Experimental Study of Almonertinib Crossing the Blood-Brain Barrier in EGFR-Mutant NSCLC Brain Metastasis and Spinal Cord Metastasis Models

Abstract

Roughly one third of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI)-sensitive mutated (EGFRm) tumors experience disease progression through central nervous system (CNS) metastases during treatment. Although EGFR-TKIs have been reported to be favored in some patients with EGFRm NSCLC CNS metastases, novel EGFR-TKIs with proven efficacy in CNS pathologies are clinically needed.To investigate whether almonertinib, a novel third-generation EGFR-TKI for NSCLC, can cross the blood-brain barrier (BBB) and deliver treatment for EGFR-mutant NSCLC brain metastases and spinal cord metastases, we constructed NSCLC brain metastasis and spinal cord metastasis models in vivo to observe the anti-tumor effects of almonertinib. Using ABCB1-MDCK and BCRP-MDCK monolayer cells as the in vitro study model, the effects of transport time and drug concentration on the apparent permeability coefficient of almonertinib and its active metabolite, HAS-719, were investigated. The results of this study show that almonertinib can significantly inhibit PC9 brain and spinal cord metastases. Pharmacokinetic studies in mice revealed that almonertinib has good BBB penetration ability, whereas the metabolite HAS-719 does not easily penetrate the BBB. Early clinical evidence of almonertinib activity in patients with EGFRm-advanced NSCLC and brain metastases has also been reported. In conclusion, almonertinib easily penetrates the BBB and inhibits advanced NSCLC brain and spinal cord metastases.

Keywords: EGFR-tyrosine kinase inhibitor; almonertinib; blood-brain barrier; brain and spinal cord metastases; non-small cell lung cancer; transmembrane resistance.

GRAPHICAL ABSTRACT

Third-generation EGFR-TKI almonertinib has a low affinity for blood-brain barrier efflux transporters ABCB1 and BCRP, and this can effectively improve the ability of almonertinib to penetrate the brain and successfully treat brain and spinal cord tumors.

FIGURE 1

Interaction between almonertinib and EGFR-T790 mutant protein. (A) Chemical structure of almonertinib; (B) molecular model diagram of interaction between almonertinib and EGFR T790M mutation; (C) determination of the affinity of EGFR T790M and almonertinib using the SPR method, the affinity constant was 11.1 μM; (D) the affinity of EGFR T790M and osimertinib was 90.8 μM.

FIGURE 2

Anti-tumor effect of almonertinib in PC9-LUC brain metastatic tumor model. (A) Establishment of a brain metastatic tumor model of non-small cell lung cancer; (B) Two weeks after the injection of PC9-LUC, the animals were treated with almonertinib or osimertinib once daily. Almonertinib 10 and 25 mg/kg (Qd) and osimertinib 10 and 25 mg/kg (Qd) were used to treat PC9-LUC brain metastatic tumor mice. Tumor bioluminescence (C), body weight (D), and overall survival rate (E) were observed. (F) The expression of Ki-67 in the brain tissue was detected by hematoxylin-eosin and immunohistochemical staining. (G,H) Images of brain metastases in mice after administration; (I,J) neurological score of mice with brain metastases. Scale bar, 50 μm. Data are presented as mean ± SEM (n = 5/group). **p < 0.01 vs. vehicle control.

FIGURE 3

Antitumor effect of almonertinib in PC9-LUC-injected mice with spinal cord metastases. (A) Establishment of a spinal cord metastasis model of non-small cell lung cancer with brain metastasis; (B) Two weeks after the injection of PC9-LUC, the animals were treated with almonertinib or osimertinib once daily. Almonertinib 5 and 25 mg/kg (Qd) and osimertinib 5 and 25 mg/kg (Qd) were used to treat PC9-LUC-injected mice with brain metastases and spinal cord metastases. Intracranial tumor bioluminescence (C), spinal cord tumor bioluminescence (D), body weight (E), and overall survival rate (F) were observed. The expression of Ki-67 in the spinal cord tissue was detected by hematoxylin-eosin staining and immunohistochemical staining. Scale bar, 50 μm. Data are presented as mean ± SEM (n = 5/group). **p < 0.01 vs. vehicle control. (G) The expression of Ki-67 in the spinal cord tissue was detected by hematoxylin-eosin staining and immunohistochemical staining.

FIGURE 4

Almonertinib and its metabolite HAS-719 in the brain tissue and plasma of mice with brain metastases were determined by liquid chromatography-mass spectrometry (LC-MS). (A) The second-order mass spectra of almonertinib and HAS-7A9; the typical LC-MS chromatograms of almonertinib (left) and HAS-7199 (right). (B) Blank plasma; (C) blank plasma mixed with standard almonertinib and HAS-719; (D) almonertinib 25 mg/kg was orally administered to mice. (E) Concentration-time profiles of almonertinib and HAS-719 after the oral administration of almonertinib in mice with brain metastases (n = 5).

FIGURE 5

Permeability of almonertinib and HAS-719 across MDCK, ABCB1-MDCK and BCRP-MDCK cell monolayers. (A) The production of ABCB1-MDCK and BCRP-MDCK cells. (B) The ABCB1-overexpressing and BCRP-overexpressing MDCK cells were identified by western blotting. (C) The effects of almonertinib on the viability of MDCK, ABCB1-MDCK, and BCRP-MDCK cells. These cells were treated with almonertinib at a gradient concentration (0–70 μM) for 4 h. (D) Permeability of almonertinib (1, 10, and 20 μM), (E) HAS-719 (1, 10, and 20 μM) across MDCK, ABCB1-MDCK, and BCRP-MDCK cell monolayers.

FIGURE 6

Almonertinib demonstrated potent anti-tumor activity in a patient with EGFR mutant NSCLC. Intracranial and extracranial response after treatment with 110 mg almonertinib in a 67-year old female patient with the EGFR mutation.