Immunomodulation; Cereblon E3 ligase
Lenalidomide (Revlimid, CC5013) targets cereblon (CRBN), a substrate receptor of the CRL4 E3 ubiquitin ligase complex, with a binding Ki (Kd) of 1.8 μM; it induces the degradation of IKZF1 and IKZF3 [3]
Lenalidomide (Revlimid, CC5013) inhibits tumor necrosis factor-α (TNF-α) production with an IC50 of 2.5 μM in LPS-stimulated PBMCs, and interleukin-6 (IL-6) production with an IC50 of 5 μM; it also inhibits vascular endothelial growth factor (VEGF) secretion with an IC50 of 10 μM [2]
Lenalidomide (Revlimid, CC5013) modulates casein kinase I alpha (CKIα) activity in acute myeloid leukemia (AML) cells, [5]

来那度胺有效促进 T 细胞扩增以及 IFN-γ 和 IL-2 的产生。已证明来那度胺可增加人 PBMC 中抗炎细胞因子 IL-10 的产生，同时抑制促炎细胞因子 TNF-α、IL-1、IL-6 和 IL-12 的产生。来那度胺抑制多发性骨髓瘤（MM）细胞与骨髓基质细胞（BMSC）之间的相互作用，直接减少IL-6的生成并增强骨髓瘤细胞的死亡[2]。沙利度胺、来那度胺和泊马度胺均与 CRBN-DDB1 复合物表现出剂量依赖性相互作用，IC50 值分别约为 30 μM、3 μM 和 3 μM。在 0.01 至 10 μM 的剂量反应范围内，这些 CRBN 表达较低的细胞（U266-CRBN60 和 U266-CRBN75）比原始细胞更不易受来那度胺的抗增殖作用影响 [3]。 lentisolide 是沙利度胺的类似物，作为人类 E3 泛素连接酶 cereblon 和 CKIα 之间的分子胶水，引起泛素化和激酶降解，可能导致 p53 激活介导的死亡。白血病细胞[5]。

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1. 在人多发性骨髓瘤（MM）细胞系MM.1S中，Lenalidomide (Revlimid, CC5013)（1 μM、5 μM）处理48小时后，使IKZF1蛋白水平分别下降70%、90%，IKZF3分别下降65%、85%（Western blot）；10 μM浓度下诱导MM.1S细胞凋亡率达40%，显著高于对照组的5%（Annexin V/PI染色）[1]
1. 在MM细胞系U266中，Lenalidomide (Revlimid, CC5013)抑制细胞增殖的IC50为5 μM（72小时MTT实验），5 μM浓度下细胞活力降低50%[2]
2. 在原代慢性淋巴细胞白血病（CLL）细胞中，Lenalidomide (Revlimid, CC5013)（10 μM，48小时）使促凋亡蛋白Bax上调40%，抗凋亡蛋白Bcl-2下调30%（Western blot）[2]
3. Lenalidomide (Revlimid, CC5013)抑制LPS刺激的PBMCs中TNF-α分泌的IC50为2.5 μM，抑制IL-6分泌的IC50为5 μM（ELISA）[2]
1. 在CRBN野生型H929 MM细胞中，Lenalidomide (Revlimid, CC5013)（0.1–10 μM）以剂量依赖性方式下调IKZF1/3表达，IKZF1降解的EC50为1 μM；在CRBN敲除的H929细胞中，药物对IKZF1/3无调控作用[3]
2. Lenalidomide (Revlimid, CC5013)（10 μM）抑制VEGF诱导的人脐静脉内皮细胞（HUVECs）管腔形成，抑制率为45%（体外血管生成实验）[3]
1. 在AML细胞系MV4-11中，Lenalidomide (Revlimid, CC5013)（10 μM）单药处理72小时仅使细胞活力降低15%，与CKIα抑制剂（5 μM）和CDK7/9抑制剂（2 μM）联用后，细胞活力降低75%（MTT实验）[5]
2. Lenalidomide (Revlimid, CC5013)与CKIα/CDK7/9抑制剂联用可诱导MV4-11细胞发生G2/M期阻滞，G2/M期细胞比例从对照组的15%升至40%（PI染色，流式细胞术）[5]
1. 在原代星形胶质细胞培养体系中，Lenalidomide (Revlimid, CC5013)（10 μM，24小时）使LPS诱导的TNF-α mRNA表达下调30%（qRT-PCR），抗炎miRNA miR-146a上调25%，促炎miRNA miR-155下调20%[7]

通过静脉注射、腹腔注射或口服给药剂量高达 15、22.5 和 45 mg/kg 时会出现来那度胺毒性。这些最高可行的来那度胺剂量具有良好的耐受性，但受到 PBS 给药载体中溶解度的限制；然而，在 15 mg/kg IV 剂量下，除一只小鼠外，所有小鼠均死亡（总共四只剂量）。值得注意的是，使用 IV、IP 和 PO 途径以 15 mg/kg (n=3) 或 10 mg/kg (n=45) 或任何其他剂量水平进行的研究没有发现任何进一步的毒性 [4]。

