ABSTRACT Genital herpes simplex virus (HSV) infections in pregnancy pose a risk for perinatal transmission of the virus to neonates, and HSV infections in the newborns are associated with severe morbidity and mortality. A standard treatment of HSV infections with nucleoside analogs such as acyclovir does not eliminate asymptomatic viral shedding and is ineffective against acyclovir-resistant HSV strains. Amenamevir and pritelivir, the helicase-primase inhibitors, represent novel antiherpesviral medications that were developed to circumvent the limitations of the nucleoside analogs. Both novel drugs completed Phase II clinical trials in treatment of genital herpes in men and nonpregnant women and demonstrated superiority over placebo and standard treatments. While amenamevir is currently indicated for the treatment of herpes zoster in Japan, pritelivir received an FDA Breakthrough Therapy designation and has entered a Phase III clinical trial. Pregnant women infected with genital herpes could benefit from amenamevir and pritelivir in suppressive or episodic treatments to prevent the transmission of HSV to neonates. The first step to determine the potential use of the novel drugs in pregnancy is to obtain preclinical data on their placental biodisposition. The focus of the proposed investigation is to determine the biodisposition of amenamevir and pritelivir by human placenta using ex vivo and in vitro experimental models. Meanwhile, assessing fetal safety for amenamevir and pritelivir, as well as their effect on placental function, is imperative in clinical development of these novel drugs for their potential use in pregnancy. While existing data from toxicity studies in animal models suggest a favorable reproductive and developmental safety profile for amenamevir, assessment of potential developmental hazards of pritelivir remains to be conducted. The specific aims are to 1) Determine the extent of bidirectional transfer of amenamevir and pritelivir across term human placenta ex vivo, the distribution of these drugs in the placental tissue and maternal and fetal circuits, and their effect on placental viability and functional parameters; 2) Determine the role of placental efflux membrane transporters in the placental disposition of amenamevir and pritelivir; and 3) Determine potential embryo-fetal developmental toxicity of pritelivir in mice. This work will provide the preclinical data needed to determine potential use of amenamevir and pritelivir in the treatment of newly acquired and reactivated genital herpes in pregnancy as well as in HSV suppressive therapy during pregnancy. Amenamevir and pritelivir could be effective alternative treatments to manage genital herpes in pregnancy to reduce the risk of mother-to-neonate HSV transmission in women with asymptomatic presentation of genital herpes at the time of delivery and for those with inadequate response to standard treatment with nucleoside analogs.