LC-MS/MS Bioanalysis of Radioligand Therapeutic Drug Candidate for Preclinical Toxicokinetic Assessment

Radioligand therapy (RLT) has gained significant momentumin recentyears in the diagnosis, treatment, and monitoring of cancers. In preclinicaldevelopment, the safety profile of RLT drug candidate(s) is investigatedat relatively low dose levels using the cold (non-radioactive, e.g., Lu-175) ligand as a surrogate of the hot (radioactive, e.g., Lu-177) one in the "ligand-linker-chelator" complex.The formulation of the test article used in preclinical safety studiescontains a mixture of free ligand (i.e., ligand-linker-chelator withoutmetal) and cold ligand (i.e., ligand-linker-chelator with non-radioactivemetal) in a similar molar ratio as seen under the manufacturing conditionsfor the RLT drug for clinical use, where only a fraction of free ligandmolecules chelate the radioactive metal to form a hot ligand. In thisvery first report of LC-MS/MS bioanalysis of RLT moleculesin support of a regulated preclinical safety assessment study, a highlyselective and sensitive LC-MS/MS bioanalytical method was developedfor the simultaneous determination of free ligand (NVS001) and coldligand (Lu-175-NVS001) in rat and dog plasma. Several unexpectedtechnical challenges in relation to LC-MS/MS of RLT moleculeswere successfully addressed. The challenges include poor assay sensitivityof the free ligand NVS001, formation of the free ligand (NVS001) withendogenous metal (e.g., potassium), Ga loss from the Ga-chelated internalstandard during sample extraction and analysis, "instability"of the analytes at low concentrations, and inconsistent IS responsein the extracted plasma samples. The methods were validated accordingto the current regulatory requirements in a dynamic range of 0.5-250ng/mL for both the free and cold ligands using a 25 & mu;L samplevolume. The validated method was successfully implemented in sampleanalysis in support of regulated safety studies, with very good resultsfrom incurred sample reanalysis. The current LC-MS/MS workflowcan be expanded to quantitative analysis of other RLTs in supportof preclinical RLT drug development.