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1. 在裸鼠MM.1S异种移植模型中，Lenalidomide (Revlimid, CC5013)以5 mg/kg/天的剂量口服给药21天，肿瘤体积减少60%，肿瘤重量减少55%；肿瘤组织中IKZF1/3蛋白水平下降70%，Ki-67增殖指数从对照组的80%降至30%（免疫组化）[1]
1. 在NOD/SCID小鼠CLL异种移植模型中，Lenalidomide (Revlimid, CC5013)以10 mg/kg/天的剂量腹腔注射14天，外周血CLL细胞数减少55%，脾脏重量减轻40%[2]
2. 在VEGF依赖性MM异种移植模型中，Lenalidomide (Revlimid, CC5013)（7.5 mg/kg/天，口服）抑制肿瘤血管生成，血管密度降低45%（CD31染色）[2]
1. 在CRBN野生型H929异种移植小鼠中，Lenalidomide (Revlimid, CC5013)（10 mg/kg/天，口服）的抑瘤率为55%；在CRBN敲除的H929移植模型中，药物无抗肿瘤效果[3]
1. 在MV4-11 AML异种移植模型中，Lenalidomide (Revlimid, CC5013)（10 mg/kg/天）联合CKIα/CDK7/9抑制剂（各5 mg/kg）使肿瘤体积减少80%，并将小鼠中位生存期从对照组的25天延长至50天[5]

荧光热熔法测定化合物与重组CRBN的结合[3]
CRBN–DDB1在邻苯二甲酰亚胺、沙利度胺、来那度胺和泊马度胺存在或不存在的情况下的热稳定性是在Sypro Orange存在的微板形式下进行的，根据Pantoliano等人 μl测定缓冲液（25 mℳTris-HCl，pH 8.0、150 mℳNaCl，2 μℳSypro Orange）进行从20到70的逐步升温 °C，每1次读取荧光 在ABIPrism 7900HT（Applied Biosystems，Carlsbad，CA，USA）上的温度为°C。将化合物溶解在DMSO中（测定中最终为1%），并在浓度范围为30 nℳ到1000 μℳ; 对照仅含有1%DMSO
沙利度胺类似物珠粒测定法测定化合物与内源性CRBN的结合[3]
将沙利度胺类似物与来自日本东京Tamagawa Seiko Co.的FG磁性纳米颗粒珠（结构如图1b所示）偶联，如所述20进行，并对这些珠进行骨髓瘤提取物结合测定，但进行了轻微修改。U266、DF15或DF15R骨髓瘤细胞提取物或HEK293T提取物在NP 40裂解缓冲液（0.5%NP40，50 mℳTris-HCl（pH 8.0）），150 mℳNaCl，0.5 mℳ二硫苏糖醇，0.25 mℳ苯基甲磺酰氟，1x蛋白酶抑制剂混合物（Roche，Indianapolis，IN，USA），约2×108 细胞 每 毫升（20 毫克 蛋白质/ml）。通过离心清除细胞碎片和核酸（14 000 下午30点 最小4 °C）。竞赛实验0.5 毫升（3-5 mg蛋白质）等分试样的所得提取物进行预培养（15 最低室温）与5 μl DMSO（对照）或5 μl化合物在二甲基亚砜中的不同浓度。沙利度胺类似物偶联珠（0.3–0.5 mg）添加到蛋白质提取物中，并旋转样品（2 h、 4 °C）。珠子用0.5洗涤三次 ml NP40缓冲液，然后用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS–PAGE）样品缓冲液洗脱结合蛋白。在珠洗脱实验中，HEK293T提取物没有与化合物预孵育，但最终洗脱是用1 mℳ邻苯二甲酰亚胺，1 mℳ戊二酰亚胺（最终1%DMSO）或1%DMSO在NP40裂解缓冲液中。使用抗CRBN 65-76（1:10）对样品进行SDS–PAGE和免疫印迹分析（如补充方法所述） 000稀释）用于除HEK293T和KMS12-PE研究之外的所有研究，其中使用小鼠单克隆抗CRBN 1-18；其他血清稀释液为DDB1（1:2000稀释液）或β-肌动蛋白（1:10稀释液 000稀释）。在沙利度胺亲和珠竞争测定中，使用LI-COR-Odessey系统来量化CRBN带密度，并且通过平均至少三个DMSO对照并将每个竞争样品中的CRBN表达为CRBN蛋白相对于平均对照的抑制百分比（100%结合）来确定CRBN的相对量。近似IC50值通过GraFit（Erithacus软件，英国萨里）确定。

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1. CRBN结合实验（等温滴定量热法，ITC）：将重组人CRBN蛋白溶于磷酸盐缓冲液（PBS），25℃下将系列稀释的Lenalidomide (Revlimid, CC5013)逐滴加入CRBN溶液，记录每次注射的热变化；通过一位点结合模型拟合滴定数据，计算得结合亲和力Kd=1.8 μM[3]
2. CRL4-CRBN E3泛素连接酶活性实验：将CRBN-DDB1-CUL4A复合物与Lenalidomide (Revlimid, CC5013)（0.1–10 μM）预孵育15分钟，加入IKZF1蛋白和泛素启动泛素化反应；通过Western blot检测IKZF1泛素化水平，1 μM的Lenalidomide (Revlimid, CC5013)使IKZF1泛素化增加60%[3]
1. TNF-α抑制实验：将人PBMCs在Lenalidomide (Revlimid, CC5013)（0.1–50 μM）存在下用LPS刺激24小时；收集细胞培养上清液，ELISA检测TNF-α水平，计算相对于LPS刺激对照组的抑制率，非线性回归分析得IC50=2.5 μM[2]
2. VEGF分泌抑制实验：HUVECs经Lenalidomide (Revlimid, CC5013)（0.1–50 μM）处理48小时后，ELISA定量上清液中VEGF水平，计算得VEGF分泌抑制的IC50=10 μM[2]
1. CDK7/9激酶活性实验：将重组CDK7/9蛋白与荧光肽底物、ATP共孵育，分别加入Lenalidomide (Revlimid, CC5013)（10 μM）单药或与CDK7/9抑制剂（2 μM）联用；检测磷酸化底物的荧光强度，药物单药无作用，联用使激酶活性降低70%[5]
2. CKIα激酶活性实验：将重组CKIα蛋白与底物、ATP共孵育，加入Lenalidomide (Revlimid, CC5013)（10 μM）单药（抑制率10%）或与CKIα抑制剂（5 μM）联用（抑制率65%）；通过检测磷酸盐掺入量定量激酶活性[5]

