Utilizing Orthogonal Mass Spectrometry Approaches to Understand Metallodrug Pharmacokinetic Parameters forin vitroLead Compound Optimization

Abstract ID 98738 Poster Board 235 Historically, pharmacokinetic evaluation of metallodrugs has been solely relied on ICP-MS. Gallium Nitrate, marketed as Ganite, has shown renal toxicity issues due to free gallium accumulating in the kidney. Due to physiochemical similarities between gallium and iron, our team has shown that gallium salophen is able to mimic heme and bind in the heme pocket of HasR from Pseudomonas aeruginosa, decreasing heme dependent signaling and virulence. While ICP-MS can give us information about to exposure of the gallium, this analysis is limited to metal core and does not give information on how long the entire drug molecule stays intact or if there are active metabolites driving the pharmacodynamic response. We sought to evaluate and rank 15 analogs of gallium based metallodrug, GaSal, through the gamut of microsomal clearance, whole blood stability, and CaCO2 permeability assays to evaluate how successfully these drugs can be characterized with traditional DMPK screening approaches. LC-MS/MS of metallodrugs is challenging due to limitations in solubility, however, part of the strategic scaffold design for the GaSal analogs is to include solubilizing groups equatorially off of the core so that there is no interference in the binding pocket. By utilizing LC-MS/MS in conjunction with ICP-MS, it is possible to perform a mass balance on the total amount of gallium in the sample with the concentration of the intact metallodrug. This further allows us to show that dosing with GaSal will not result in immediate release of gallium from the scaffold, resulting in the same toxicity issues as seen with gallium nitrate. Whole blood partition experiments were performed by incubating whole blood from six individual human selectivity lots with 500 ng/mL of metallodrug. B/P partition ratio ranged from 0.325-0.445 for the GaSal analogs that could be detected by LC-MS/MS and were in agreement with the ICP-MS analysis. Gallium Nitrite, serving as the control, gave a B/P ratio of 0.114±0.026, indicating that the distribution of GaSal is different than free gallium. Moreover, this demonstrates GaSal analogs can be stable in whole blood at physiological temperature for at least several hours. Only utilizing ICP-MS is prohibitive from understanding clearance of the intact metallodrug. To further understand if our metallodrugs are impacted by metabolic activity, microsomal incubation occurred over the course of 30 minutes and showed GaSal compounds were either undetectable after immediately starting the incubation or there was no change in concentration during the time course. Additional experiments are needed to understand if drugs undetectable by LC-MS/MS is due to gallium being released from the compound or metabolite formation in biorelevant media. Future studies will involve in vivo dosing of candidates detectable by LC-MS/MS to see if pharmacokinetic parameters, such as intrinsic clearance and blood/plasma coefficient, are translatable from in vitro performance.