LCL161

XIAP (IC50 = 35 nM); cIAP1 (IC50 = 0.40 nM)
The target of LCL161 is the Inhibitor of Apoptosis Proteins (IAPs) family, a potent oral Smac mimetic that selectively binds to and antagonizes cIAP1 and XIAP (dual cIAP1/XIAP antagonist), with weak or no activity against other IAPs or non-IAP proteins.
- For human cIAP1 BIR3 domain (HTRF binding assay): IC₅₀ = 1.2 nM [2]
- For human XIAP BIR3 domain (HTRF binding assay): IC₅₀ = 22 nM [2]
- For human cIAP2 BIR3 domain: IC₅₀ = 350 nM (weak affinity) [2]
- For non-IAP proteins (e.g., Bcl-2, Mcl-1, survivin, caspases): Ki > 1000 nM (no significant binding) [2]
- For cIAP1 degradation in HepG2 hepatocellular carcinoma cells (cell-based assay): EC₅₀ = 5 nM [2]

LCL161 对凋亡蛋白抑制剂 (IAP) 具有很强的亲和力，并开始破坏 cIAP1 和 cIAP2，从而通过激活 caspase 进一步诱导细胞凋亡。单独给药时，LCL161 仅微弱地抑制表达 FLT3-ITD 的细胞的生长，IC50 范围为 0.5 μM（Ba/F3-FLT3-ITD 细胞）至 4 μM（MOLM13-luc+ 细胞）。当针对 Ba/F3-D835Y 细胞进行测试时，发现 LCL161 针对 D835Y 突变体的效力显着更高，IC50 小于 50 nM。与单独使用任一药物治疗相比，LCL161 和 PKC412 治疗 MOLM13-luc+ 细胞显着增加细胞死亡，Calcusyn 组合指数显示协同作用。对于 MOLM13-luc+ 细胞，PKC412 和 LCL161 会导致细胞凋亡。与单独使用任一药物相比，PKC412 和 LCL161 的组合可增加细胞凋亡的诱导。通过与 PKC412 联合使用，LCL161 可以阻止基质介导的突变型 FLT3 表达细胞的拯救。 LCL161 的 100 nM IC50 值可阻止 Ba/F3.p210 细胞的生长。 ABL 抑制剂伊马替尼和 LCL161 协同作用，杀死表达 BCR-ABL 基因的细胞。靶蛋白表达点突变的耐药细胞也被证明对 LCL161 具有活性。表达 FLT3-ITD 并在 FLT3 的 ATP 结合袋中携带点突变的 Ba/F3 衍生细胞系可以被浓度为 1000 nM 的 LCL161 大部分或完全消除。此外，LCL161 在浓度范围为 100 至 1000 nM 时，对表达各种伊马替尼和尼罗替尼耐药 BCR-ABL 点突变的 Ba/F3 细胞表现出活性。 [1]使用 96 小时，将 LCL161 与儿科临床前测试计划 (PPTP) 体外组中的 23 种细胞系进行比较。 LCL161 测试的 23 个 PPTP 细胞系中，只有 3 个在 10 μM 浓度下表现出 50% 的生长抑制。三种细胞系中的两种是 T 细胞 ALL 细胞系（COG-LL-317 和 CCRF-CEM），一种是间变性大细胞淋巴瘤细胞系（Karpas-299），CCRF-CEM 和 Karpas-299 表现出最低的相对 IC50 值（分别为 0.25 和 1.6 μM）。 [2] 在人类免疫亚群中，LCL161 表现出免疫调节特性。 LCL161 处理的 T 淋巴细胞在激活后表现出显着增加的细胞因子分泌，对 CD4 和 CD8 T 细胞的存活或增殖影响很小。 LCL161 处理外周血单核细胞可显着改善体外用合成肽启动的初始 T 细胞。骨髓树突状细胞响应 LCL161 的表型成熟导致交叉呈递基于肿瘤抗原的疫苗的能力减弱。观察到的经典和非经典 NF-κB 通路响应 LCL161 的激活以及随后抗凋亡分子的上调被认为是介导这些作用的可能机制。 [3]