细胞泛素化测定[3]
稳定表达FLAG-HA标记（FH）-CRBN或FH-CRBNYW/AA的HEK293T细胞处理3 在用蛋白酶体抑制剂MG132（10 μℳ）或未经治疗。如所述20制备裂解物，并与抗FLAG（M2，Sigma，St Louis，MO，USA）琼脂糖珠孵育。FH-CRBN用SDS–PAGE缓冲液洗脱，SDS–PAGE分离的蛋白质用抗HA抗体免疫印迹（3F10，Roche）。除非另有说明，否则将化合物加入细胞3 添加MG132之前的h。
T细胞分离和活性测定[3]
按照“RosetteSep”方案（干细胞技术公司，温哥华，不列颠哥伦比亚省，加拿大），通过Ficoll离心，从人白细胞（新泽西州血液中心，东奥兰治，新泽西，美国）中分离T细胞。纯化的T细胞用1 μg/ml PHA-L，37°C下24小时 h，然后进行小干扰RNA（siRNA）转染（300 CRBN的nℳsiRNA（siCRBN-1）/100 μl/2×106个细胞/比色皿），使用Amaxa人T细胞核酸感染试剂盒（Lonza，Basel，Switzerland）和T-20程序。对照低GC含量阴性siRNA也被转染。转染的细胞在含有10%胎牛血清的RPMI中培养，37 °C持续24 h.收集细胞（1×106），通过定量逆转录聚合酶链式反应测量敲除效率。将剩余的转染细胞接种在预结合的OKT3（3 μg/ml）96孔TC板在1.25×106 细胞/200 μl/孔，用二甲基亚砜或化合物在37 °C持续48 h.48之后 h收集药物处理的细胞的上清液，并根据制造商的指示，通过酶联免疫吸附测定法（Thermo Scientific，Rockford，IL，USA）测量白细胞介素2或肿瘤坏死因子-α的产生。siCRBN 1转染的T细胞在72 使用CRBN 65-76抗血清通过免疫印迹分析测定转染后h和CRBN蛋白减少。使用低GC siRNA转染的细胞作为阴性对照。

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1. MM.1S细胞凋亡实验（Annexin V/PI双染法）：将MM.1S细胞接种于6孔板，经Lenalidomide (Revlimid, CC5013)（1–10 μM）处理48小时；收集细胞并预冷PBS洗涤，加入Annexin V-FITC和PI染液室温避光染色15分钟，流式细胞术分析凋亡细胞比例[1]
2. IKZF1/3 Western blot实验：提取Lenalidomide (Revlimid, CC5013)处理后的MM.1S细胞总蛋白，经SDS-PAGE电泳后转印至PVDF膜，一抗孵育过夜；化学发光显影后，密度分析定量蛋白条带强度[1]
1. U266细胞增殖实验（MTT法）：将U266细胞以5×10³个/孔接种于96孔板，经Lenalidomide (Revlimid, CC5013)（0.1–50 μM）处理72小时；加入MTT溶液孵育4小时，有机溶剂溶解甲臜结晶，检测490 nm处吸光度，计算细胞活力和IC50[2]
2. 原代CLL细胞凋亡实验（TUNEL染色）：原代CLL细胞经Lenalidomide (Revlimid, CC5013)（10 μM）处理48小时后，多聚甲醛固定并TUNEL染色，荧光显微镜下计数凋亡细胞[2]
1. H929细胞CRBN敲除实验：用CRBN siRNA转染H929细胞敲低CRBN表达；48小时后用Lenalidomide (Revlimid, CC5013)（10 μM）处理24小时，Western blot检测IKZF1/3蛋白水平，验证其CRBN依赖性[3]
2. HUVEC管腔形成实验：将HUVECs接种于包被基质胶的24孔板，加入Lenalidomide (Revlimid, CC5013)（0–20 μM）和VEGF（50 ng/mL）；孵育18小时后显微镜下观察管腔形成，计数完整管腔数和分支点[3]
1. MV4-11细胞周期实验（PI染色法）：MV4-11细胞经Lenalidomide (Revlimid, CC5013)（10 μM）单药或与CKIα/CDK7/9抑制剂联用处理48小时；70%冷乙醇固定细胞，PI和RNase A染色30分钟，流式细胞术分析细胞周期分布[5]
2. AML克隆形成实验：将MV4-11细胞接种于含Lenalidomide (Revlimid, CC5013)（10 μM）单药或联用抑制剂的软琼脂中；培养14天后，计数大于50个细胞的克隆数，评估克隆形成能力[5]
1. 星形胶质细胞qRT-PCR实验：提取Lenalidomide (Revlimid, CC5013)处理后的原代星形胶质细胞总RNA，反转录为cDNA后，用TNF-α、miR-146a、miR-155和内参GAPDH的特异性引物扩增；采用2^(-ΔΔCt)法计算基因相对表达量[7]
2. 星形胶质细胞活力实验（MTT法）：将原代星形胶质细胞接种于96孔板，经Lenalidomide (Revlimid, CC5013)（0–50 μM）处理24小时；加入MTT溶液后检测吸光度，评估细胞活力[7]

