PO.ET02.10 · 实验与分子治疗
将奈必洛尔与CPI-613重新定位用于三阴性乳腺癌：将体外协同作用与体内暴露相关联
Repurposing nebivolol with CPI-613 for triple-negative breast cancer: Linking in-vitro synergy to in-vivo exposure
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
三阴性乳腺癌（TNBC）是一种侵袭性亚型，缺乏ER、PR和HER2表达，使化疗成为默认的标准治疗方案，但复发率高且存在迟发性毒性。药物重定位为利用具有已知安全性的药物开发更安全、基于机制的疗法提供了一条快速、经济高效的途径。奈必洛尔（NEB）是一种FDA批准的第三代β₁受体阻滞剂，在临床前模型中显示出抗癌活性，被认为部分通过抑制氧化磷酸化和血管生成发挥作用，尽管其确切机制仍未完全阐明；而CPI-613（CPI）是一种硫辛酸类似物，靶向丙酮酸和α-酮戊二酸脱氢酶，破坏线粒体代谢。我们假设双重代谢靶向将抑制TNBC生长。以固定比例用NEB（0.5-16 µM）和CPI（5-250 µM）处理小鼠4T1细胞。MTT实验（72 h）和Chou-Talalay分析确定4-8 µM NEB + 5-50 µM CPI为最佳协同比例（抑制率>90%，CI < 0.2）。体内实验中，通过乳腺脂肪垫注射在BALB/c小鼠中建立原位4T1-Luc肿瘤，并用溶媒（N=5）、NEB（10 mg/kg QD）（N=5）、CPI（25 mg/kg BID）（N=5）或NEB + CPI（NC）（N=6）治疗，每周5天，持续3周。在第28天，所有治疗组相比溶媒组均显著降低肿瘤生长（p < 0.01-0.001）。平均肿瘤体积（mm³ ± SD）为：溶媒组1484.9 ± 492.4；NEB组595.8 ± 134.6；CPI组634.7 ± 116.9；NEB + CPI组702.5 ± 352.0。肿瘤重量呈现相同模式（p < 0.0001），所有治疗组均显著低于溶媒组，但NEB、CPI和联合组之间无差异。LC-MS/MS确证了NEB和CPI的瘤内浓度（NEB 42.2 ± 20.4 ng/g；CPI 22.7 ± 6.6 ng/g；联合组NEB 39.3 ± 39.8，CPI 16.8 ± 8.5 ng/g）。各组的全身PK相似，但NC中的CPI相比单用CPI显示出分布容积降低（p = 0.0368）。给药后23 h采集的肿瘤用于计算摩尔浓度（µM = [ng/g × ρ_肿瘤 (g/mL)] / MW），其中密度（ρ = 重量/体积）由卡尺测量的体积推导。以µM计的肿瘤浓度为：NEB 0.093 ± 0.051，CPI 0.043 ± 0.011；联合组NEB 0.067 ± 0.080，CPI 0.031 ± 0.022——低于体外协同范围且未达到最佳比例，这可能解释了联合用药相比单药缺乏获益的原因。PK/PD分析显示中等程度、无统计学意义的趋势（r = 0.26-0.70）。NEB和CPI抑制了TNBC生长，但联合用药并未优于单药。亚协同、比例不匹配的肿瘤暴露可能是这一结果的原因；即将开展的肿瘤PK研究将确定在给药间隔内是否达到协同比例，以指导剂量优化。
查看英文原文 English abstract
Triple-negative breast cancer (TNBC) is an aggressive subtype lacking ER, PR, and HER2 expressions, making chemotherapy the default standard of care with high relapse and late-onset toxicities. Drug repurposing offers a rapid, cost-effective path to safer, mechanism-based therapies using agents with known safety. Nebivolol (NEB), an FDA-approved third-generation beta₁-blocker, shows anti-cancer activity in preclinical models and is believed to act in part by inhibiting oxidative phosphorylation and angiogenesis, although its precise mechanism remains incompletely defined, while CPI-613 (CPI), a lipoate analog that targets pyruvate and alpha-ketoglutarate dehydrogenases, disrupts mitochondrial metabolism. We hypothesized that dual metabolic targeting would suppress TNBC growth. Murine 4T1 cells were treated with NEB (0.5-16 µM) and CPI (5-250 µM) in fixed ratios. MTT assays (72 h) and Chou-Talalay analysis identified 4-8 µM NEB + 5-50 µM CPI as the optimal synergistic ratio (>90% inhibition, CI < 0.2) . In vivo , orthotopic 4T1-Luc tumors were established by mammary fat-pad injection in BALB/c mice and treated with vehicle (N=5), NEB (10 mg/kg QD) (N=5), CPI (25 mg/kg BID) (N=5), or NEB + CPI (NC) (N=6), 5 days/week for 3 weeks. All treatments significantly reduced tumor growth versus vehicle on day 28 (p < 0.01-0.001). Mean tumor volumes (mm³ ± SD) were: Vehicle 1484.9 ± 492.4; NEB 595.8 ± 134.6; CPI 634.7 ± 116.9; NEB + CPI 702.5 ± 352.0. Tumor weights showed the same pattern (p < 0.0001), with all treated groups significantly lower than vehicle but no difference among NEB, CPI, and combination. LC-MS/MS confirmed intratumoral concentrations of NEB and CPI (NEB 42.2 ± 20.4 ng/g; CPI 22.7 ± 6.6 ng/g; combo NEB 39.3 ± 39.8, CPI 16.8 ± 8.5 ng/g). Systemic PK was similar across groups, though CPI in NC showed reduced volume of distribution vs single CPI (p = 0.0368). Tumors collected 23 h post-dose were used to calculate molar concentrations (µM = [ng/g × ρ_tumor (g/mL)] / MW), where density (ρ = weight/volume) was derived from caliper volume. Tumor concentrations in µM were NEB 0.093 ± 0.051, CPI 0.043 ± 0.011; combo NEB 0.067 ± 0.080, CPI 0.031 ± 0.022-below the in-vitro synergistic range and not reaching the optimal ratio, which may explain the lack of combination benefit over single agents. PK/PD analyses showed moderate, non-significant trends (r = 0.26-0.70). NEB and CPI inhibited TNBC growth, but the combination did not outperform single agents. Sub-synergistic, ratio-mismatched tumor exposure may underlie this outcome; upcoming tumor PK studies will determine whether the synergistic ratio is reached within the dosing interval to guide dose optimization.
利益披露 Disclosure
M. Skillman, None..
A. Li, None..
J. Park, None..
A. Mohammad-Gholizadeh, None..
F. Dagher, None..
M. Trivedi, None..
B. Kaiparettu, None..
D. S. Chow, None.