Simple SummaryProstate cancer (PC) remains fourth in terms of new cases of cancer in the world, and most deaths are caused by metastatic PC, for which limited therapeutic options are available. Faced with a lack of effective treatment, it is, therefore, urgent to dedicate more effort to the development of new innovative drugs to reverse this trend. Having developed an original molecule with promising antiproliferative activity on various cancer cell types, namely the aminosteroid derivative RM-581, its evaluation was extended to other PC models, the androgen-dependent LAPC-4 cells, and tumors. The most important result was its ability to completely block tumor growth in mice when given orally at a dose as low as 3 mg/kg. Another important result was its non-toxicity in mice during a 28-day experiment and a 7-week dose-escalation study, whose last RM-581 dose was 720 mg/kg given orally, suggesting a favorable safety window for this new promising drug.AbstractThe aminosteroid derivative RM-581 blocks with high potency the growth of androgen-dependent (AR+) prostate cancer VCaP, 22Rv1, and LAPC-4 cells. Notably, RM-581 demonstrated superior antiproliferative activity in LAPC-4 cells compared to enzalutamide and abiraterone, two drugs that exhibited a synergistic effect in combination with RM-581. These findings suggest that RM-581 may have an action that is not directly associated with the hormonal pathway of androgens. Furthermore, RM-581 completely blocks tumor growth in LAPC-4 xenografts when given orally at 3, 10, and 30 mg/kg in non-castrated (intact) nude mice. During this study, an accumulation of RM-581 was observed in tumors compared to plasma (3.3–10 folds). Additionally, the level of fatty acids (FA) increased in the tumors and livers of mice treated with RM-581 but not in plasma. The increase was greater in unsaturated FA (21–28%) than in saturated FA (7–11%). The most affected FA were saturated palmitic acid (+16%), monounsaturated oleic acid (+34%), and di-unsaturated linoleic acid (+56%), i.e., the 3 most abundant FA, with a total of 55% of the 56 FA measured. For cholesterol levels, there was no significant difference in the tumor, liver, or plasma of mice treated or not with RM-581. Another important result was the innocuity of RM-581 in mice during a 28-day xenograft experiment and a 7-week dose-escalation study, suggesting a favorable safety window for this new promising drug candidate when given orally.