Akt1 (IC50 = 5 nM); Akt3 (IC50 = 8 nM); Akt2 (IC50 = 18 nM); PKA (IC50 = 3100 nM)

当针对大量 230 种激酶（PRKG1α、PRKG1β 和 p70S6K，IC50 值分别为 98 nM、69 nM 和 860 nM）进行测试时，GDC-0068 在 1 μM 浓度下仅抑制 3 种激酶 >70%。 GDC-0068 的 IC50 为 3.1 μM，对 Akt 的选择性是 PKA 的 100 倍以上。 GDC-0068 处理可抑制 LNCaP、PC3 和 BT474M1 细胞中 Akt 底物 PRAS40 的磷酸化，IC50 值分别为 157 nM、197 nM 和 208 nM。此外，GDC-0068 选择性抑制肿瘤抑制因子 PTEN 缺陷、PIK3CA 致癌突变和 HER2 扩增的 Akt 信号驱动癌细胞系，其中 HER2+ 和 Luminal 亚型的效果最强。 [1-3]

GDC-0068 口服给药会导致 PC3 前列腺肿瘤异种移植模型中 p-PRAS40 下调。 BT474-Tr 异种移植物中的 GDC-0068 治疗可降低 pS6 和 peIF4G 水平，将 FOXO3a 重新定位到细胞核，并导致 HER3 和 pERK 的反馈上调。在许多异种移植肿瘤模型中，包括 PTEN 缺陷型前列腺癌模型 LNCaP 和 PC3、PIK3CA H1047R 突变乳腺癌模型 KPL-4 和 MCF7-neo/HER2 肿瘤模型，GDC-0068 的给药表现出强大的抗肿瘤功效。 [1-3]

Enzymatic Assays/酶实验[1]
用于测定Akt1/2/3和PKA激酶活性的测定采用IMAP荧光偏振（FP）磷酸化检测试剂来检测已被相应激酶磷酸化的荧光标记肽底物。这些研究中使用的Akt酶由重组杆状病毒表达、氨基末端、聚组氨酸标记、全长、野生型人类形式组成，并从商业上获得。这些研究中使用的PKA酶由商业上获得的大肠杆菌中表达的重组未标记的PKA人分离催化亚基组成。在环境温度下，将抑制剂、酶（9 nM Akt1或100 pM PKA）和底物（100 nM Crosstide）与5μM ATP在测定缓冲液（10 mM Tris-HCl（pH 7.2）、10 mM MgCl2、0.1%BSA（w/v）、最终DMSO 2%（v/v））中在5μL反应体积中孵育60分钟。通过向ATP溶液中加入酶+肽底物引发反应。加入IMAP结合试剂（15μL）终止反应，并将停止的反应在室温下孵育至少30分钟。

在以5000个细胞/孔的密度镀在黑色透明底96孔板上的LNCaP细胞中测量细胞存活率的抑制，随后在37°C和5%CO2下用0-10μM 28（ipatasertib）处理72小时。细胞增殖的程度是通过使用560nm的激发波长和590nm的发射波长测量制造商方案中所述的刃天青还原为再硫酸镁来确定的。使用四参数逻辑斯蒂模型生成剂量-反应曲线，并根据这些曲线拟合确定50%抑制浓度（IC50）值。[1]

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简而言之，在ipatasertib处理后，HCT116 WT或p53-/-用1%甲醛固定，并在温SDS裂解缓冲液中裂解。获得基因组DNA，并通过在冰上超声处理将其剪切至200-1000bp。样品用蛋白A-琼脂糖/鲑鱼精子DNA（50%浆液）在4°C下搅拌预清洗1小时。然后加入抗FoxO3a抗体或抗p65抗体，在4°C的摇床上孵育过夜。正常兔IgG用作阴性对照。然后加入蛋白质琼脂糖/鲑鱼精子DNA（50%浆液）珠，以沉淀抗体/蛋白质/DNA复合物。用连续洗涤缓冲液洗涤后，用洗脱缓冲液（1%SDS，0.1 M NaHCO3）从珠粒中洗脱DNA-蛋白质免疫复合物30分钟。最后，通过在65°C下与0.2 M NaCl孵育4小时，逆转蛋白质-DNA交联以释放DNA[3]。