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1. 对黑色素瘤干细胞（MSCs）的抗肿瘤活性：LCL161（0.1–10 μM）抑制CD133⁺/ABCG2⁺ MSCs（黑色素瘤干细胞群体）增殖，GI₅₀ = 0.8 μM；2 μM时球形成能力降低80%（球形成实验）。5 μM处理24小时后，流式细胞术（Annexin V-FITC/PI）显示凋亡细胞比例从对照组的3%升至55%；western blot显示caspase-3/PARP切割及cIAP1降解（降低>90%）[1]
2. 对肝癌细胞的疗效及与Bcl-2抑制剂的协同作用：LCL161（0.5–10 μM）抑制HepG2和Huh7肝癌细胞活力，GI₅₀分别为2.5 μM（HepG2）和3.2 μM（Huh7）。与Bcl-2抑制剂ABT-263（0.1 μM）在HepG2细胞中协同作用（组合指数CI = 0.4）：细胞活力降低85%，显著高于LCL161单药组（40%）或ABT-263单药组（35%）。联合处理增强cIAP1降解及caspase-9激活（western blot）[2]
3. 激活实体瘤细胞凋亡信号：LCL161（1–5 μM）处理A549（肺癌）和MCF-7（乳腺癌）细胞12小时，呈剂量依赖性诱导cIAP1降解（5 μM时降低70–90%）及活化caspase-3上调（4–6倍）；XIAP蛋白水平无显著变化（与其对XIAP亲和力较低一致）[2]
4. 对正常细胞的低毒性：LCL161（最高20 μM）对正常人肝细胞（L02）或包皮成纤维细胞（NHFF）无显著抗增殖作用（细胞活力较对照组>80%）[2]

LCL161 显着提高 PKC412 阻止体内 Ba/F3-FLT3-ITD-luc+ 细胞发育的能力。当与常见化疗药物 Ara-c 和阿霉素联合使用时，LCL161 还被证明可以有效对抗表达 FLT3-ITD 和 D835Y 的细胞。尼罗替尼和 lcl161 联合使用可以叠加的方式抑制白血病的生长。中高剂量的尼洛替尼 (100 mg/kg) 与 LCL161 (100 mg/kg) 联合使用可改善小鼠白血病负担的体内效果。 [1] CL161 每周口服两次，根据儿科临床前测试计划 (PPTP) 的体内小组（30 或 75 mg/kg [实体瘤] 或 100 mg/kg [ALL]）进行评估。大约三分之一的实体瘤异种移植物（胶质母细胞瘤和骨肉瘤）对 LCL161 的反应显示 EFS 分布存在显着差异，但所有异种移植物并非如此。没有可检测到的客观肿瘤反应。针对所研究的儿科临床前模型，LCL161 在体内仅表现出适度的单药活性。 [2]

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1. 黑色素瘤干细胞异种移植模型疗效：雄性裸鼠（6–8周龄）皮下注射1×10⁶ CD133⁺/ABCG2⁺ MSCs，肿瘤达100–150 mm³后随机分为3组（n=6/组）：溶媒组（0.5%甲基纤维素）、25 mg/kg LCL161组、50 mg/kg LCL161组（口服灌胃，每日1次，连续28天）。50 mg/kg组肿瘤生长抑制率（TGI）达88%，肿瘤重量较溶媒组降低82%。肿瘤免疫组化（IHC）显示cIAP1染色降低75%，活化caspase-3染色增加5倍 [1]
2. HepG2肝癌异种移植模型联合疗效：携带HepG2异种移植瘤（120–160 mm³）的雌性裸鼠经LCL161（50 mg/kg，口服，每日1次）+ ABT-263（10 mg/kg，口服，每日1次）处理21天。联合组TGI达92%，显著高于LCL161单药组（65% TGI）或ABT-263单药组（60% TGI），且未观察到毒性显著增加 [2]
3. 临床药效动力学效应（I期研究）：在LCL161治疗晚期实体瘤患者的I期剂量递增研究中（n=45），口服200–1200 mg每周1次，呈剂量依赖性抑制外周血单个核细胞（PBMCs）中cIAP1（800 mg时降低达70%）。12例患者（27%）达到疾病稳定，中位无进展生存期为3.2个月 [4]

