Purpose: RadioLigand Theragnostics (RLTs) are a type of precision nuclear medicine designed to administer radiation to cancer cells in a restrict unnecessary exposure to surrounding normal healthy tissues. The targeted approach of radioligands has both diagnostic and therapeutic functions which are attained by selective biodistribution of radioactive atoms either to reveal tumor locations or to selectively damage cancer cells. Two major components of RLTs are: a ligand, essential to look for target cells with specific surface receptors and a radioisotope, which delivers radiation to either diagnose (image) or kill cancerous cells. Additional components needed for RLTs include a chelator, designed to hold different radioisotopes, and a linker which is used to attach the chelator to the ligand. RLTs are designed in a manner that if not delivered to target cancer cells, they will rapidly be eliminated in the urine, thus limiting unnecessary radiation exposure to the patients. This makes it important to understand the in vivo stability and clearance of the RLT early in drug discovery (since both can influence its biodistribution), to guide design strategies and selection of development candidates. Discovery and optimization phase in vivo experiments can be conducted with non-radioactive metal containing RLTs, which are, in general, easier to perform experiments than those conducted with their radioactive counterparts. Therefore, we applied ICP-MS in an innovative manner to detect non-radioactive metal (Galium-69/71 and Lutetium-175) containing RLTs to evaluate these critical properties. Methods: ICP-MS was applied in two different configurations; 1) Flow injection (FIA-ICP-MS), for quantitative measurement of total metal in urine, and 2) LC-ICP-MS, where LC was integrated with the ICP-MS, to enable the separation of the dosed compound from its metabolites on a reverse phase LC column. To achieve the desired level of sensitivity and consistent performance in the LC-ICP-MS configuration, we optimized several critical interface parameters such as nebulizer flow, spray chamber temperature, percent oxygen flow, etc. A surrogate RLT with Ga-69/71 was used as an internal standard to prevent co-elution and interference during the analysis of the dosed Lu-175 containing RLTs and their metabolites. The coupling of reverse phase LC with the ICP-MS cause varying instrument response with the constantly changing ICP plasma conditions.Therefore, the method included the post-column infusion of Thulium-169, to allow for correction of the effect of the gradient elution used in the chromatographic run. The technology was applied to assess the in-vivo stability and urinary excretion of three promising RLT compounds to guide selection and prioritization of a candidate with minimal ADME liabilities. In-life experiments were designed as single dose excretion studies with Lu-175 containing non-radioactive RLT compounds, where compounds were administered discretely by intravenous injection to rats and dogs housed in metabolic cages. Blood, urine, and feces samples were collected up to 24 h after the dose was administered. Conventional LC-MS/MS analysis was also performed for the quantitation of dosed compound to measure the total percent dose excreted unchanged in the urine and feces.In addition, when appropriate, LC-HRMS was also used to determine the identity of metabolites that were identified by LC-ICP-MS. Results: The LC-MS/MS analysis of dosed RLTs revealed 30-40% and 30-70% total recovery (urine,feces) of unchanged drug in rat and dog, respectively, with minimal excretion in the feces, indicative of either potential metabolism or accumulation in the body. We then applied the FIA-ICP-MS approach to measure total Lu-175 excretion in the urine, as LC-MS/MS data indicated urine as the major route of excretion. The results showed 70-100% total urinary recovery of Lu-175 in both rat and dog, within 24 hours post-dose, signifying that the RLTs underwent metabolism and did not have significant accumulation in the body. To investigate further, the LC-ICP-MS metabolite profiling was performed in different matrices. The blood profiles for all three compounds showed predominantly one major Lu-175 peak corresponding to the dosed compound and a few additional minor peaks. However, the urine profiles showed 1-2 major and a few minor metabolite peaks in addition to the dosed RLT. Taken together, the FIA-ICP-MS and LC-ICP-MS data revealed that 1) the major portion of Lu dose was excreted in the urine, and 2) dosed RLTs were indeed metabolized in the body with minimal circulating metabolites in the blood and majorly in the urine, indicative of potential metabolism occurring during excretion via kidney. Conclusion: The ICP-MS technology was successfully implemented in two different configurations for the analysis of non-radioactive RLTs to gain early insight into their in vivo stability and excretion. We also established a gated-workflow strategy to use LC-MS/MS, FIA-ICP-MS, LC-ICP-MS, and LC-HRMS in sequence to probe different questions pertaining to candidates’ in vivo ADME properties. Collectively, the data can be used to guide the compound and study design strategies as well as for the selection and prioritization of compounds for subsequent hot biodistribution and GLP tox studies.
