Cereblon E3 ligase
Avadomide targets cereblon (CRBN) with a Ki value of 0.5 nM [2]
Avadomide modulates CRBN-mediated ubiquitination pathway [1]

体外活性：C-122 是一种治疗 DLBCL 的新型药物，具有抗肿瘤和免疫调节活性。在 DLBCL 细胞系中，它结合 CRBN 并诱导 Aiolos 和 Ikaros 的降解或短发夹 RNA 介导的敲低，这与干扰素 (IFN) 刺激的基因转录增加相关，与 IFN-α、-β 和 -γ 的产生无关和/或分泌并导致活化 B 细胞 (ABC) 和生发中心 B 细胞 DLBCL 细胞系凋亡。 CRBN 是 CC-122 的分子靶标，CC-122 与 CRBN 结合将 Aiolos/Ikaros 招募到 CRL4CRBN，并且 E3 连接酶活性对于 Aiolos 和 Ikaros 泛素化以及 CC-122 诱导的蛋白酶体降解是必需的。 CC-122 诱导 IFN 调节蛋白，其对 IFN 通路的介导作用独立于自分泌 I 型和 II 型 IFN 分泌和信号转导。激酶测定：CC-122 是一种新型 DLBCL 药物，具有抗肿瘤和免疫调节活性。CC-122 结合 CRBN 并降解 Aiolos 和 Ikaros，从而模拟 DLBCL 中的 IFN 信号传导和细胞凋亡。体外：CC122 抑制 ABC 和 GCB DLBCL 的增殖并诱导细胞凋亡。在 DLBCL 细胞系中，CC122 诱导的 Aiolos 和 Ikaros 降解或短发夹 RNA 介导的敲低与干扰素 (IFN) 刺激基因的转录增加相关，与 IFN-α、-β 和 -γ 的产生和/或分泌无关，并且导致活化 B 细胞 (ABC) 和生发中心 B 细胞 DLBCL 凋亡。细胞测定：弥漫性大 B 细胞淋巴瘤在含有 10-20% 胎牛血清、1% 青霉素/链霉素和 1 mM 丙酮酸钠的 RPMI-1640 中培养。每孔 2×104 个细胞接种于含有 DMSO 或不同浓度 CC-122 的培养基中。将细胞在 37 摄氏度下培养 5 天，然后将氚化胸苷添加到细胞培养物中，持续最后 6 小时。随后将细胞收获到过滤板上。板干燥后，将闪烁液添加到板中并在 Top-count 读数器上读取。

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在弥漫性大B细胞淋巴瘤（DLBCL）细胞系（OCI-Ly3、SU-DHL-4、SU-DHL-6、WSU-DLCL2）中，阿瓦度胺（Avadomide）具有抗增殖活性，IC50值范围为0.3-3.1 nM；可诱导细胞G0/G1期周期阻滞和凋亡，表现为切割型caspase-3和PARP蛋白水平升高 [2]
- 阿瓦度胺（Avadomide）可上调DLBCL细胞中干扰素（IFN）刺激基因（ISGs）的表达，包括IRF7、MX1和OAS1，通过激活JAK-STAT通路模拟IFN应答；同时可增强DLBCL细胞表面MHC I类分子的表达 [2]
- 在多种晚期恶性肿瘤细胞系（包括实体瘤和血液系统恶性肿瘤）中，阿瓦度胺（Avadomide）表现出温和的抗增殖活性，实体瘤细胞系的IC50值大多高于10 nM [1]
- 阿瓦度胺（Avadomide）可诱导DLBCL细胞中CRBN底物Ikaros（IKZF1）和Aiolos（IKZF3）的降解，这一过程有助于其抗肿瘤作用的发挥 [2]

CC-122 可减少由 ABC- 和 GCB-DLBCL 细胞系建立的异种移植模型中的肿瘤生长，并刺激原代 T 细胞中 IL-2 的产生。此外，在单臂 CC-122 临床试验中，暴露于 CC-122 使每位患者的 Aiolos 和 Ikaros 表达水平降低了 25% 至 50%，证明了这 2 种蛋白质作为 CC-122 药效学标志物的效用

