Cocrystallization of Gefitinib Potentiate Single-Dose Oral Administration for Lung Tumor Eradication via Unbalancing the DNA Damage/Repair

Abstract

Gefitinib (GEF) is a clinical medication for the treatment of lung cancer targeting the epidermal growth factor receptor (EGFR). However, its efficacy is remarkably limited by low solubility and dissolution rates. In this study, two cocrystals of GEF with co-formers were successfully synthesized using the recrystallization method characterized via Powder X-ray Diffraction, Fourier Transform Infrared Spectroscopy, and 2D Nuclear Overhauser Effect Spectroscopy. The solubility and dissolution rates of cocrystals were found to be two times higher than those of free GEF. In vitro cytotoxicity studies revealed that the cocrystals enhanced the inhibition of cell proliferation and apoptosis in A549 and H1299 cells compared to free GEF. In mouse models, GEF@TSBO demonstrated targeted, safe, and effective antitumor activity with only one-dose administration. Mechanistically, the GEF cocrystals were shown to increase the cellular levels of damaged DNA, while potentially downregulating PARP, thereby impairing the DNA repair machinery and leading to an imbalance between DNA damage and restoration. These findings suggest that the cocrystallization of GEF could serve as a promising adjunct to significantly enhance the physicochemical and biopharmaceutical performance for lung cancer treatment, providing a facial strategy to improve GEF anticancer efficiency with high bioavailability that can be orally administrated with only one dose.

Keywords: DNA damage and repair; co-formers; cocrystal; gefitinib; oral administration.

Figure 1

The schematic illustration represents (top) the formulation of cocrystal by the noncovalent hydrogen bonding interaction between API (GEF, the EGFR-TK inhibitor) and (bottom) co-former in crystal lattice and oral drug administration, leading to the imbalance of DNA damage and repair and inhibiting of cancer cell growth by increased cell apoptosis.

Figure 2

Represents the P-XRD pattern comparison of (A) GEF@TSBO and (B) GEF@NCA with the respective material of the cocrystal.

Figure 3

Molecular docking study of GEF cocrystals: (I) Chem draw illustration highlighting noncovalent hydrogen bonding between the GEF and co-former; (II) Optimized geometric crystal structure with space filling and ball and stick; (III) Cocrystal binding mode analysis of top poses in 1xkk protein active sites showing ligand-protein binding and ligand-residue interaction of (A) GEF@TSBO and (B) GEF@NCA cocrystal.

Figure 4

In vitro cytotoxic effect and cellular uptake. (A) Cytotoxic effects of GEF, GEF@NCA, and GEF@TSBO on lung cancer cells A549 and (B) H1299 in vitro at different concentrations. (C) Quantitative uptake of GEF, GEF@NCA, and GEF@TSBO by A549 and (D) H1299 cells using flow cytometry.

Figure 5

In vitro apoptosis assessment of A549 cells (A) and H1299 cells (B) treated with GEF, GEF@NCA, and GEF@TSBO.

Figure 6

(A) Cell clonogenic assay of different treatments in A549 cells and H1299 cells. Western blot analysis and correlated quantitation (bottom bar charts) of protein PAPR and γ-H2AX in (B) A549 cells and (C) H1299 cells treated with GEF, GEF@TSBO, and GEF@NCA after incubation. GAPDH was used as a control. Statistical significances between every two groups were calculated via paired student t-test. * p < 0.05, ** p < 0.01, ns stands for no-significance.

Figure 7

The studies of anticancer effect in vivo. (A) Tumor volume of mice after one dose of treatment, the arrow indicates dose administration at day 11. (B) Tumor photographs obtained from BALB/c-nuc mice bearing A549 of different groups after treatment, red circles mean those tumors eradicated at the endpoint. (C) Tumor growth curves in each group. (D) Body weight of mice throughout treatment. (E) Weight of mouse tumors. (F) H&E staining image of tumor after treatment. Scale: 200 μm. Data are expressed as standard deviations ± averages. Statistical significances between every two groups were calculated via paired student t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns stands for no-significance.

Figure 8

H&E staining of major organs (heart, liver, spleen, lungs, and kidneys). Scale bars: 200 μm.