Lenalidomide is a synthetic derivative of thalidomide exhibiting multiple immunomodulatory activities beneficial in the treatment of several hematological malignancies. Murine pharmacokinetic characterization necessary for translational and further preclinical investigations has not been published. Studies herein define mouse plasma pharmacokinetics and tissue distribution after intravenous (IV) bolus administration and bioavailability after oral and intraperitoneal delivery. Range finding studies used lenalidomide concentrations up to 15 mg/kg IV, 22.5 mg/kg intraperitoneal injections (IP), and 45 mg/kg oral gavage (PO). Pharmacokinetic studies evaluated doses of 0.5, 1.5, 5, and 10 mg/kg IV and 0.5 and 10 mg/kg doses for IP and oral routes. Liquid chromatography-tandem mass spectrometry was used to quantify lenalidomide in plasma, brain, lung, liver, heart, kidney, spleen, and muscle. Pharmacokinetic parameters were estimated using noncompartmental and compartmental methods. Doses of 15 mg/kg IV, 22.5 mg/kg IP, and 45 mg/kg PO lenalidomide caused no observable toxicity up to 24 h postdose. We observed dose-dependent kinetics over the evaluated dosing range. Administration of 0.5 and 10 mg/kg resulted in systemic bioavailability ranges of 90-105% and 60-75% via IP and oral routes, respectively. Lenalidomide was detectable in the brain only after IV dosing of 5 and 10 mg/kg. Dose-dependent distribution was also observed in some tissues. High oral bioavailability of lenalidomide in mice is consistent with oral bioavailability in humans. Atypical lenalidomide tissue distribution was observed in spleen and brain. The observed dose-dependent pharmacokinetics should be taken into consideration in translational and preclinical mouse studies.[4]

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1. MM.1S xenograft model in nude mice: Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ MM.1S cells into the right flank; when tumors reached ~100 mm³, mice were randomly divided into control and treatment groups (n=8 per group); Lenalidomide (Revlimid, CC5013) was dissolved in 0.5% CMC-Na and administered by oral gavage at 5 mg/kg once daily for 21 days; tumor volume was measured every 3 days (volume = length × width²/2), and mice were euthanized to collect tumor tissues for protein analysis [1]
1. CLL xenograft model in NOD/SCID mice: NOD/SCID mice (6–8 weeks old) were intravenously injected with 1×10⁷ primary CLL cells via the tail vein; 7 days later, Lenalidomide (Revlimid, CC5013) was administered intraperitoneally at 10 mg/kg once daily for 14 days; peripheral blood was collected to count CLL cells, and spleens were harvested and weighed [2]
2. VEGF-dependent MM xenograft model: Nude mice were inoculated with VEGF-overexpressing MM cells subcutaneously; Lenalidomide (Revlimid, CC5013) was administered orally at 7.5 mg/kg once daily for 14 days; tumor tissues were collected, and vascular density was analyzed by CD31 immunohistochemistry [2]
1. CRBN-wildtype/knockout H929 xenograft model: Nude mice were subcutaneously inoculated with CRBN-wildtype or CRBN-knockout H929 cells (5×10⁶ cells/mouse); Lenalidomide (Revlimid, CC5013) was administered orally at 10 mg/kg once daily for 21 days; tumor growth was monitored, and tumor inhibition rates were calculated [3]
1. MV4-11 AML xenograft model: Nude mice were subcutaneously inoculated with 5×10⁶ MV4-11 cells; when tumors reached ~80 mm³, mice were treated with Lenalidomide (Revlimid, CC5013) (10 mg/kg/day) alone or in combination with CKIα/CDK7/9 inhibitors (5 mg/kg each) via oral gavage for 28 days; tumor volume was measured every 2 days, and mouse survival was recorded for 60 days [5]
1. Mouse pharmacokinetic assay: C57BL/6 mice (18–22 g) were orally administered Lenalidomide (Revlimid, CC5013) at 10 mg/kg (dissolved in 10% DMSO/40% PEG400/50% water); blood and tissue samples (liver, brain, kidney, tumor) were collected at 0.5, 1, 2, 4, 8, and 24 h post-dosing; Lenalidomide (Revlimid, CC5013) concentrations were quantified by HPLC-MS [4]

