Application of preclinical absorption, distribution, metabolism, elimination in vitro techniques for the...

Multidrug-resistant pathogens are an emerging threat to public health. Metal-based drugs have shown antimicrobial properties. As a result, metallotherapeutics have an untapped potential to combat antibiotic-resistant infections. Characterization of additional physiochemical attributes is needed to progress metallodrugs in the clinical pipeline. In order to fully characterize a modest library of compounds based on novel therapeutic gallium salophen (GaSal), target binding affinity (dissociation constant, K_d), lipophilicity (octanol-water partition coefficient, logP), protein binding (fraction of protein bound compound, K_b), Caco-2 permeability, microsomal stability, and blood/plasma partitioning (blood-plasma partition coefficient, K_RBC/PL) experiments were performed to help inform lead optimization. Analogs with 2 identical solubilizing groups had a lower theoretical LogP than analogs with only one solubilizing group; however, the experimental data showed the inverse to be true. All of the analogs tested were highly plasma protein bound (>98%). Caco-2 permeability showed that the apparent permeability from apical to basolateral had limited permeability. Apparent permeability calculated from the basolateral to apical side, however, resulted in a high efflux ratio for all compounds. The addition of inhibition cocktails for P-glycoprotein and organic cation transporter 1 did not vastly impact the efflux ratio, indicating that further investigation is needed to determine the transporter involved in drug distribution. Microsomal stability results indicated that GaSal analogs do not undergo cytochrome P450 metabolism, likely are metabolized by enzymes found in the S9 liver fraction (S9), and can potentially be cytochrome P450 inhibitors. This study also provides insight into optimizing liquid chromatography and mass spectrometry parameters because metallodrugs show unique ionization and physiochemical properties. Finally, to our knowledge, this article is the first to detail Ga³⁺ blood/plasma partitioning because this is not a common metal found in diet or environmental exposure. During partitioning experiments, analogs with polar acidic functional groups portioned heavily into the red blood cells compared to other analogs. Herein, we determine in vitro physiochemical properties in order to characterize absorption, distribution, metabolism, elimination parameters useful for subsequent generations of GaSal analogs as metallotherapeutics. SIGNIFICANCE STATEMENT: Few studies detail drug metabolism and pharmacokinetic (DMPK) library screening for metal-based therapeutics, and there is a large literature gap in metallodrug preclinical development. Establishing the relationship between inductively coupled plasma mass spectrometry and liquid chromatography tandem mass spectrometry analysis is critical during preclinical development to ensure in vitro pharmacokinetic parameters are accurately reported. The information from this work is important for optimizing gallium salophen analogs as potential metallotherapeutics against multidrug-resistant pathogens.