Experimental Study Of Aumolertinib Crossing The Blood-brain Barrier In EGFR-Mutant NSCLC Brain Transplant Tumor And Spinal Cord Metastasis Tumor Models

Lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world,and the incidence of NSCLC accounts for about 85% of the total lung cancer.EGFR-TKI are currently recognized as the drug of choice for the treatment of NSCLC with EGFR activating mutations,including exon 19 deletion（Del19）and L858 R mutations.Treatment with first-generation（eg,gefitinib or erlotinib）and second-generation（eg,afatinib or dacomitinib）EGFR-TKIs significantly improved survival in patients with advanced EGFR-mutant NSCLC.Roughly one third of NSCLC patients with EGFR-TKI-sensitive mutated（EGFRm）tumors experience disease progression through 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.Objective:To investigate whether Aumolertinib,a novel third-generation EGFR-TKI for NSCLC,can cross the BBB and deliver treatment for EGFR-mutant NSCLC brain metastases and spinal cord metastases Patients,we constructed NSCLC brain transplant tumor and spinal cord metastasis tumor models in vivo to observe the anti-tumor effects of Aumolertinib.Methods:Using molecular docking and surface plasmon resonance to detect the interaction of Aumolertinib with EGFR-T790 M mutant protein.Orthotopic NSCLC brain transplant tumor and NSCLC spinal cord metastases tumor models were constructed in Balb/c nude mice by in situ injection technology with a brain stereotaxic instrument,and the antitumor activity of Aumolertinib was studied in vivo.The drug concentration in brain tissue and plasma of mice with brain transplant tumor was detected within 24 hours after multiple consecutive administrations to verify the ability of Aumolertinib to penetrate the BBB.ABCB1-MDCK and BCRP-MDCK cells stably overexpressing ABCB1 and BCRP genes were constructed as BBB research models in vitro,and the effects of transit time and drug concentration on the apparent permeability of Aumolertinib and its active metabolite HAS-719 were investigated.Results:1.Interaction Between Aumolertinib and EGFR T790 M Mutant ProteinThe molecular docking results showed that Aumolertinib can flexibly bind to small molecule pockets on the EGFR-T790 M mutant protein with a better geometrical match.Aumolertinib can flexibly bind to the small molecule pocket on the EGFR-T790 M mutant protein with good geometric matching,and the binding energy between the two is-7.03 kcal/mol.At the same time,Aumolertinib showed a higher affinity for T790M（KD = 11.1 μM）than for Osimertinib（KD = 90.8 μM）.2.Effects of Aumolertinib on Proliferation,Invasion and Migration of NSCLC cellsAumolertinib inhibits the proliferation,invasion and migration of PC-9 and H1975 cells,and the mechanism may work by inhibiting the EMT process in tumor cells and suppressing the expression of MMP.3.Aumolertinib Shows Anti-tumor Efficacy in an EGFRm Brain Transplant Tumor ModelIn this experiment,10 and 25 mg/kg Aumolertinib and Osimertinib effectively inhibited the growth of intracranial tumors.Correspondingly,we performed a sticky dot test and a vertical test on mice to score neurological function and to evaluate motor and sensory nerve function.The mice with brain transplant tumor in the Aumolertinib 25 mg/kg group showed a significant improvement in neurological function compared with those in the control group.4.Aumolertinib Shows Anti-tumor Efficacy in an EGFRm Spinal Cord Metastases Tumor ModelCompared with the control group,the biofluorescence signal of the high-dose Aumolertinib group and the high-dose Osimertinib group was significantly reduced,showing inhibited growth and metastasis of intracranial and spinal cord tumor cells.The biofluorescence signal of the Aumolertinib and Osimertinib low-dose groups did not change significantly compared with that of the control group,and the growth of tumor cells was the same as that before administration.After treatment,the spinal cords of the high-dose Aumolertinib and control mice were dissected for H＆E and Ki-67 staining.The high-dose Aumolertinib group showed a significant inhibition in the proliferation of tumor cells.5.Aumolertinb can Cross the BBB and Enter the Brain to Distribute in the Tumor TissueIn the PC9-LUC brain transplant tumor model,Aumolertinib demonstrated excellent BBB penetration.The maximum concentration of the compound was detected in the brain at 4 h after a single dose of 25 mg/kg.According to the curve of blood concentration at each time point,the plasma concentration of Aumolertinib increased rapidly within 0.5 h after oral administration,decreased rapidly after 1 h,and reached a maximum at 4 h.Aumolertinib could still be detected in the plasma and brain tissue at 24 h.At the same time,we observed that the concentration of Aumolertinib metabolite HAS-719 in the brain tissue was considerably lower than that in the plasma.6.Aumolertinib is a Substrate of ABCB1 and BCRP TransportersThere are a series of expatriate transporter proteins on the BBB,and they can further restrict the entry of therapeutic drugs into the brain.Among all expatriate transporters expressed on the BBB,two（ABCB1 and BCRP）are mainly responsible for transporting anticancer drugs that penetrate the brain parenchyma,and back to the circulating blood.To verify whether Aumolertinib is a substrate of efflux transporters such as ABCB1 and BCRP,we measured the net efflux ratio（ER）of ABCB1 or BCRP at different concentrations of Aumolertinib and HAS-719.Based on the positive substrate criteria of the efflux ratio（ER >2）and the finding of a previous study,the relative efflux ratios of Aumolertinib or its metabolite HAS-719 between ABCB1-MDCK and normal MDCK cells and between BCRP-MDCK and MDCK cells suggest that these agents are ABCB1 and BCRP transporter substrates.7.Aumolertinib Demonstrates Potent Anti-Tumor Activity in a Patient With EGFRm NSCLCA patient with EGFR 19 del mutant lung adenocarcinoma with brain metastases was treated with Aumolertinib 110 mg po qd as a first-line treatment.After 6 weeks of treatment,the patient’s intracranial tumor partially responded.During the treatment,the patient had no other abnormalities.Conclusion:The results of this study showed that Aumolertinib significantly inhibited PC-9brain transplant tumor and spinal cord metastases tumor.Pharmacokinetic studies in mice have shown that Aumolertinib has good BBB penetration,while the metabolite HAS-719 does not readily penetrate the BBB.Early clinical evidence of activity of Aumolertinib in patients with EGFRm advanced NSCLC brain metastases patient was also reported.In conclusion,Aumolertinib readily penetrates the BBB and inhibits advanced NSCLC brain tumor and spinal cord metastases tumor.