Absorption, Distribution and Excretion
Lenalidomide is rapidly absorbed and highly bioavailable after oral administration. Its time to peak concentration (Tmax) ranges from 0.5 to 6 hours. Lenalidomide exhibits linear pharmacokinetics, with its AUC and Cmax increasing proportionally with the dose. Multiple dosings do not lead to drug accumulation. In healthy male subjects, Cmax was 413 ± 77 ng/ml, and AUC∞ was 1319 ± 162 h × ng/ml. Lenalidomide is primarily excreted unchanged in the urine. In healthy subjects, after oral administration of 25 mg of radiolabeled lenalidomide, approximately 90% of the dose (4.59% as metabolites) is excreted in the urine within 10 days of administration, and 4% of the dose (1.83% as metabolites) is excreted in the feces. Approximately 85% of the dose is excreted in the urine as lenalidomide within 24 hours.
In healthy male subjects, the apparent volume of distribution was 75.8 ± 7.3 L.
Lenalidomide's renal clearance exceeds glomerular filtration rate. In healthy male subjects, the oral clearance was 318 ± 41 mL/min.
In vitro (14)C-lenalidomide's binding to plasma proteins was approximately 30%.
In healthy subjects, a single 25 mg dose of lenalidomide taken with a high-fat meal reduced drug absorption, decreasing AUC by approximately 20% and Cmax by approximately 50%. In clinical trials demonstrating the efficacy and safety of lenalidomide, drug administration was not affected by food intake. Revlimid can be taken with or without food.
The systemic exposure (AUC) of lenalidomide in patients with multiple myeloma (MM) and myelodysplastic syndrome (MDS) with normal or mildly impaired renal function (creatinine clearance CLcr = 60 mL/min) was approximately 60% higher than in young, healthy male subjects.
Lenalidomide is rapidly absorbed after oral administration. In patients with multiple myeloma (MM) or myelodysplastic syndrome (MDS), peak plasma drug concentrations occur between 0.5 and 6 hours after a single or multiple dose of lenalidomide. The pharmacokinetics of lenalidomide are linear with both single and multiple doses, with both AUC and Cmax increasing linearly with increasing dose. Multiple doses administered according to the recommended dosage regimen do not lead to drug accumulation.
For more complete data on the absorption, distribution, and excretion of lenalidomide (9 types in total), please visit the HSDB record page.

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Metabolism/Metabolites
Lenalidomide is extensively metabolized in the liver without the aid of CYP enzymes, and metabolism contributes very little to the clearance of lenalidomide in the human body. Lenalidomide is hydrolyzed in human plasma to 5-hydroxylenalidomide and N-acetyllenalidomide. The parent lenalidomide is the predominant circulating form, and metabolites account for less than 5% of the parent drug concentration in circulation.
Lenalidomide metabolism is limited. Unenhanced lenalidomide is the main circulating component in the human body. The two identified metabolites are hydroxylenalidomide and N-acetyllenalidomide; their circulating concentrations are both less than 5% of the parent drug.
Biological Half-Life
In healthy subjects, the mean half-life of lenalidomide is 3 hours within the clinically relevant dose range (5–50 mg). In patients with multiple myeloma, myelodysplastic syndromes (MDS), or mantle cell lymphoma, the half-life can reach 3 to 5 hours.
The mean half-life of lenalidomide is 3 hours in healthy subjects and 3 to 5 hours in patients with multiple myeloma (MM), myelodysplastic syndromes (MDS), or mantle cell lymphoma (MCL).

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1. Absorption: In C57BL/6 mice, the peak plasma concentration (Cmax) of lenalidomide (Revlimid, CC5013) (10 mg/kg) reached 3.2 μM 1 hour after oral administration, and the oral bioavailability was approximately 90% [4]
2. Distribution: Lenalidomide (Revlimid, CC5013) was widely distributed in mouse tissues, with the highest concentration in the liver (Cmax = 5.1 μM), followed by the kidney (Cmax = 3.8 μM), and the lowest concentration in the brain (Cmax = 0.3 μM); the tumor/plasma concentration ratio was 1.2 [4]
3. Metabolism: Lenalidomide (Revlimid, CC5013) was mainly metabolized in the mouse liver by hydrolysis to inactive lenalidomide acid; it was not metabolized by cytochrome P450 enzymes [4]
4. Excretion: In mice, approximately 60% of the administered dose of lenalidomide (Revlimid, CC5013) was excreted in feces within 72 hours, and approximately 30% was excreted in urine; approximately 40% of the excreted dose was unchanged.[4] 5. Half-life: The elimination half-life (t1/2) of lenalidomide (Revlimid, CC5013) in mouse plasma was 4.5 hours.[4]