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在接种OCI-Ly3或SU-DHL-4 DLBCL细胞的SCID小鼠异种移植模型中，阿瓦度胺（Avadomide）以0.3、1或3 mg/kg的剂量每日口服给药21天，呈剂量依赖性抑制肿瘤生长；与溶媒对照组相比，3 mg/kg剂量组对OCI-Ly3和SU-DHL-4移植瘤的生长抑制率分别达到78%和65% [2]
- 首次人体研究中，阿瓦度胺（Avadomide）在晚期恶性肿瘤患者中显示出初步抗肿瘤活性，40例可评估患者中25%达到疾病稳定，1例非霍奇金淋巴瘤患者达到部分缓解 [1]
- 阿瓦度胺（Avadomide）治疗DLBCL异种移植小鼠后，肿瘤组织中ISGs和MHC I类分子的表达水平升高，与体外实验结果一致 [2]

采用均相时间分辨荧光（HTRF） assay检测阿瓦度胺（Avadomide）与CRBN的结合亲和力；将重组CRBN蛋白与不同浓度的阿瓦度胺（Avadomide）共同孵育，通过荧光共振能量转移（FRET）信号检测结合相互作用，基于剂量-反应曲线计算Ki值 [2]
- 采用激酶活性实验评估阿瓦度胺（Avadomide）对JAK-STAT通路激酶的影响；将纯化的JAK1、JAK2、STAT1和STAT3蛋白与阿瓦度胺（Avadomide）及其相应底物共同孵育，通过ELISA检测磷酸化底物以测定激酶活性；结果显示阿瓦度胺（Avadomide）以CRBN依赖的方式增强JAK-STAT磷酸化 [2]

CC-122抑制ABC和GCB DLBCL细胞增殖并诱导细胞凋亡[1]
为了探讨CC-122在DLBCL细胞株中的抗增殖活性，在CC-122作用5天后，对一组DLBCL细胞株进行胸苷掺入试验。4个ABC-DLBCL细胞系（TMD8、U2932、Riva和OCI-LY10）和5个GCB-DLBCL细胞系（Karpas 422、WSU-DLCL2、SUDHL-4、OCI-LY19和Pfeiffer）与0.01 ~ 10000 nM CC-122暴露5天，细胞增殖明显降低（图1A）。ABC细胞株比GCB细胞株更敏感(ABC 50%抑制浓度范围为8 nM ~ 6 μM；GCB 50%抑制浓度范围，1 μM ~ >10 μM)。

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CC-122促进CRBN-Ikaros相互作用及随后Aiolos和Ikaros的蛋白酶体降解[1]
最近研究表明，CRBN的沙利度胺结合域包含一个疏水性口袋，其中3个色氨酸残基控制着沙利度胺、来那度胺和波马度胺与谷氨酰胺的结合由于CC-122的化学结构中含有一个戊二胺环，我们探索CC-122是否与CRBN结合。如图2A所示，来自U266多发性骨髓瘤细胞提取物的CRBN与连接到固定化沙利度胺类似物（DMSO控制通道）的FG亲和珠相互作用。此外，随着游离CC-122浓度的增加，该复合物的培养导致CRBN从沙利度胺类似物固定的微球中移位，这与CC-122与沙利度胺竞争结合CRBN一致。此外，对纯化的CRBN C端片段（氨基酸321-440）的荧光猝灭研究证实了CC-122与CRBN的直接结合（补充图1）。

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DLBCL细胞系在添加胎牛血清和抗生素的RPMI 1640培养基中培养；将细胞以5×10³个/孔接种到96孔板中，用0.01-100 nM浓度的阿瓦度胺（Avadomide）处理72小时；采用基于线粒体脱氢酶活性的比色法评估细胞活力，从剂量-反应曲线计算IC50值 [2]
- 细胞周期分析中，DLBCL细胞用1 nM 阿瓦度胺（Avadomide）处理48小时，经乙醇固定、碘化丙啶染色后，通过流式细胞术测定G0/G1、S和G2/M期细胞的比例 [2]
- 采用Annexin V-FITC/PI双染色法评估凋亡；DLBCL细胞用1 nM 阿瓦度胺（Avadomide）处理72小时，经Annexin V-FITC和PI染色后，通过流式细胞术定量凋亡细胞（Annexin V阳性） [2]
- 采用蛋白质印迹法（Western blot）检测蛋白表达；细胞用RIPA缓冲液裂解，蛋白经SDS-PAGE分离后转移至PVDF膜，用针对IKZF1、IKZF3、切割型caspase-3、PARP、STAT1、磷酸化STAT1和内参蛋白GAPDH的抗体进行孵育；通过化学发光法显影免疫反应条带 [2]
- 采用实时定量PCR（qPCR）检测ISG表达；从阿瓦度胺（Avadomide）处理的DLBCL细胞中提取总RNA，逆转录为cDNA后，用IRF7、MX1、OAS1和内参基因GAPDH的特异性引物进行扩增；采用ΔΔCt法计算相对基因表达量 [2]

