KRAS(G12C)

在细胞测定中，Sotorasib (AMG-510) 共价修饰 KRAS G12C 并抑制 KRAS G12C 信号，通过所有 KRAS p.G12C 突变细胞系中的 ERK1/2 (p-ERK) 磷酸化来测量[2]。 Sotorasib (AMG-510；1-10 μM；72 小时) 对 NCI-H358 和 MIA PaCa-2 中的细胞活力也有效损害，IC50 分别为 0.006 μM 和 0.009 μM。非 KRASG12C 细胞系对 Sotorasib (IC50>7.5 μM细胞活力测定[3] 细胞系：NCI-H358 和 MIA PaCa-2 细胞 浓度：1-10 μM 孵育时间：72 小时 结果：NCI-H358 和 MIA PaCa 中的细胞活力明显受损-2（IC50分别约为0.006μM和0.009μM）。

在临床前肿瘤模型中，Sotorasib (AMG-510) 快速且不可逆地结合 KRAS G12C，持久抑制丝裂原激活蛋白偶联 (MAPK) 信号放大器。Sotorasib (脸部；每天一次) 能够在 KRAS G12C 癌症模型中中诱导肿瘤消退[3]。

RAS激活突变是癌症最常见的致癌驱动突变。半胱氨酸在第12位取代甘氨酸的单个氨基酸（KRASG12C）在实体恶性肿瘤中很常见，特别是在肺腺癌（约13%）、结直肠腺癌（3%）和胰腺癌（约1%）中。最近已经证明，KRASG12C可以用共价小分子抑制剂靶向，这些抑制剂与开关II口袋（SIIP）附近的突变半胱氨酸反应，将KRAS锁定在其非活性的GDP结合状态。我们在这里描述了AMG 510的发现和体外表征，AMG 510是KRASG12C的共价抑制剂，具有强大的生化和细胞活性，以及强大的体内功效。AMG 510抑制了重组突变体KRASG12C/C118A的SOS1催化的核苷酸交换，但对KRASC118A的影响很小，KRASC118B是12位的野生型。AMG 510共价修饰KRASG12C的观察到的速率常数（kinact/Ki）通过质谱和细胞环境（kobs/[I]）进行生化测定。用AMG 510处理的细胞的半胱氨酸蛋白质组分析表明，只有KRAS的含G12C的肽被共价修饰。AMG 510抑制了所有测试的KRAS p.G12C细胞系中通过ERK磷酸化测量的KRAS信号传导，但在缺乏KRAS p.G2C突变的细胞系中没有抑制ERK的磷酸化。通过质谱法测定AMG 510对KRASG12C的细胞占有率，并与ERK磷酸化的抑制密切相关。AMG 510还选择性地损害KRAS p.G12C突变系的存活率。AMG 510与其他细胞信号通路抑制剂的联合治疗显示出对细胞存活率的协同作用的证据。用共价KRASG12C抑制剂处理KRAS p.G12C系增加了HLA的表达。为了测试KRASG12C抑制对体内免疫监视的影响，我们产生了一种适用于检测AMG 510与检查点抑制剂联合治疗的同基因肿瘤细胞系，并在体外对该系进行了表征。AMG 510目前正在一项针对携带KRAS p.G12C突变的实体瘤患者的I期研究中进行评估[1]。

细胞系：NCI-H358 和 MIA PaCa-2 细胞
浓度：1-10 μM
孵育时间：72 小时
结果：NCI-H358 和 MIA PaCa-2 中的细胞活力均严重受损（IC50）分别约为 0.006 μM 和 0.009 μM）。

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RAS家族成员的体细胞激活突变是估计21%的癌症中发现的肿瘤驱动突变。G12、G13和Q61残基的致癌KRAS突变是实体恶性肿瘤中最常见的RAS突变。KRAS p.G12C肿瘤的患病率约为肺腺癌（包括非小细胞肺癌）的13%，结直肠癌（CRC）的3%，以及许多其他实体瘤的1%-2%，这代表了未满足的医疗需求。我们开发了AMG 510，这是一种口服生物可利用的KRASG12C共价抑制剂，具有强大的生化和细胞活性，并具有强大的体内疗效。AMG 510抑制了重组突变体KRASG12C/C118A的SOS催化核苷酸交换，但对KRASC118A的影响很小，KRASC118B是12位的野生型。在细胞检测中，AMG 510共价修饰了KRASG12C，并在所有测试的KRAS p.G12C突变细胞系中通过ERK1/2（p-ERK）的磷酸化来抑制KRASG12B信号传导，但在具有各种其他KRAS突变的细胞系中没有抑制p-ERK。AMG 510还选择性地损害KRAS p.G12C突变细胞系的存活率，但不影响具有其他KRAS突变的细胞系[5]。

Female ICR-SCID mice
100 mg/kg
o.g.

