Positron emission tomography (PET) is a highly sensitive, non-invasive, imaging technique used for quantification of biological targets. The accuracy of quantification is influenced by the tracer metabolism and plasma protein binding (PPB). This thesis focuses on the development of novel methodologies for radiometabolite analysis (RMA) and PPB assessment of the P2X7 receptor binding PET tracer [¹¹C]SMW139 in both preclinical and clinical research.

Key challenges in PET imaging include the rapid metabolism of tracers, which generates radiometabolites that may have altered binding properties and biodistribution. These radiometabolites can accumulate in tissues in critical quantification areas, affecting PET signal quantification and interpretation. To address this, robust thin-layer chromatography (TLC), high-performance TLC (HPTLC), and high-performance liquid chromatography (HPLC) methods were developed and optimised for quantification of the unchanged tracer and its radiometabolites in plasma and brain tissue. Additionally, an ultrafiltration PPB analysis method was developed to simultaneously determine the plasma protein-free fraction of [¹¹C]SMW139 and of its radiometabolites. The free fraction of the radiometabolites had not been analysed before.

The results demonstrate that [¹¹C]SMW139 undergoes rapid metabolism in both mouse and human plasma, the parent and its radiometabolites are able to penetrate the blood-brain-barrier (BBB). However, suitable compartment modelling with corrections for BBB penetrating radiometabolites enabled robust quantification. Moreover, [¹¹C]SMW139 was found to be highly bound to plasma proteins, while the radiometabolites had a much higher free fraction enhancing their abundant penetration through the BBB. The findings provide essential insights into the pharmacokinetics of [¹¹C]SMW139 and its radiometabolites, enabling improved PET data interpretation and tracer evaluation for brain imaging. Standardisation of RMA and PPB analysis methodologies and especially their detailed reporting are recommended for enhancing the reliability of RMA and PPB studies in future studies.