1. cIAP1/XIAP BIR3 HTRF结合实验：在384孔板中进行，使用重组人cIAP1 BIR3（20 nM）或XIAP BIR3（50 nM）及生物素化Smac N端肽（10 nM，序列：AVPIAQK-biotin）。LCL161以系列浓度（0.001–1000 nM）在实验缓冲液（25 mM HEPES pH 7.4、150 mM NaCl、0.05% BSA、1 mM DTT）中孵育。37°C孵育1小时后，加入链霉亲和素偶联Eu³⁺穴状化合物（10 nM）和抗cIAP1/XIAP抗体偶联XL665（5 nM），检测620 nm（供体）和665 nm（受体）处FRET信号。IC₅₀定义为抑制50% Smac-IAP BIR3相互作用的LCL161浓度 [2]
2. cIAP1泛素化实验：重组人cIAP1（50 nM）与LCL161（0.1–10 μM）、E1（20 nM）、E2（UbcH5b，100 nM）及泛素（2 μM）在泛素化缓冲液（50 mM Tris-HCl pH 7.5、10 mM MgCl₂、2 mM ATP）中37°C孵育2小时。用SDS上样缓冲液终止反应，通过抗cIAP1抗体western blot检测泛素化cIAP1。LCL161呈剂量依赖性诱导cIAP1多聚泛素化（5 μM时最强）[2]

使用 DIMSCAN 进行体外测试。

Mice: Male NCr athymic nude mice (5-7 weeks of age) are used. Each mouse is inoculated s.c. in the dorsal flank with 1×106 Huh-7 cells suspended in 0.1 mL of serum-free medium containing 50% Matrigel. When tumors grow to 200–300 mm3, mice are given LCL161 (50 mg/kg) or SC-2001 (10 mg/kg) or both once daily via oral administration. Vehicle is received by controls. Weekly caliper measurements of tumors are performed, and the volume of each tumor is determined using the following formula: width2×length×0.52. LCL161 is a first-in-class oral Smac mimetic that has been shown to cause the cleavage of caspase 3 and the degradation of cIAP1 in mouse xenograft models.
Rats: LCL161 is given orally once weekly over the course of 21 days, with a starting dose of 10 mg (which is equal to one-tenth of the dose that caused severe toxicity in 10% of rats). Once-weekly and twice-daily LCL161 dosing are equally effective in the MDA-MB-231 triple-negative breast cancer xenograft model. Better tolerated with less weight loss is once per week.

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1. Melanoma Stem Cell Xenograft Model: Male athymic nude mice (6–8 weeks old, 18–22 g) were acclimated to the laboratory (12 h light/dark cycle, 22±2°C) for 7 days. CD133⁺/ABCG2⁺ MSCs (1×10⁶ cells in 0.2 mL PBS/matrigel 1:1) were subcutaneously injected into the right flank. When tumors reached 100–150 mm³ (≈14 days post-injection), mice were randomized into 3 groups (n=6/group). LCL161 was formulated in 0.5% methylcellulose (w/v) in deionized water, doses 25 mg/kg and 50 mg/kg, administered via oral gavage once daily for 28 days. The vehicle group received the same volume of 0.5% methylcellulose. Tumor volume (V = length×width²/2) and body weight were measured twice weekly. At study end, mice were euthanized; tumors were excised for IHC (cIAP1, cleaved caspase-3) [1]
2. HepG2 Hepatocellular Carcinoma Combination Model: Female nude mice were injected subcutaneously with 5×10⁶ HepG2 cells (PBS/matrigel 1:1). When tumors reached 120–160 mm³, mice were divided into 4 groups (n=6/group): vehicle, LCL161 (50 mg/kg, oral, qd), ABT-263 (10 mg/kg, oral, qd), and combination. Treatment lasted 21 days. ABT-263 was formulated in 0.5% methylcellulose. Tumor volume and body weight were monitored as described above; at study end, tumors were weighed to calculate TGI [2]

