Repurposing nebivolol with CPI-613 for triple-negative breast cancer: Linking in-vitro synergy to in-vivo exposure

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.