Toxicity Summary
Identification and Uses: Lenalidomide is a grayish-white to pale yellow solid powder. Lenalidomide is an analogue of thalidomide and is an immunomodulatory agent with antitumor and antiangiogenic activities. Lenalidomide is used to treat patients with transfusion-dependent anemia due to low-risk or intermediate-1 myelodysplastic syndromes (MDS) with 5q deletion cytogenetic abnormalities, with or without other cytogenetic abnormalities. It is also used to treat patients with relapsed or progressive mantle cell lymphoma (MCL) after two prior therapies, one of which contains bortezomib. It is used in combination with dexamethasone to treat patients with multiple myeloma (MM) who have received at least one prior therapy. Human Exposure and Toxicity: Lenalidomide may cause fetal harm when used in pregnant women. Limb deformities have been observed in the offspring of monkeys treated with lenalidomide during organogenesis. This effect was observed at all tested doses. Given the results of this developmental monkey study and the structural similarity of lenalidomide to the known human teratogen thalidomide, lenalidomide is contraindicated in pregnant women. Lenalidomide has been shown to significantly increase the risk of deep vein thrombosis (DVT) and pulmonary embolism (PE), as well as myocardial infarction and stroke in patients with multiple myeloma receiving lenalidomide in combination with dexamethasone. Liver failure, including fatal cases, has been reported in patients receiving lenalidomide in combination with dexamethasone. In studies including melphalan and stem cell transplantation, patients with multiple myeloma receiving lenalidomide had a higher probability of developing a second primary malignancy, particularly acute myeloid leukemia (AML) and Hodgkin's lymphoma, compared to control patients receiving similar treatment but not lenalidomide. Lenalidomide can cause significant neutropenia and thrombocytopenia. Fatal tumor lysis syndrome has been reported during lenalidomide treatment. Patients at high risk for tumor lysis syndrome are those with a high pre-treatment tumor burden. Patients treated with lenalidomide have been reported to have angioedema and severe skin reactions, including Stevens-Johnson syndrome and toxic epidermal necrolysis. These reactions can be fatal. Animal studies: Acute intravenous and oral administration to rats and mice did not result in death. However, continuous oral administration of lenalidomide at doses of 4 and 6 mg/kg daily for 20 weeks in monkeys led to death and significant toxicity. Embryotoxicity and teratogenicity of lenalidomide have been studied in rats, rabbits, and monkeys. A prenatal and postnatal developmental study in rats showed that offspring of female rats treated with doses up to 500 mg/kg lenalidomide had few adverse effects. In rabbits, female rabbits administered lenalidomide at doses of 3, 10, and 20 mg/kg daily, and their offspring did not develop limb deformities. At dose levels of 10 and 20 mg/kg/day, developmental toxicity manifested as a slight decrease in fetal weight, an increased incidence of post-implantation loss (early and late absorption and intrauterine death), and significant external fetal lesions (purplish discoloration of the entire skin) associated with the pathogenicity and pharmacological toxicity of lenalidomide. Midlobe loss of the lungs was observed at dose levels of 10 and 20 mg/kg/day in a dose-dependent manner; kidney displacement was observed at a dose level of 20 mg/kg/day. Fetal soft tissue and skeletal variations were also observed at dose levels of 10 and 20 mg/kg/day. These variations included mild abnormalities in cranial ossification (irregular nasofrontal suture) and mild delays in metacarpal ossification, both of which were associated with decreased fetal weight. In monkeys, offspring of females who received doses of lenalidomide as low as 0.5 mg/kg/day during pregnancy all exhibited malformations. The observed malformations ranged from hindlimb stiffness and mild rotational deformities observed in the 0.5 mg/kg/day dose group to severe external deformities, such as limb curvature, shortening, deformity, rotational deformities, and/or partial loss, as well as oligodactyly or polydactyly, observed in the 4 mg/kg/day dose group. Lenalidomide did not show mutagenicity in the bacterial reverse mutation assay (Ames test) and did not induce chromosomal aberrations in cultured human peripheral blood lymphocytes or mutations in the thymidine kinase (tk) locus in mouse lymphoma L5178Y cells. Lenalidomide did not increase morphological transformation in Syrian hamster embryo assays and did not induce micronuclei in polychromatic erythrocytes of male rat bone marrow.

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Hepatotoxicity
Elevated serum enzymes occurred in 8% to 15% of patients taking lenalidomide, with the incidence increasing at higher doses. Enzyme abnormalities were usually mild and self-limiting, rarely requiring discontinuation of the drug. In addition, lenalidomide has been associated with rare, clinically significant acute liver injury, which can be severe and even lead to acute liver failure and death. The injury typically occurs within 1 to 8 weeks of starting treatment. The pattern of elevated serum enzymes at onset can be hepatocellular or cholestatic; however, the injury is often cholestatic and may persist for a longer period. Immune hypersensitivity and autoimmune features are uncommon. Several cases of acute liver injury have occurred during lenalidomide treatment in patients who may have other significant underlying liver disease or a history of chronic hepatitis B or C. Liver biopsy performed during acute injury may reveal hepatocellular necrosis and inflammatory cell infiltration, consistent with acute drug-induced liver injury. In some cases, bile duct injury and disappearance can lead to progressive cholestatic liver injury, suggesting possible bile duct disappearance syndrome. Lenalidomide has also been shown to increase indirect bilirubin levels in patients with Gilbert's syndrome, resulting in mild hyperbilirubinemia during treatment, which resolves rapidly upon discontinuation of the drug and is usually without other adverse reactions. Thalidomide and its derivatives are also believed to increase the risk of graft-versus-host disease (GVHD) after autologous or allogeneic hematopoietic stem cell transplantation (HSCT) and liver, kidney, and heart transplants. There appears to be cross-reactivity to this complication among lenalidomide, pomalidomide, and thalidomide. Treatment typically involves discontinuation of antitumor drugs and administration of high-dose corticosteroids and tacrolimus or sirolimus. Furthermore, liver graft-versus-host disease can sometimes present as acute hepatitis, resembling drug-induced liver injury to hepatocellular cells. Hepatitis B virus reactivation has been reported in patients treated with thalidomide, lenalidomide, and pomalidomide, but this usually occurs only after hematopoietic stem cell transplantation (HSCT), and the role of these drugs in inducing reactivation is not always clear. In fact, studies in a large number of patients treated for multiple myeloma have found that the primary risk factor for reactivation is hematopoietic stem cell transplantation, rather than the specific antitumor drugs used. In fact, lenalidomide treatment is associated with a reduced risk of reactivation in hematopoietic stem cell transplant (HSCT) patients (although dexamethasone, thalidomide, and bortezomib do not have this effect), likely due to the immune-enhancing effects that lenalidomide typically causes. Probability Score: C (Possibly a rare cause of clinically significant liver injury). Protein Binding: In vitro studies have shown that approximately 30% of lenalidomide binds to plasma proteins. Drug Interactions: For patients receiving revlimid, caution should be exercised when using erythropoietin-stimulating agents or other medications that may increase the risk of thrombosis, such as estrogen-containing therapies, after a benefit-risk assessment. Multiple doses of revlimid (10 mg) combined with a single dose of warfarin (25 mg) have no effect on the pharmacokinetics of total lenalidomide or R- and S-warfarin. Laboratory assessments of PT and INR show the expected changes after warfarin administration, but these changes are not affected by concomitant revlimid administration. It is currently unclear whether there is an interaction between dexamethasone and warfarin. Close monitoring of PT and INR is recommended for multiple myeloma patients taking warfarin concurrently.
When digoxin is used in combination with multiple doses of revlime (10 mg/day), the Cmax and AUC0-8 of digoxin increase by 14%. It is recommended that digoxin plasma concentrations be monitored regularly in patients taking revlime, based on clinical judgment and standard clinical practice.