On the first day of these studies, the female SCID mice (CB17/Icr-Prkdcscid, Charles River) were 8 weeks old and weighed between 15.0 and 23.2 g. Each SCID mouse received a subcutaneous injection of 5x106 OCI-LY10 cells (0.2 ml cell suspension) in the right flank. In order to track growth, tumors were classified in two dimensions as their mean volume got closer to 100–150 mm3. Mice were divided into treatment groups (n=10) after tumor cell implantation, either twenty-one days (OCI-LY10) or fourteen days (WSU-DLCL2). Throughout the study, tumors were called in twice a week.In 0.5% carboxymethyl cellulose:0.25% Tween-80 in de-ionized water, apadomide (CC122) was suspended. For twenty-eight days (qd x28), avadomide (CC122) and the vehicle were each given by oral gavage (p.o.) once daily. [1]

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Female SCID mice (6-8 weeks old) were subcutaneously inoculated with 5×10⁶ OCI-Ly3 or SU-DHL-4 DLBCL cells into the right flank; when tumors reached a volume of ~100 mm³, mice were randomly divided into 4 groups (n=6 per group) [2]
- Avadomide was formulated in 0.5% methylcellulose and 0.1% Tween 80 in water; mice were administered Avadomide orally at doses of 0.3, 1, or 3 mg/kg once daily, or vehicle control, for 21 consecutive days [2]
- Tumor volume was measured twice weekly using calipers (volume = length × width² / 2), and body weight was recorded to monitor toxicity; at the end of the study, mice were euthanized, tumors were excised and weighed, and tumor tissues were collected for protein and RNA analysis [2]
- For the first-in-human study, patients with advanced malignancies received Avadomide orally once daily in escalating doses (0.15 to 2.5 mg/day) in 28-day cycles; dose escalation followed a 3+3 design to evaluate safety and tolerability [1]

Pharmacokinetics, pharmacodynamics, and biomarkers[1]
At all dose levels, the avadomide plasma concentration versus time profiles were characterized by a rapid absorption phase and similar median time to maximum concentration (Fig. 1). After attainment of maximum observed concentration in plasma, avadomide appeared to decrease in a monophasic manner at all dose levels. By visual inspection of mean plasma concentrations versus time profiles, avadomide plasma exposures increased in a dose-dependent manner across the 0.5- to 3.5-mg dose range. All 7 dose levels showed mild-to-moderate accumulation of avadomide plasma exposure after multiple doses. Supplementary Table S1 summarizes avadomide plasma pharmacokinetic parameters by day and dose level. In general, as assessed from the geometric coefficient of variation percentage, interpatient variability was noted for both avadomide area under the concentration–time curve and maximum observed concentration in plasma. The mean total recovery of avadomide in urine within 24 hours ranged from 18% to 35% across the 0.5- to 3.5-mg dose range. The mean avadomide renal clearance ranged from 0.53 to 1.31 L/h across the 0.5- to 3.5-mg dose range. The t1/2 ranged from 7.68 to 27.91 hours.

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In humans, after oral administration of Avadomide, peak plasma concentrations (Cmax) were achieved at a median time (Tmax) of 2-4 hours; Cmax and area under the plasma concentration-time curve (AUC) increased proportionally with doses from 0.15 to 2.5 mg/day [1]
- The terminal elimination half-life (t1/2) of Avadomide in humans was approximately 12-16 hours [1]
- Plasma protein binding of Avadomide was 97-99% in human plasma, as determined by equilibrium dialysis [1]
- In preclinical studies (mice), Avadomide showed good oral bioavailability (~70%) and distributed widely into tissues, with tumor tissue concentrations approximately 2-3 times higher than plasma concentrations [2]
- Avadomide was primarily metabolized by cytochrome P450 (CYP) 3A4 in human liver microsomes; no major active metabolites were identified [1]

Most patients (85%) had ≥1 TEAE that was suspected by the investigators of being related to avadomide. Across all cohorts, the most common TEAEs (≥15%) were fatigue (44%), neutropenia (29%), and diarrhea (15%). Avadomide-related grade ≥3 TEAEs occurred in 14 patients (41%). The most common grade ≥3 TEAEs were neutropenia (2 patients in the 1.0-mg cohort; 1 patient each in the 1.5-, 2.0-, 2.5-, and 3.5-mg cohorts; and 3 patients in the 3.0-mg cohort) and pneumonia (2 patients in the 3.0-mg cohort). Table 2 summarizes the TEAEs in the treated population. One death occurred within 28 days of the last dose of avadomide; 1 patient with pancreatic carcinoma in the 3.5-mg cohort died due to disease progression.