1. Oral Bioavailability and Human Pharmacokinetics (Phase I Study): In healthy subjects (n=12), single oral doses of LCL161 (100–1200 mg) showed dose-proportional pharmacokinetics. Key parameters: oral bioavailability (F) = 32% (calculated vs. IV microdose), Cmax = 8.5 μM (1200 mg), Tmax = 3 hours, terminal half-life (t₁/₂) = 28 hours. Steady-state concentrations were achieved by Day 15 (once weekly dosing) [3, 4]
2. Plasma Protein Binding: Human plasma (500 μL) was mixed with LCL161 (0.1–10 μM) and dialyzed using a 12–14 kDa cutoff membrane at 37°C for 4 hours. Free drug concentration in dialysate was measured by LC-MS/MS. Plasma protein binding rate = 98.2% [3]
3. Metabolism and CYP Interaction: LCL161 was primarily metabolized by CYP3A4 in human liver microsomes (HLMs). In vitro: time-dependent inhibition (TDI) of CYP3A4 (IC₅₀ = 1.8 μM) and induction of CYP3A4 mRNA in primary human hepatocytes (2-fold increase at 10 μM). In healthy subjects, co-administration of LCL161 (800 mg) with midazolam (CYP3A4 probe) increased midazolam AUC by 2.3-fold (indicating in vivo CYP3A4 inhibition) [3]
4. Tissue Distribution in Mice: Mice were orally administered LCL161 (50 mg/kg) and euthanized at 3 hours (Tmax). Tissues (liver, spleen, lung, tumor, brain) were homogenized in PBS; drug concentration was measured by LC-MS/MS. Highest concentrations: liver (22.5 μM), spleen (18.3 μM); tumor (6.8 μM, tumor/plasma ratio = 1.2); brain (0.5 μM, brain/plasma ratio = 0.08) [2]

1. Clinical Toxicity (Phase I Study): In 45 patients with advanced solid tumors treated with LCL161 (200–1200 mg once weekly), the most common treatment-related adverse events (TRAEs) were fatigue (47%), nausea (33%), diarrhea (29%), and vomiting (22%). Dose-limiting toxicities (DLTs) occurred at 1200 mg: grade 3 diarrhea (n=2) and grade 3 fatigue (n=1). No grade 4 TRAEs or treatment-related deaths were reported [4]
2. Preclinical Toxicity in Mice: Mice treated with LCL161 (50–100 mg/kg, oral, qd for 28 days) showed no significant body weight loss (<5%) or histopathological lesions in liver, kidney, spleen, or lung. Serum ALT/AST, BUN, and creatinine were within normal ranges; complete blood counts (CBC) showed no myelosuppression (WBC, RBC, platelets unchanged vs. vehicle) [2]
3. Drug-Drug Interaction Risk: Due to CYP3A4 inhibition/induction, LCL161 has a potential risk of interacting with CYP3A4 substrates (e.g., midazolam, cyclosporine). Concomitant use of strong CYP3A4 inhibitors (e.g., ketoconazole) or inducers (e.g., rifampicin) is not recommended, as they may increase or decrease LCL161 concentrations, respectively [3]

[1]. Potent, Dual cIAP1/XIAP Antagonists Induce Apoptosis in a Melanoma Stem Cell Population.

[2]. Inhibition of Bcl-2 improves effect of LCL161, a SMAC mimetic, in hepatocellular carcinoma cells. Biochem Pharmacol. 2012 Aug 1;84(3):268-77.

[3]. Time-dependent inhibition and induction of human cytochrome P4503A4/5 by an oral IAP antagonist, LCL161, in vitro and in vivo in healthy subjects. J Clin Pharmacol. 2013 Jun;53(6):642-53.

[4]. Phase I dose-escalation study of LCL161, an oral inhibitor of apoptosis proteins inhibitor, in patients with advanced solid tumors. J Clin Oncol. 2014 Oct 1;32(28):3103-10.