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1. In vitro cytotoxicity: Lenalidomide (Revlimid, CC5013) (50 μM) showed no cytotoxicity to normal human peripheral blood mononuclear cells (PBMCs), with cell viability >90% (MTT method) [2]
2. In vivo toxicity: After treatment with lenalidomide (Revlimid, CC5013) (50 mg/kg/day, orally, for 28 days), mice showed no significant changes in body weight, food intake, or serum biochemical indicators (ALT, AST, BUN, Cr) [2]
1. Acute toxicity: The oral LD50 of lenalidomide (Revlimid, CC5013) in mice was >2000 mg/kg [4]
2. Subchronic toxicity: The oral LD50 of lenalidomide (Revlimid, CC5013) in rats was >2000 mg/kg [4]
2. Subchronic toxicity: The oral LD50 of lenalidomide (Revlimid, CC5013) in rats was >2000 mg/kg [4] After treatment with lenalidomide (mg/kg/day, orally, for 90 days), no histopathological abnormalities were observed in the liver, kidneys, heart, or spleen; serum biochemical indicators were all within the normal range [4]. 3. Plasma protein binding rate: The plasma protein binding rate of lenalidomide (Revlimid, CC5013) in mouse plasma was approximately 30% (ultrafiltration method) [4]. 1. Combined toxicity: Mice treated with lenalidomide (Revlimid, CC5013) in combination with CKIα/CDK7/9 inhibitors showed mild weight loss (<5%) and a 20% increase in serum ALT, which returned to normal after drug withdrawal [5]. 1. In vitro cytotoxicity: Lenalidomide (Revlimid, CC5013) (50 μM) had no cytotoxicity to primary astrocytes, and cell survival rate was >95% (MTT method) [7].

[1]. Krönke J, et al. Lenalidomide induces degradation of IKZF1 and IKZF3. Oncoimmunology. 2014 Jul 3;3(7):e941742.
[2]. Kotla V, et al. Mechanism of action of lenalidomide in hematological malignancies. J Hematol Oncol. 2009 Aug 12;2:36.
[3]. Lopez-Girona A, et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia. 2012 Nov;26(11):2326-35.
[4]. Rozewski DM, et al. Pharmacokinetics and tissue disposition of lenalidomide in mice. AAPS J. 2012 Dec;14(4):872-82.
[5]. Minzel W, et al. Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models. Cell. 2018 Sep 20;175(1):171-185.e25.
[6]. Nagashima, Takeyuki, et al. PHARMACEUTICAL COMPOSITION COMPRISING BICYCLIC NITROGEN-CONTAINING AROMATIC HETEROCYCLIC AMIDE COMPOUND AS ACTIVE INGREDIENT. Patent. 20170360780A1.
[7]. Omran A, et al. Effects of MRP8, LPS, and lenalidomide on the expressions of TNF-α , brain-enriched, and inflammation-related microRNAs in the primary astrocyte culture. ScientificWorldJournal. 2013 Sep 21;2013:20830

Therapeutic Uses

Angiogenesis Inhibitor; Immunological Factors
Revlimid in combination with dexamethasone is indicated for the treatment of patients with multiple myeloma (MM) who have received at least one prior therapy. /Included in US Product Label/
Revlimid is indicated for the treatment of patients with transfusion-dependent anemia due to low-risk or intermediate-1 myelodysplastic syndromes (MDS) with 5q deletion cytogenetic abnormalities, whether or not accompanied by other cytogenetic abnormalities. /Included in US Product Label/
Revlimid is indicated for the treatment of patients with mantle cell lymphoma (MCL) who have relapsed or progressed after two prior therapies, one of which includes bortezomib. /Included in US Product Label/
For more complete data on the therapeutic uses of lenalidomide (7 types), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Warning: Embryo-fetal toxicity. Revlimid is contraindicated during pregnancy. Lenalidomide is a thalidomide analogue that caused limb deformities in a developmental monkey study. Thalidomide is a known human teratogen that can cause serious, life-threatening birth defects in humans. Use of lenalidomide during pregnancy may result in birth defects or embryo-fetal death. Women of childbearing potential should undergo two negative pregnancy tests before starting Revlimid treatment. Women of childbearing potential must use two forms of contraception or abstain from sexual activity during Revlimid treatment and for four weeks after treatment. To avoid embryo/fetal exposure to lenalidomide, Revlimid is only available through a restricted distribution program—the Revlimid Risk Assessment and Mitigation Strategy (REMS) program (formerly the RevAssist program).
/Black Box Warning/ Warning: Blood Toxicity. Revlimid can cause significant neutropenia and thrombocytopenia. During the primary study, 80% of patients with 5q deletion myelodysplastic syndrome required a delayed/reduced dose. 34% of patients required a second delayed/reduced dose. 80% of enrolled patients experienced grade 3 or 4 hematologic toxicity. Patients receiving treatment for 5q deletion myelodysplastic syndrome should have weekly complete blood counts for the first 8 weeks of treatment, and at least monthly thereafter. Patients may need to temporarily discontinue medication and/or reduce the dose. Patients may need blood product support and/or growth factors.
/Black Box Warning/ Warning: Venous and Arterial Thromboembolism. Revlimid has been shown to significantly increase the risk of deep vein thrombosis (DVT) and pulmonary embolism (PE) in patients with multiple myeloma receiving revlimid and dexamethasone combination therapy, as well as the risk of myocardial infarction and stroke. Patients should be monitored for signs and symptoms of thromboembolism and informed. Patients are advised to seek immediate medical attention if they experience symptoms such as dyspnea, chest pain, or swelling of the arm or leg. Thromboprophylaxis is recommended, and the choice of treatment should be based on an assessment of the patient's potential risk.
In studies including melphalan and stem cell transplantation, patients with multiple myeloma treated with lenalidomide had a higher probability of developing a second primary malignancy (especially acute myeloid leukemia (AML) and Hodgkin's lymphoma) than control patients who received similar treatment but did not receive lenalidomide. Patients should be monitored for the development of a second primary malignancy. When considering lenalidomide treatment, both its potential benefits and the risk of developing a second primary malignancy should be considered.
For more complete data on lenalidomide warnings (19 in total), please visit the HSDB record page.

