(M1130-01-01) Repurposing Neratinib for the Treatment of Triple-Negative Breast Cancer by Nanocrystals Formulation Approach

Purpose: Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, highly metastatic and difficult to treat. It is characterized by a lack of estrogen receptor, progesterone receptor, and HER2 expression and is frequently of high histologic grade. Although TNBC lacks these receptors, it has an overexpression of the epidermal growth factor receptor (EGFR), and its inhibition leads to cancer cell death (1). Neratinib is a kinase inhibitor indicated for adult patients with early-stage HER2-overexpressed breast cancer. Neratinib also irreversibly binds with EGFR showing antitumor activity; however, this indication is not approved due to its limited activity. The aim of this study is to repurpose neratinib for the treatment of TNBC via nanocrystal formulation by improving its pharmacokinetic parameters and targeted activity by enhanced permeability and retention effect.

Methods: A Liquid Chromatography -mass spectrometry (LC-MS/MS) method is developed for the quantitation neratinib in electrospray ionization (ESI) positive mode, controlled by Analyst 1.6.1 software was used for this study. The mobile phase consisted of 0.1% (v/v) formic acid in water (30%): acetonitrile (70%) at 0.25 ml/min flow rate. Mass transitions of 557.1 – 111.9, 557.1 – 419.2, 557.1 – 446.2, 557.1 – 464.2, and 557.1 – 512.0 were monitored and 557.1 – 111.9 exhibited better linearity than other mass transitions. The calibration range was 7.81- 1000 ng/mL, and the injection volume was 0.5 µl. The nanocrystals (NCs) formulations were prepared with some modifications to the previously reported method (2). Briefly, neratinib (0.1% w/v) was dispersed in deionized water containing 1% (w/v) PF127. Primary size reduction was achieved by probe sonication (QSonica sonicators, Newtown, CT) at 30% amplitude on pulse mode (10 sec on- 5 sec off) for 1 hour under an ice bath. Further size reduction was performed on 2070 bars using a high-pressure homogenizer (HPH) (EmulsiFlex®-B15, Avestin, Ottawa, Canada) at different HPH cycles. The optimized formulation was freeze-dried using 10% (w/v) glucose as a cryoprotectant. The mean particle size (z-average) and the polydispersity index (PDI) were determined by dynamic light scattering (Malvern Zeta Sizer, Malvern Instruments, UK). The characterization studies of optimized formulations include scanning Electron Microscopy (SEM), physical stability on storage at room temperature and 4°C, aqueous equilibrium solubility, and a cell viability study in MDA-MB-231 TNBC cells by MTT assay. Student's test was applied to compare the data from MTT assay.

Results: The standard curve of neratinib was linear in the concentration range of 7.81- 1000 ng/mL with r² 0.9998 and the method was specific for neratinib. The formulation was optimized, and the particle size characteristics are reported in Figure 1. The optimized formulation (20 HPH cycles) had a particle size of 174 ± 1.7 nm and a PDI value of 0.13 ± 0.01, further HPH cycles did not reduce the particle size. The addition of a cryoprotectant provided sufficient stabilization to the NCs since only a slight increase in the particle size (187.7 ± 1.9 nm) and PDI (0.17 ± 0.03) was observed. The LC-MS method was applied in the determination of aqueous equilibrium solubility, crude neratinib had 1.63 ± 0.20 µg/mL solubility, while in the NCs form it was increased by 87 folds with 143.19 ± 8.22 µg/mL. The SEM analysis revealed that the NCs were spherical to oval with an average particle size of around 150 nm. The storage stability study suggested that the formulation is stable for at least 2 weeks at 4°C (Table 2). The cytotoxicity studies provided critical information about the therapeutic potential of neratinib. The investigation of neratinib NCs on MDA-MB-231 cells after 48 h -treatment expressed a dose-dependent increase in cytotoxicity than the free drug (Figure 2). NCs formulation exhibited 3.17- a fold increase in cytotoxicity (IC50 value 1.78 ± 0.55 μM) than free drug (IC50 value 5.65 ± 3.06 μM). The vehicle control, containing only PF127 at the same concentrations as in the NCs formulation, did not exhibit any cytotoxicity.

Conclusion: The LC-MS method for the quantitation of neratinib in analytical samples is developed. Probe sonication followed by high-pressure homogenization enabled the formulation of neratinib NCs and the SEM analysis revealed the formation of NCs. The optimized formulation is more stable when stored at 4°C and it enhanced the solubility of neratinib by 87-fold, because of the reduction in particle size and increase in surface area than its crude form. The cytotoxicity study demonstrated that the NCs enhance the cytotoxicity of neratinib than its free form since the formulation had a significantly lower IC50 value (*P < 0.05). Based on the results of these preliminary studies we postulate that the developed NCs formulation has the potential to enhance the cytotoxic activity of neratinib against TNBC, further characterization and efficacy studies are being performed.

References: 1. Wahba HA, El-Hadaad HA. Current approaches in treatment of triple-negative breast cancer. Cancer Biol Med. 2015;12(2):106.
2. Agrawal S, Dwivedi M, Ahmad H, Chadchan SB, Arya A, Sikandar R, Kaushik S, Mitra K, Jha RK, Dwivedi AK. CD44 targeting hyaluronic acid coated lapatinib nanocrystals foster the efficacy against triple-negative breast cancer. Nanomedicine: Nanotechnology, Biology and Medicine. 2018 Feb 1;14(2):327-37.

Acknowledgements: The authors are grateful to the Creighton University Department of Chemistry and Biochemistry for the use of a Thermo Scientific Phenom Pharos Desktop SEM (model PW-100-60), and Erik D. Moore, Department of Pharmacy Sciences, Creighton University for technical support. Funding and other Disclosure: The authors gratefully thank the Department of Pharmacy Sciences, School of Pharmacy and Health Professions, Creighton University for funding this project. Conflict of Interest Statements: We confirm that there are no conflicts of interest associated with this work and there has been no significant financial support for this work that could have influenced its outcome.

Figure 1: Optimization of the neratinib nanocrystals formulation-effect of sonication followed by high-pressure homogenization on (A) particle size and (B) Polydispersity index.

Figure 2: Percent cell viability of MDA-MB-231 cells after incubation for 48 h with free neratinib and NCs formulation.

Table 1: Physical stability on storage of the optimized Neratinib-NCs formulation.