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The RAS gene family encodes the small GTPase proteins NRAS, HRAS, and KRAS, which play an essential role in cellular growth and proliferation. KRAS is one of the most frequently mutated oncogenes in human cancer, with KRAS p.G12D, p.G12V, and p.G12C constituting the major mutational subtypes across lung, colon, and pancreatic cancers. Despite more than three decades of research, indirect approaches targeting KRAS mutant cancers have largely failed to show clinical benefit, and direct approaches have been stymied by the apparently ‘undruggable’ nature of KRAS. Cysteine-12 of KRASG12C has recently emerged as a unique vulnerability in KRAS-mutant cancers, and a small number of cysteine-reactive inhibitory tool molecules have been disclosed. We here report independent efforts to identify cysteine-reactive molecules capable of selectively inhibiting KRASG12C. Through iterative screening and structural biology efforts, we identified a novel Cys12-reactive inhibitor scaffold that derived its potency from occupancy of a previously unknown cryptic pocket induced by side-chain motion of the His95 residue of KRAS. Employing a scaffold-hopping approach, we leveraged knowledge of this cryptic pocket to design a series of N-aryl quinazolin-2(1H)-one-based inhibitors that demonstrated significantly enhanced potency relative to prior tool compounds. Extensive optimization of these leads led to the identification of a highly potent, selective, and well-tolerated inhibitor of KRASG12C, which was nominated for clinical development as AMG 510. In preclinical tumor models, AMG 510 rapidly and irreversibly binds to KRASG12C, providing durable suppression of the mitogen-activated protein kinase (MAPK) signaling pathway. Dosed orally (once daily) as a single agent, AMG 510 is capable of inducing tumor regression in mouse models of KRASG12C cancer. AMG 510 is, to the best of our knowledge, the first direct KRASG12C therapeutic to reach human clinical testing and is currently in a Phase I clinical trial evaluating safety, tolerability, PK, and efficacy in subjects with solid tumors bearing the KRAS p.G12C mutation (NCT03600883).

Absorption, Distribution and Excretion
A 960 mg once daily dose of sotorasib reaches a Cmax of 7.50 µg/mL, with a median Tmax of 2.0 hours, and an AUC0-24h of 65.3 h\*µg/mL.
Sotorasib is 74% eliminated in the feces and 6% eliminated in the urine. 53% of the dose recovered in the feces and 1% of the dose recovered in the urine is in the form of the unchanged parent compound.
The volume of distribution of sotorasib is 211 L.
Sotorasib has an apparent clearance at steady state of 26.2 L/h.

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Metabolism / Metabolites
Sotorasib is predominantly metabolized through conjugation or by CYP3As.

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Biological Half-Life
Sotorasib has a terminal elimination half life of 5.5 ± 1.8 hours.

Hepatotoxicity
In the prelicensure clinical trials of sotorasib in patients with solid tumors harboring KRAS G12C mutations, liver test abnormalities were frequent although usually self-limited and mild. Some degree of ALT elevations arose in 38% of sotorasib treated patients and were above 5 times the upper limit of normal (ULN) in 6% to 7%. In these trials that enrolled approximately 427 patients, sotorasib was discontinued early due to increased AST or ALT in 8% of patients. In addition, a small proportion of patients developed significant hepatotoxicity requiring sotorasib discontinuation and treatment with corticosteroids. The liver test abnormalities had a median onset of 9 weeks after initiation of therapy. While serum aminotransferase elevations were occasionally quite high (5 to 20 times upper limit of normal), there was no accompanying elevations in serum bilirubin and no patient developed clinically apparent liver injury with jaundice. The product label for sotorasib recommends monitoring for routine liver tests before, at 3 week intervals during the first 3 months of therapy, and monthly thereafter as clinically indicated.
Strikingly, the more severe elevations of serum aminotransferase levels during therapy with sotorasib occurred among patients who had recently received checkpoint inhibitor therapy (usually anti-PD-L1) in the 1 to 3 months before starting sotorasib. Furthermore, the elevations tended to respond quickly to corticosteroid therapy and sometimes did not recur when sotorasib was restarted several months later. These findings suggest that the aminotransferase elevations during sotorasib therapy are due to a delayed immune-mediated hepatotoxicity triggered by the previous checkpoint inhibitor therapy.
Likelihood score: D (possible but infrequent cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of sotorasib during breastfeeding. Because sotorasib is 89% bound to plasma proteins, the amount in milk is likely to be low. However, because of its potential toxicity in the breastfed infant, the manufacturer recommends that breastfeeding be discontinued during sotorasib therapy and for 1 week after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Sotorasib is 89% protein bound in plasma.

[1]. Cancer Res (2019) 79 (13_Supplement): 4484.

[2]. Cancer Res (2019) 79 (13_Supplement): 4455.

[3]. Cancer Commun (Lond) . 2022 Aug;42(8):716-749.

[4]. Cancer Res Commun . 2022 May;2(5):342-352.

[5]. Cancer Res (2019) 79 (13_Supplement): 3090.

[6]. Nature. 2019 Nov;575(7781):217-223.

