Project Summary Our twelve years of research has led to the discovery of triptonide (TN) as a promising non- hormonal male contraceptive agent (Nature Communications, 2021, 12:1253). In addition to its excellent potency and proven oral bioavailability, TN does not cause discernable toxic effects in either mice or monkeys at its minimum effective dose. All available data strongly suggest that TN is worth further investment to develop it as a viable non-hormonal male contraceptive drug candidate. Taking advantage of the R61/R33 funding mechanism, we propose to conduct several Investigational New Drug (IND)-enabling studies to collect data required by the FDA for official IND application. Specifically, we will conduct non-GLP toxicity and safety pharmacology studies to identify the maximum tolerated dose (MTD) followed by a six-month repeat dose toxicity study using MTD and minimum effective doses (MED) in male Sprague-Dawley rats in the R61 phase. In addition, deep learning-based pathological and RNA-seq-based transcriptomic analyses will also be performed. The data will not only guide and corroborate the GLP-compliant toxicity and safety pharmacology studies proposed in the R33 phase, but also provide molecular insights for any potential phenotypes observed. In the R33 phase, we will work with Pharmaron Inc. to perform non-GLP toxicity studies in dogs, in vitro secondary pharmacology, and GLP-compliant repeat-dose toxicity and safety pharmacology studies all as part of a package of studies intended to move triptonide into a Phase I clinical trial.