The underlying mechanisms of lorlatinib penetration across the blood‐brain barrier and the distribution characteristics of lorlatinib in the brain

Abstract

Objective: To clarify the distribution of lorlatinib in the brain and elucidate the molecular mechanisms of lorlatinib penetration across the blood-brain barrier (BBB). Methods: Cytological experiments were performed to investigate the growth inhibitory effect of lorlatinib on different cells (endothelial cells HUVEC, HMEC-1, and HCMEC/D3) and to investigate the protective effect of lorlatinib on neuronal cells after SH-SY5Y hypoxia/reoxygenation injury. Furthermore, rat brain tissue was sequenced, and the differentially expressed genes (secreted phosphoprotein 1 (SPP1), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β), Claudin, ZO-1 and P-gp) in several different drug treatment groups were verified by Real-Time PCR. Lorlatinib brain distribution was predicted by physiologically based pharmacokinetics (PBPK). Results: Lorlatinib and crizotinib both had inhibitory effects on endothelial cells, however lorlatinib inhibited the growth of HCMEC/D3 more efficaciously than crizotinib. In the SH-SY5Y hypoxia model, lorlatinib had a greater protective effect on nerve cell damage caused by hypoxia and reoxygenation than crizotinib. The expression of SPP1, VEGF, TGF-β, and Claudin in brain tissue was significantly downregulated after lorlatinib administration, and the expression level of early growth transcription factor 1 (Egr-1) was significantly increased. The PBPK model successfully described lorlatinib concentrations in blood and brain tissue in the mouse model and gave a brain tissue partition coefficient of 0.7. Conclusion: Lorlatinib can increase the permeability of the blood-brain barrier whereby we suggest its underlying working mechanism is related to downregulating SPP1, inhibiting VEGF, TGF-β, and Claudin subsequently reducing the number of tight junctions between BBB cells. Lorlatinib plays a protective role on injured nerve cells and does not change the amount of P-gp expression in brain tissue, which may be important for its ability to be efficacious across the BBB with a low incidence of resistance.

A Comprehensive Analysis of Metabolomics and Transcriptomics Reveals Novel Biomarkers and Mechanistic Insights on Lorlatinib Crosses the Blood-Brain Barrier

“…P-glycoprotein, an ATP-binding cassette (ABC) transporter, which is a major pump that transports promiscuous xenobiotics out of cells, associates with multidrug resistance (MDR) due to overexpression ( Cannon et al, 2012 ; Mollazadeh et al, 2018 ; Ren and Gray, 2019 ). In a previous study, through RNA sequencing, we confirmed that lorlatinib did not exhibit a significant regulatory effect on the p-glycoprotein via mRNA transcription ( Chen et al, 2020 ). However, sphingolipid, signaling via S1P and acting via S1PR1, appears to induce a fast and reversible regulatory effect resulting in low p-glycoprotein pump activity level and an improvement in the delivery of small-molecule compounds to the brain ( Cannon et al, 2012 ).…”

“…In the preliminary experiment, we sequenced the RNA of the control group and the lorlatinib group mice ( Chen et al, 2020 ). By using DEGseq algorithm, |log2Fold Change| ≥1 and Adjusted p value ≤0.001 as the screening criteria, 126 differentially expressed genes were obtained.…”

“…To further clarify the explicit effect and underlying mechanism of lorlatinib, especially regarding its intracranial activity, metabolomic profiles were investigated and combined with previous transcriptomics research ( Chen et al, 2020 ), rendering a panoramic view of the interaction between lorlatinib and the body. In this research project, 9 noteworthy differential metabolites contributing to the altered metabolic profiles of experimental groups were identified, and they were enriched in 4 major metabolic pathways, namely, Sphingolipid metabolism, Glycerophospholipid metabolism, Thiamine metabolism and Synthesis and degradation of ketone bodies.…”

“…In our previous study, SD rats were randomly divided into groups ( Chen et al, 2020 ). After cardiac perfusion with saline, the brain tissue of rats in the control group and in the lorlatinib administration group were taken for sequencing, which was completed at the BGI-Shenzhen.…”

“…Lorlatinib has an active role in the treatment and prevention of CNS metastasis in ALK-positive NSCLC patients ( Bauer et al, 2020 ). In addition to the possible mechanism of minimizing p-glycoprotein-mediated efflux of relatively large (>400 Da) hydrophobic drugs ( Schinkel, 1999 ; Seelig, 2020 ), our previous research showed that downregulating SPP1 and inhibiting VEGF, TGF-β may also be potential mechanisms for lorlatinib’s characteristics of effective brain penetration ( Chen et al, 2020 ).…”