Female nude mice bearing LNCaP, PC3, KPL-4, or MCF7 tumor xenografts
~100 mg/kg/day
Orally

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For in vivo tumor xenograft studies, female nu/nu (nude) mice were inoculated subcutaneously in the right hind flank with PC3 cells suspended in Hank’s balanced salt solution (HBSS). When tumors reached a mean volume of 150 mm3, the animals were size matched and distributed into treatment groups consisting of 10 animals/group. Tumor volume was calculated as follows: tumor size (mm3) = (longer measurement × (shorter measurement)2) × 0.5. Following data analysis, p values were determined using Dunnett’s t test with JMP statistical software, version 7.0 (SAS Institute). Mouse body weights were recorded twice weekly using an Adventura Pro AV812 scale (Ohaus Corp.). Mice were promptly euthanized when the tumor volume exceeded 2000 mm3 or if body weight loss was ≥20% of the starting weight per IACUC protocol guidelines.[1]

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For PK/PD studies, blood and tumor samples were collected at 1, 3, 8, and 24 h after a single dose of ipatasertib from PC3 tumor bearing mice. Blood samples (approximately 800 μL) were collected from each animal at the scheduled sample collection time by terminal cardiac puncture into tubes containing K2EDTA as an anticoagulant and centrifuged at 1500–2000g to isolate plasma. The concentration of ipatasertib in each plasma sample was determined by a nonvalidated LC/MS/MS assay in the DMPK Bioanalytical Department at Genentech. The assay lower limit of quantitation (LLOQ) was 0.005 μM. Tumor samples were dissociated in Tris lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100. Protein concentrations were determined using the BCA Protein Assay Kit. The human enzyme-linked immunosorbent assay (ELISA) kits were used to determine the levels of total PRAS40 and PRAS40 phosphorylated at Thr246 (p-PRAS40). The assay quantifies protein levels on the basis of measurements of absorbance. The colored product is directly proportional to the concentration of p-PRAS40 and tPRAS40 present in the specimen. The Meso Scale Discovery Multi-Spot Biomarker Detection System was used to determine the levels of total S6RP and S6RP phosphorylated at Ser235/236 (pS6RP). These assays quantify protein levels on the basis of measurements of electrochemiluminescence intensity. Levels of phosphorylated protein were normalized to total protein levels in ipatasertib-treated tumors and compared to the vehicle control.[1]

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In vivo efficacy was evaluated in multiple tumor cell line- and patient-derived xenograft models. Cells or tumor fragments were implanted subcutaneously into the flank of immunocompromised mice. Female or male nude (nu/nu) or severe combined immunodeficient mice (SCID)/beige mice were used. For the MCF7-neo/HER2 model, 17β-estradiol pellets (0.36 mg/pellet, 60-day release, no. SE-121) were implanted into the dorsal shoulder before cell inoculation. Male mice were castrated before implantation of tumor fragments. After implantation of tumor cells or fragments into mice, tumors were monitored until they reached mean tumor volumes of 180 to 350 mm3 and distributed into groups of 8 to 10 animals/group. ipatasertib/GDC-0068 was formulated in 0.5% methylcellulose/0.2% Tween-80 (MCT) and administered daily (QD), via oral (per os; PO) gavage.[2]

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HCT116 WT and PUMA−/− were harvested, and 1 × 10~6 cells in 0.2 ml of medium were implanted subcutaneously into the back of athymic nude female mice. Female 5-week-old nude mice were housed in a sterile environment with microisolator cages and allowed access to water and chow ad libitum. Mice were treated daily with ipatasertib/GDC-0068 at 40 mg/kg by oral gavage for 21 days treatment after 7 days. Calipers were used to monitor the tumor growth, volume was calculated by the formula: 0.5 × length × width2. Mice were euthanized when tumors reached ~1.0 cm3 in size. Tumors were dissected and fixed in 10% formalin and embedded in paraffin.[3]

[1]. Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors. J Med Chem. 2012 Sep 27;55(18):8110-27.