Sotorasib is a pyridopyrimidine that is pyrido[2,3-d]pyrimidin-2(1H)-one substituted by 4-methyl-2-(propan-2-yl)pyridin-3-yl, (2S)-2-methyl-4-(prop-2-enoyl)piperazin-1-yl, fluoro and 2-fluoro-6-hydroxyphenyl groups at positions 1, 4, 6 and 7, respectively. It is approved for the treatment of patients with non-small cell lung cancer having KRAS(G12C) mutations. It has a role as an antineoplastic agent. It is a member of acrylamides, a N-acylpiperazine, a pyridopyrimidine, a member of monofluorobenzenes, a member of methylpyridines, a tertiary carboxamide, a tertiary amino compound and a member of phenols.
Sotorasib, also known as AMG-510, is an acrylamide-derived KRAS inhibitor developed by Amgen. It is indicated in the treatment of adult patients with KRAS G12C mutant non-small cell lung cancer. This mutation makes up >50% of all KRAS mutations. Mutant KRAS discovered in 1982 but was not considered a druggable target until the mid-2010s. It is the first experimental KRAS inhibitor. The drug [MRTX849] is also currently being developed and has the same target. Sotorasib was granted FDA approval on May 28, 2021, followed by the European Commission's approval on January 10, 2022.
Sotorasib is a small molecule inhibitor of the KRAS G12C mutant protein which is found in up to 13% of refractory cases of non-small cell lung cancer. Serum aminotransferase elevations are common during therapy with sotorasib, and a proportion of patients develop clinically apparent liver injury that can be severe.
Sotorasib is an orally available inhibitor of the specific KRAS mutation, p.G12C, with potential antineoplastic activity. Upon oral administration, sotorasib selectively targets, binds to and inhibits the activity of the KRAS p.G12C mutant. This may inhibit growth in KRAS p.G12C-expressing tumor cells. The KRAS p.G12C mutation is seen in some tumor cell types and plays a key role in tumor cell proliferation.

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Drug Indication
Sotorasib is indicated in the treatment of KRAS G12C-mutated locally advanced or metastatic non-small cell lung cancer (NSCLC) in adults who have received at least one prior systemic therapy.
Lumykras as monotherapy is indicated for the treatment of adults with advanced non-small cell lung cancer (NSCLC) with KRAS G12C mutation and who have progressed after at least one prior line of systemic therapy.

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Mechanism of Action
Normally GTP binds to KRAS, activating the protein and promoting effectors to the MAP kinase pathway. GTP is hydrolyzed to GDP, and KRAS is inactivated. KRAS G12C mutations impair hydrolysis of GTP, leaving it in the active form. Sotorasib binds to the cysteine residue in KRAS G12C mutations, holding the protein in its inactive form. The cysteine residue that sotorasib targets is not present in the wild type KRAS, which prevents off-target effects. This mutation is present in 13% of non small cell lung cancer, 3% of colorectal and appendix cancer, and 1-3% of solid tumors.

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Pharmacodynamics
Sotorasib is indicated in the treatment of adults with KRAS G12C mutant non small cell lung cancer. It has a moderate duration of action as it is given daily. Patients should be counselled regarding the risks of hepatotoxicity, interstitial lung disease and pneumonitis; and to avoid breastfeeding during treatment and up to 1 week after the last dose.

C30H30F2N6O3

560.5944

560.23

C, 64.28; H, 5.39; F, 6.78; N, 14.99; O, 8.56

2296729-00-3

Sotorasib racemate; 2252403-56-6; Sotorasib isomer; Sotorasib-d7; 2296729-66-1; 2387559-45-5

137278711

White to yellow solid powder

4

102Ų

1

7

5

41

1030

1

C[C@H]1CN(CCN1C2=NC(=O)N(C3=NC(=C(C=C32)F)C4=C(C=CC=C4F)O)C5=C(C=CN=C5C(C)C)C)C(=O)C=C

NXQKSXLFSAEQCZ-SFHVURJKSA-N

InChI=1S/C30H30F2N6O3/c1-6-23(40)36-12-13-37(18(5)15-36)28-19-14-21(32)26(24-20(31)8-7-9-22(24)39)34-29(19)38(30(41)35-28)27-17(4)10-11-33-25(27)16(2)3/h6-11,14,16,18,39H,1,12-13,15H2,2-5H3/t18-/m0/s1

6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(4-methyl-2-propan-2-ylpyridin-3-yl)-4-[(2S)-2-methyl-4-prop-2-enoylpiperazin-1-yl]pyrido[2,3-d]pyrimidin-2-one

AMG-510; sotorasib; AMG 510; AMG510; trade names: Lumakras; Lumykras;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50~100 mg/mL (89.2~178.4 mM)
Ethanol: ~13 mg/mL

配方 1 中的溶解度: ≥ 2.08 mg/mL (3.71 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 (3.71 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 (3.71 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%PEG300+ 5%Tween 80+ 50%ddH2O: 5.0mg/ml (8.92mM)

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配方 5 中的溶解度: 10 mg/mL (17.84 mM) in 20% HP-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。
*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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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。