Background: Oral AA is a standard of care for metastatic prostate cancer. The recommended daily AA 1000 mg dose produces high peak and low trough plasma concentrations that are associated with safety issues (e.g., hepatotoxicity) and inadequate efficacy, respectively. PRL-02 is a novel abiraterone prodrug undergoing evaluation in patients with advanced prostate cancer (NCT04729114). In chemically-castrate monkeys, single doses of PRL-02 suppressed androgens for 14 weeks at much lower plasma exposures than AA (Moore et al, ASCO GU 2021). The present study in intact male rats evaluated the systemic exposures and activity of PRL-02 compared to daily oral AA at 7 and/or 14 days post-treatment start. Methods: Sexually mature male rats (n=4 per timepoint) were administered a single IM dose of PRL-02 (90 mg/kg), daily AA (90 mg/kg) or IM/oral vehicle (VEH). Biological samples were collected on Day 7 (PRL-02/VEH; 0h) or Day 14 (PRL-02/AA/VEH; 2, 6 and 24h) post-dosing. Drug and androgen concentrations were determined by LC-MS/MS. Testicular CYP17 hydroxylase activity was measured ex vivo on Day 14. Results: The Day 14 plasma exposure from abiraterone was greater from AA (5.6 - 210 ng/mL) than from PRL-02 (1.15 – 1.37 ng/mL). However, for Day 14 tissue exposures (AUC_0-24) from ‘total abiraterone equivalents’ (TAE; concentration of free abiraterone plus abiraterone from prodrug) were greater from PRL-02 in adrenal, testes, lymph node and bone target tissues than from oral AA; exposures from AA were only greater in non-target tissues such as liver and brain (Table). There was no measurable Day 14 CYP17 hydroxylase activity in testes following PRL-02 or AA dosing; inhibition appeared irreversible from PRL-02 and AA. Compared to VEH, a single IM dose of PRL-02 resulted in 81% and 75% reductions in plasma testosterone (T) on Days 7 and 14 post-dose, and an 80% reduction in testicular T on Day 14. Daily AA resulted in a 98% reduction in plasma T after 14 days. Conclusions: Single-dose IM PRL-02 blocked testicular CYP17 enzyme activity as effectively as daily AA in intact male rats and produced a durable large reduction in plasma T. The pharmacological activity, tissue and plasma distribution of TAE from IM PRL-02 in the rat model, along with results from prior monkey model studies, suggest the potential for a greater therapeutic index than from daily oral AA and support the ongoing evaluation of IM PRL-02 in patients with advanced prostate cancer.