[2]. Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models. Clin Cancer Res. 2013 Apr 1;19(7):1760-72

[3]. Ipatasertib, a novel Akt inhibitor, induces transcription factor FoxO3a and NF-κB directly regulates PUMA-dependent apoptosis. Cell Death Dis. 2018 Sep 5;9(9):911.

(2S)-2-(4-chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]-1-piperazinyl]-3-(propan-2-ylamino)-1-propanone is a N-arylpiperazine.
Ipatasertib has been used in trials studying the treatment of Cancer, Neoplasms, Solid Cancers, Breast Cancer, and Gastric Cancer, among others.
Ipatasertib is an orally bioavailable inhibitor of the serine/threonine protein kinase Akt (protein kinase B) with potential antineoplastic activity. Ipatasertib binds to and inhibits the activity of Akt in a non-ATP-competitive manner, which may result in the inhibition of the PI3K/Akt signaling pathway and tumor cell proliferation and the induction of tumor cell apoptosis. Activation of the PI3K/Akt signaling pathway is frequently associated with tumorigenesis and dysregulated PI3K/Akt signaling may contribute to tumor resistance to a variety of antineoplastic agents.

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Drug Indication
Treatment of breast cancer , Treatment of prostate cancer

C24H32CLN5O2

457.9962

457.224

C, 62.94; H, 7.04; Cl, 7.74; N, 15.29; O, 6.99

1001264-89-6

Ipatasertib dihydrochloride;1396257-94-5; Ipatasertib;1001264-89-6; 1489263-16-2 (HCl); 1491138-24-9; 1491138-23-8 (besylate)

24788740

White to light yellow solid powder

1.3±0.1 g/cm3

669.4±55.0 °C at 760 mmHg

358.7±31.5 °C

0.0±2.1 mmHg at 25°C

1.603

1.71

81.59

2

6

6

32

622

3

ClC1C([H])=C([H])C(=C([H])C=1[H])[C@@]([H])(C([H])([H])N([H])C([H])(C([H])([H])[H])C([H])([H])[H])C(N1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C1C2=C([C@@]([H])(C([H])([H])[C@@]2([H])C([H])([H])[H])O[H])N=C([H])N=1)=O

GRZXWCHAXNAUHY-NSISKUIASA-N

InChI=1S/C24H32ClN5O2/c1-15(2)26-13-19(17-4-6-18(25)7-5-17)24(32)30-10-8-29(9-11-30)23-21-16(3)12-20(31)22(21)27-14-28-23/h4-7,14-16,19-20,26,31H,8-13H2,1-3H3/t16-,19-,20-/m1/s1

(2S)-2-(4-chlorophenyl)-1-[4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]piperazin-1-yl]-3-(propan-2-ylamino)propan-1-one

GDC0068; GDC 0068; GDC-0068; RG-7440; 1001264-89-6; Ipatasertib (GDC-0068); RG7440; (S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one; RG-7440; GDC0068; RG 7440; RG7440; Ipatasertib

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)

DMSO: ~92 mg/mL (~200.9 mM)
Water: <1 mg/mL
Ethanol: ~92 mg/mL (~200.9 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.54 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 (4.54 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 (4.54 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: 5% DMSO+40% PEG 300+5%Tween80+ 50%ddH2O: 92mg/ml

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配方 5 中的溶解度: 10 mg/mL (21.83 mM) in 0.5% MC 0.5% Tween-80 (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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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。