PK 11195

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are significant burdens on global health. Remimazolam (REM), a novel sedative, has shown potential in its anti-inflammatory effects. However, a lack of evidence currently hinders our ability to determine if REM can improve ALI/ARDS.

We initially evaluated REM's impact on lung injury in a lipopolysaccharide (LPS)-induced ALI mouse model. Subsequently, a network pharmacology (NP) strategy and ribonucleic acid-sequencing (RNA-seq) technique were used to investigate the potential molecular mechanisms underlying REM's action against ALI. Finally, we carried out in vivo and in vitro experiments to validate our findings on these mechanisms.

REM effectively mitigated lung injury in the mouse model. NP and RNA-seq analyses revealed significant enrichment of apoptosis-related pathways. Both in vivo and in vitro experiments revealed that REM significantly reduced levels of cleaved cysteine-aspartic acid-specific protease/proteinases 7 and 3 (cleaved Caspases-7 and -3) and cytochrome c (Cyt c) while enhancing the B-cell lymphoma 2 (Bcl-2)/Bcl-2-like protein 4 (Bax) ratio and phosphorylated protein kinase B (P-AKT) levels in lung tissue, endothelial cells, and epithelial cells. Furthermore, in vitro experiments confirmed that inhibiting the phosphoinositide 3-kinase (PI3K)/AKT pathway with LY294002 weakened REM's antiapoptotic effects. In addition, pretreatment with PK11195 (the ligand of 18-kDa translocator protein [TSPO]) attenuated REM's upregulation of the PI3K/AKT pathway and antiapoptotic effect in LPS-induced endothelial cells.

This study presents novel findings elucidating the beneficial effect of REM in ALI. This effect can be attributed to REM's ability to inhibit apoptosis by activating of the PI3K/AKT pathway in endothelial and epithelial cells. Additionally, REM targeted TSPO to regulate this pathway in endothelial cells. These results suggested a potential protective role for REM in ALI/ARDS management.

New approach methodologies (NAMs) aim to accelerate the pace of chemical risk assessment while simultaneously reducing cost and dependency on animal studies. High Throughput Transcriptomics (HTTr) is an emerging NAM in the field of chemical hazard evaluation for establishing in vitro points-of-departure and providing mechanistic insight. In the current study, 1201 test chemicals were screened for bioactivity at eight concentrations using a 24-h exposure duration in the human- derived U-2 OS osteosarcoma cell line with HTTr. Assay reproducibility was assessed using three reference chemicals that were screened on every assay plate. The resulting transcriptomics data were analyzed by aggregating signal from genes into signature scores using gene set enrichment analysis, followed by concentration-response modeling of signatures scores. Signature scores were used to predict putative mechanisms of action, and to identify biological pathway altering concentrations (BPACs). BPACs were consistent across replicates for each reference chemical, with replicate BPAC standard deviations as low as 5.6 × 10^-3 μM, demonstrating the internal reproducibility of HTTr-derived potency estimates. BPACs of test chemicals showed modest agreement (R² = 0.55) with existing phenotype altering concentrations from high throughput phenotypic profiling using Cell Painting of the same chemicals in the same cell line. Altogether, this HTTr based chemical screen contributes to an accumulating pool of publicly available transcriptomic data relevant for chemical hazard evaluation and reinforces the utility of cell based molecular profiling methods in estimating chemical potency and predicting mechanism of action across a diverse set of chemicals.