD45, and PERCP-conjugated anti-murine CD45 antibodies. Dead cells and debris are excluded via DAPI staining. A BD LSR II flow cytometer is used for analysis. Flow cytometry analysis is performed with FlowJo software. More than 100,000 DAPI-negative events are collected. Engraftment of AmL is said to be present if a single population of mCD45^–hCD45⁺CD33⁺CD19^– cells is present without accompanying mCD45^–hCD45⁺CD33−CD19⁺cells^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Di Tullio A, et al. The combination of CHK1 inhibitor with G-CSF overrides cytarabine resistance in human acute myeloid leukemia. Nat Commun. 2017 Nov 22;8(1):1679.

  [2]. Oo ZY, et al. Endogenous Replication Stress Marks Melanomas Sensitive to CHEK1 Inhibitors In Vivo. Clin Cancer Res. 2018 Jun 15;24(12):2901-2912.