Transiently associating amines with therapeutic agents through the formation of ion-pairs has been established both in vitro and in vivo as an effective means to systemically direct drug delivery to the lung via the polyamine transport system (PTS). However, there remains a need to better understand the structural traits required for effective PTS uptake of drug ion-pairs. This study aimed to use a structurally related series of amine counterions to investigate how they influenced the stability of theophylline ion-pairs and their active uptake in A549 cells. Using ethylamine (mono-amine), ethylenediamine (di-amine), spermidine (tri-amine) and spermine (tetra-amine) as counterions the ion-pair affinity was shown to increase as the number of protonated amine groups in the counterion structure increased. The mono and diamines generated a single hydrogen bond and the weakest ion-pair affinities (pKFTIR: 1.32 ± 0.04 and 1.43 ± 0.02) whereas the polyamines produced two hydrogen bonds and thus the strongest ion-pair affinities (pKFTIR: 1.93 ± 0.05 and 1.96 ± 0.04). In A549 cells depleted of endogenous polyamines using α-difluoromethylornithine (DFMO), the spermine-theophylline uptake was significantly increased (p
Ion-pairing a lifesaving drug such as theophylline with a targeting moiety could have a significant impact on medical emergencies such as status asthmaticus or COVID-19 induced pneumomediastinum. However, to achieve rapid drug targeting in vivo the ion-pair must be protected against breakdown before the entry into the target tissue. This study aims to investigate if inserting theophylline, when ion-paired to the polyamine transporter substrate spermine, into a cyclodextrin (CD), to form a triplex, could direct the bronchodilator to the lungs selectively after intravenous administration. NMR demonstrates that upon the formation of the triplex spermine protruded from the CD cavity and this results in energy-dependent uptake in A549 cells (1.8-fold enhancement), which persists for more than 20 min. In vivo, the triplex produces a 2.4-fold and 2.2-fold increase in theophylline in the lungs 20 min after injection in rats and mice, respectively (p