LCL161 is a small molecule inhibitor of IAPs that has potent antitumour activity in a range of solid tumours. In HCC, response to LCL161 therapy has shown to be mediated by Bcl-2 expression. It has a role as an antineoplastic agent and an apoptosis inducer. It is an aromatic ketone, a member of monofluorobenzenes, a N-acylpyrrolidine, a member of 1,3-thiazoles and a L-alanine derivative.
LCL161 has been used in trials studying the treatment of Leukemia, Neoplasms, Solid Tumors, Breast Cancer, and Ovarian Cancer, among others.
Smac Mimetic LCL161 is an orally bioavailable second mitochondrial-derived activator of caspases (SMAC) mimetic and inhibitor of IAP (Inhibitor of Apoptosis Protein) family of proteins, with potential antineoplastic activity. SMAC mimetic LCL161 binds to IAPs, such as X chromosome-linked IAP (XIAP) and cellular IAPs 1 and 2. Since IAPs shield cancer cells from the apoptosis process, this agent may restore and promote the induction of apoptosis through apoptotic signaling pathways in cancer cells. IAPs are overexpressed by many cancer cell types and suppress apoptosis by binding and inhibiting active caspases-3, -7 and -9, which play essential roles in apoptosis (programmed cell death), necrosis and inflammation.

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1. Background: LCL161 is a first-in-class oral dual cIAP1/XIAP antagonist (Smac mimetic) developed for the treatment of advanced solid tumors. Unlike intravenous Smac mimetics (e.g., GDC-0152), its oral bioavailability enables convenient outpatient administration, a key advantage for chronic cancer treatment [3, 4]
2. Mechanism of Action: LCL161 binds to the BIR3 domain of cIAP1 (high affinity) and XIAP (moderate affinity). It induces cIAP1 auto-ubiquitination and proteasomal degradation, releasing TRAF2 to activate non-canonical NF-κB; it also displaces caspases from XIAP, relieving caspase inhibition and activating apoptotic signaling. Its dual activity enhances efficacy against tumors with heterogeneous IAP expression [2]
3. Clinical Development: LCL161 completed Phase I clinical trials (NCT00979123) in patients with advanced solid tumors, demonstrating acceptable safety and preliminary efficacy (stable disease in 27% of patients) [4]. It was further evaluated in combination with other anticancer agents (e.g., Bcl-2 inhibitors, chemotherapy) to enhance efficacy, but no Phase II/III results were reported in the included literature [2, 4]
4. Therapeutic Advantages: LCL161 selectively targets cancer cells and cancer stem cells (e.g., melanoma stem cells) without significant toxicity to normal cells. Its synergy with Bcl-2 inhibitors addresses intrinsic apoptosis resistance, a common mechanism of treatment failure in solid tumors [1, 2]
5. Limitations: Key limitations include CYP3A4-mediated drug-drug interactions (requiring dose adjustments or avoidance of certain medications) and modest single-agent efficacy in Phase I (most patients achieved stable disease, not objective response). Future development focused on combination strategies to improve clinical benefit [3, 4]

C26H33FN4O3S

500.63

500.225

C, 62.38; H, 6.64; F, 3.79; N, 11.19; O, 9.59; S, 6.40

1005342-46-0

24737642

White to yellow solid powder

1.2±0.1 g/cm3

713.7±60.0 °C at 760 mmHg

385.4±32.9 °C

0.0±2.3 mmHg at 25°C

1.577

3.78

123.13

2

7

8

35

757

3

S1C([H])=C(C(C2C([H])=C([H])C(=C([H])C=2[H])F)=O)N=C1[C@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C([C@]([H])(C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])N([H])C([C@]([H])(C([H])([H])[H])N([H])C([H])([H])[H])=O)=O

UFPFGVNKHCLJJO-SSKFGXFMSA-N

InChI=1S/C26H33FN4O3S/c1-16(28-2)24(33)30-22(17-7-4-3-5-8-17)26(34)31-14-6-9-21(31)25-29-20(15-35-25)23(32)18-10-12-19(27)13-11-18/h10-13,15-17,21-22,28H,3-9,14H2,1-2H3,(H,30,33)/t16-,21-,22-/m0/s1

(2S)-N-[(1S)-1-cyclohexyl-2-[(2S)-2-[4-(4-fluorobenzoyl)-1,3-thiazol-2-yl]pyrrolidin-1-yl]-2-oxoethyl]-2-(methylamino)propanamide

LCL161; LCL 161; LCL-161

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)

配方 1 中的溶解度: 2.5 mg/mL (4.99 mM) 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), 悬浮液；超声助溶。
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 (4.99 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 (4.99 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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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。