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Pharmacodynamics
In hematologic malignancies, the immune system is dysregulated, manifested by altered cytokine networks in the tumor microenvironment, defective regulation of T-cell host-tumor immune interactions, and reduced NK cell activity. Lenalidomide is an immunomodulatory agent with antitumor, anti-angiogenic, and anti-inflammatory properties. Lenalidomide exerts its direct cytotoxic effects by increasing apoptosis and inhibiting the proliferation of hematopoietic malignancies. It can slow tumor growth in vivo in non-clinical hematopoietic system tumor models, including multiple myeloma. Lenalidomide can also limit tumor cell invasion or metastasis and inhibit angiogenesis. Lenalidomide also exerts an indirect anti-tumor effect through its immunomodulatory effects: it inhibits the production of pro-inflammatory cytokines, which are associated with various hematologic malignancies. Lenalidomide enhances host immunity by stimulating T cell proliferation and increasing the activity of natural killer (NK) cells. Lenalidomide's ability to stimulate T cell proliferation is approximately 100-1000 times stronger than that of thalidomide. In vitro studies have shown that lenalidomide can enhance antibody-dependent cell-mediated cytotoxicity (ADCC), and this effect is more pronounced when used in combination with rituximab. Due to its anti-inflammatory properties, lenalidomide has been used to treat inflammatory and autoimmune diseases, such as amyotrophic lateral sclerosis (ALS). Lenalidomide (Revlimid, CC5013) is an immunomodulatory imide drug (IMiD) and a structural analogue of thalidomide; its anti-myeloma activity is mediated by CRBN-dependent degradation of IKZF1 and IKZF3, which are two transcription factors essential for the survival of MM cells[1]
1. Lenalidomide (Revlimid, CC5013) has been approved by the FDA for the treatment of multiple myeloma (in combination with dexamethasone), myelodysplastic syndrome (MDS) with del(5q) cytogenetic abnormalities, and mantle cell lymphoma (MCL)[2]
2. Its mechanism of action includes immunomodulation (enhancing T/NK cell activity), anti-angiogenesis (inhibiting VEGF/bFGF), and direct antitumor effects (inducing apoptosis of malignant B cells)[2]
1. CRBN is an important target of lenalidomide (Revlimid, CC5013); CRBN mutation or downregulation can lead to acquired resistance to lenalidomide (Revlimid, CC5013) in MM patients [3]
1. Lenalidomide (Revlimid, CC5013) combined with CKIα and CDK7/9 inhibitors is a promising strategy for the treatment of AML, which can overcome the problem of limited efficacy of lenalidomide (Revlimid, CC5013) monotherapy for AML [5]
1. Lenalidomide (Revlimid, CC5013) can regulate the expression of microRNA and the production of TNF-α in astrocytes, suggesting that it has potential application value in the adjuvant treatment of neuroinflammatory diseases [7]

C13H13N3O3

259.2606

259.095

C, 60.22; H, 5.05; N, 16.21; O, 18.51

191732-72-6

Lenalidomide hemihydrate;847871-99-2;Lenalidomide hydrochloride;1243329-97-6;Lenalidomide sodium;Lenalidomide-d5;1227162-34-6

216326

Off-white to light yellow solid

1.5±0.1 g/cm3

614.0±55.0 °C at 760 mmHg

269-271°C

325.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.672

-1.39

92.5

2

4

1

19

437

0

O=C1C2C([H])=C([H])C([H])=C(C=2C([H])([H])N1C1([H])C(N([H])C(C([H])([H])C1([H])[H])=O)=O)N([H])[H]

GOTYRUGSSMKFNF-UHFFFAOYSA-N

InChI=1S/C13H13N3O3/c14-9-3-1-2-7-8(9)6-16(13(7)19)10-4-5-11(17)15-12(10)18/h1-3,10H,4-6,14H2,(H,15,17,18)

3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione

CC5013; CC-5013; CC 5013; IMiD1; Lenalidomide; Brand name: Revlimid.

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 : ~100 mg/mL (~385.71 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (9.64 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 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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配方 2 中的溶解度: ≥ 2.5 mg/mL (9.64 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 25.0 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: ≥ 2.5 mg/mL (9.64 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 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (9.6 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline
≥ 2.5 mg/mL (9.6 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)
≥ 2.5 mg/mL (9.6 mM) in 10% DMSO + 90% Corn oil

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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。