PEGylation has been used extensively for improving the stability and half-life of biological molecules and has been applied recently for small-molecule drugs. (Park et al. 2019). Recently, we introduced the pegylated imidazodiazepine (PI320) and found that the addition of a pentaethylene glycol substitution did only marginally reduce the affinity of GABA(A) receptors (Zahn et al. 2022). The compound quickly relaxed substance P constricted Guinea Pig trachea and reduced airway hyperresponsiveness in several mouse asthma models. P320-induced peripheral airway relaxation in mouse precision cut lung slices (PCLS) induced by methacholine, which coincided with reduction of Ca2+ oscillations (Perez-Zoghbi et al. 2022). in the smooth muscle cells of peripheral airways in PCLS. PI320 also reduced constriction in PCLS if trigged by intracellular activation of the IP3 receptor.
We synthesized a library of PI320 analogs changing substitution and the length of the polyethylene glycol chain to identify more potent compounds (Kowalczyk et al. 2025). We found that compound 15 relaxed substance P constricted Guinea Pig trachea rings and reduced airway hyperresponsiveness induced by methacholine in mice comparable to albuterol. Compound 15 metabolized to compound 29 in the lung and both compounds increased the bronchodilation induced by isoproterenol for acetylcholine contracted mouse trachea. Thus, airway smooth muscle relaxation was achieved by activating two distinct cellular pathways for increasing intracellular calcium namely activation of β-adrenergic receptors increasing intercellular cAMP (Gi) and GABAAR activation increasing chloride flux across the cell membrane.