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In the first-in-human study, the most common treatment-related adverse events (TRAEs) were fatigue (45%), nausea (35%), diarrhea (30%), and decreased appetite (25%); the majority of TRAEs were grade 1-2 [1]
- Grade 3 TRAEs included neutropenia (10%), anemia (5%), and thrombocytopenia (5%) [1]
- Avadomide did not cause significant changes in liver function tests (ALT, AST, bilirubin) or renal function tests (creatinine, BUN) in patients [1]
- In preclinical mouse studies, Avadomide at doses up to 3 mg/kg/day for 21 days did not cause significant weight loss (>10%) or gross pathological changes in major organs (liver, kidney, heart, lung, spleen) [2]
- No drug-drug interaction potential was identified for Avadomide based on in vitro CYP inhibition and induction studies [1]

[1].A First-in-Human Study of Novel Cereblon Modulator Avadomide (CC-122) in Advanced Malignancies. Clin Cancer Res. 2019 Jan 1;25(1):90-98.

[2].CC-122, a pleiotropic pathway modifier, mimics an IFN response and has antitumor activity in DLBCL.Blood.Aug 6;126(6):779-89.

Avadomide is under investigation in clinical trial NCT02031419 (Novel Combinations of CC-122, CC-223, CC-292, and Rituximab in Diffuse Large B-cell Lymphoma and Follicular Lymphoma).
Avadomide is a novel, small molecule cereblon-modulating agent with potential antineoplastic, antiangiogenic and immunomodulatory activities. Upon oral administration, avadomide binds to and modulates cereblon to promote recruitment of the hematopoietic transcription factors Aiolos and Ikaros to the Cullin-4 RING E3 ubiquitin ligase complex. This binding results in the ubiquitination and rapid proteasomal degradation of Aiolos and Ikaros and the derepression of interferon (IFN)-stimulated genes, including DDX58 and IRF7, leading to apoptosis of certain tumor cells. Additionally, Aiolos degredation leads to derepression of the IL2 gene, thereby enhancing interleukin-2 production, costimulation of T-lymphocytes and IL-2-induced T-cell proliferation. Avadomide may also promote the activation of natural killer (NK) cells, potentially enhancing tumor cell killing. Aiolos and Ikaros are transcriptional repressors known to play an important role in normal B- and T-cell function.

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Drug Indication
Treatment of mature B-cell neoplasms

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Avadomide is a novel oral cereblon modulator with pleiotropic antitumor activities, including direct antiproliferation, induction of apoptosis, modulation of the immune response, and enhancement of antitumor immunity [1][2]
- The antitumor mechanism of Avadomide involves CRBN-mediated degradation of IKZF1 and IKZF3, activation of the JAK-STAT pathway, and induction of an IFN-like response, which synergistically inhibit tumor cell growth [2]
- The first-in-human study enrolled patients with advanced solid tumors, non-Hodgkin lymphoma, and multiple myeloma who had failed prior standard therapies [1]
- Avadomide showed promising activity in patients with relapsed/refractory non-Hodgkin lymphoma, supporting further development in this indication [1]

C14H14N4O3

286.29

286.107

C, 58.74; H, 4.93; N, 19.57; O, 16.77

1015474-32-4

(S)-Avadomide-d1;1620055-10-8

24967599

White to gray solid powder

1.174

107.08

2

5

1

21

530

0

O=C1C([H])(C([H])([H])C([H])([H])C(N1[H])=O)N1C(C2=C(C([H])=C([H])C([H])=C2N=C1C([H])([H])[H])N([H])[H])=O

RSNPAKAFCAAMBH-UHFFFAOYSA-N

InChI=1S/C14H14N4O3/c1-7-16-9-4-2-3-8(15)12(9)14(21)18(7)10-5-6-11(19)17-13(10)20/h2-4,10H,5-6,15H2,1H3,(H,17,19,20)

3-(5-amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione

Avadomide; CC 122; CC-122; 1015474-32-4; 3-(5-Amino-2-methyl-4-oxoquinazolin-3(4H)-yl)piperidine-2,6-dione; Avadomide [USAN]; Avadomide(CC-122); CC 122; CC122

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 : 33.33~57 mg/mL ( 116.42~199.09 mM )
Ethanol : ~1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.73 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 25.0 mg/mL澄清DMSO储备液加入到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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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));
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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；
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