Abstract P4-07-08: Budesonide and loperamide do not impact the cytotoxicity of neratinib or HER2-directed monoclonal antibodies in HER2+ breast cancer cell lines
Authors: Collins DM , Gaynor N , Conlon N , Gullo G , Eustace AJ , Crown J 
Published: May 2019

Abstract
Background: Neratinib is an irreversible pan-HER tyrosine kinase inhibitor with demonstrated clinical activity in HER2+ and HER2-mutated breast cancers. The main toxicity of neratinib is diarrhea, which is common in the absence of prophylaxis. Preclinical models suggest that neratinib-associated diarrhea may involve inflammatory, bile acid malabsorption and secretory factors. The phase II CONTROL study is currently investigating the prophylactic efficacy of the opioid receptor antagonist loperamide in combination with budesonide (a corticosteroid used for inflammatory gastrointestinal conditions) or colestipol (bile acid sequestrant) on neratinib-associated diarrhea in early-stage HER2+ breast cancer (NCT02400476). This in vitro study examines the impact of loperamide and budesonide on the anti-proliferative activity of neratinib or trastuzumab and pertuzumab in HER2+ or HER2-low breast cancer cell lines.
Methods: HER2+ breast cancer cell lines SKBR3 (estrogen receptor [ER]–), BT474 (ER+), HCC1569 (ER–) and HER2-low, pertuzumab-sensitive MDA-MB-175-VII (ER+) breast cancer cells were investigated using a 5-day acid phosphatase-based proliferation assay to determine the concentrations required to inhibit growth by 50% (IC50). Fixed ratios of drugs were utilised in combination assays to generate Combination Index (CI) values (Calcusyn®) where available. Clinically relevant levels of neratinib, trastuzumab and pertuzumab were utilised in all experiments. Physiologically relevant levels of budesonide (˜4.2 nM) and loperamide (˜2.5 nM) were exceeded to provide IC50 values for these compounds.
Results: All cell lines tested had neratinib IC50 values in the nM range (Table). Trastuzumab and the trastuzumab/pertuzumab combination did not exceed 50% inhibition in the HER2+ cell lines. In the HER2+ breast cancer cell lines tested, loperamide had no impact on neratinib activity in BT474, enhanced neratinib activity in SKBR3, and the combination of loperamide and neratinib proved synergistic in HCC1569 (CI = 0.77 +/– 0.2). Budesonide produced strong synergism in combination with neratinib in SKBR3 (CI = 0.27 +/– 0.03), had no impact on neratinib activity in BT474 and improved response to neratinib in HCC1569. Loperamide and budesonide improved the activity of trastuzumab and pertuzumab in all three HER2+ models tested, and had no impact on pertuzumab activity in MDA-MB-175-VII. Interestingly, neratinib proved synergistic in combination with pertuzumab in MDA-MB-175-VII (CI = 0.75 +/– 0.5 nM).
Table.Anti-proliferative effects of agents testedBreast cancer cell lineNeratinib IC50, nMLoperamide IC50, nMBudesonide IC50, nMPertuzumab IC50, nMTrastuzumab (% inhibition, 2.5μg/ml)Trastuzumab/Pertuzumab (% inhibition, 2.5μg/ml)SKBR32.8 +/– 0.47.7 +/– 0.52.7 +/– 0.2NA26.3 +/– 1.3NABT4741.4 +/– 0.12.6 +/– 0.27 +/– 0.6NA40.1 +/– 4.3NAHCC156917.3 +/– 0.79.3 +/– 1.728.7 +/– 0.5No effectNo effect26.1 +/– 2.8MDA-MB-175-VII3 +/– 0.3&gt; 20&gt; 201.2 +/– 0.2NANANA = not acquired.
Conclusions: Our preclinical results suggest that budesonide and loperamide do not antagonise the anti-proliferative activity of neratinib or HER2-directed monoclonal antibodies in HER2+ breast cancer cell lines.
Citation Format: Collins DM, Gaynor N, Conlon N, Gullo G, Eustace AJ, Crown J. Budesonide and loperamide do not impact the cytotoxicity of neratinib or HER2-directed monoclonal antibodies in HER2+ breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-08.