Abstract

The theranostic concept has revolutionized personalized medicine by integrating diagnostic and therapeutic radiopharmaceuticals targeted at the same molecular biomarker. Preclinical small-animal molecular imaging is a critical checkpoint in translating novel theranostic agents from bench to bedside. This review evaluates the rational applications, methodological standards, and recent translational success stories of preclinical micro-single-photon emission computed tomography (SPECT) and micro-positron emission tomography (PET) systems in the development of novel radiopharmaceuticals. Micro-imaging in theranostic development is utilized to demonstrate in vivo stability, map biodistribution, determine tumor kinetics, and estimate critical organ dosimetry. Optimization of imaging protocols (dynamic vs. serial static), system calibrations, the necessity of high specific activity, injection routes (intravenous, intraperitoneal, intranasal), and the impact of anesthetic agents on pharmacokinetics constitute the cornerstones of preclinical standardization. In the literature, the role of these preclinical phases is prominent in the clinical translation of prostate-specific membrane antigen (PSMA) (PSMA-617) and fibroblast activation protein (FAP) (FAPI-46) targeted agents, while novel targets such as Trop-2 and Nectin-4 are currently emerging. Preclinical PET and SPECT imaging are the most reliable modalities to predict the in vivo behavior of potential theranostic agents prior to human translation. Early detection of in vivo instability (e.g., free iodine-124 detachment) prevents futile clinical trials, whereas successful preclinical designs pave the way for future routine clinical agents.