Identification of the uterine‐relaxant effect of mundulone as a natural‐product for therapeutic control of preterm labor

Abstract

Introduction Preterm birth rate continues to rise with over 15 million cases/year and remains the greatest contributor to neonatal morbidities and mortalities. Currently, there are no FDA‐approved tocolytics for the management of preterm labor due to the off‐target side effects and short duration of benefit of current off‐label therapeutics. High‐throughput screening of oxytocin‐induced Ca2+‐mobilization in uterine myometrial cells identified mundulone and mundulone acetate (MA) as hit‐antagonists. The aim of this work was to:1) examine the uterine‐selectivity of mundulone and MA by counterscreening vascular smooth muscle cells (VSMCs)—the major off‐target limiting the use of current tocolytics, 2) determine cytotoxic effects, 3) identify synergistic combinations of mundulone (and MA) with current tocolytics to increase efficacy and/or potency to decrease off‐target side effects and 4) determine the ex vivo tocolytic efficacy and potency on human myometrial tissue and confirm uterine selectivity at the tissue level by evaluating their effect on constriction of fetal ductus arteriosus (DA), a major off target of known tocolytics. Methods Primary human myometrial cells were isolated from tissue collected at the time of cesarean delivery from women at term (≥39 weeks) pregnancy. A phenotypic high‐throughput Ca2+‐ mobilization assay was performed to compare concentration‐response (10‐ point, 3‐fold dilutions) between myometrial and aorta VSMCs (Emax and IC50). The high‐throughput Ca2+‐mobilization assay was adapted for combination screening in 8×8 concentration‐response matrix format. Current tocolytics used for combination included: atosiban, indomethacin and nifedipine. Three models (Bliss independence, HSA and Loewe additivity) were used to determine synergy using Combenefit software. Cytotoxicity of mundulone and MA were determined using WST‐1 cell viability assay using myometrial, hepatic and renal cells. Ex vivo organ bath studies using myometrial tissue and isolated term fetal DA vessels were performed to examine concentration‐response on contractility and vessel diameter, respectively. Results MA was found to display a greater selectivity towards myometrial cells when counter screened against aorta VSMCs. Fold change (FC) in efficacy (Emax) was 13.5 and FC in potency was >4.5 for MA while fold change in potency for mundulone was only >2.3. We found that mundulone displayed synergism with two current tocolytics (atosiban and nifedipine), while MA displayed synergistic efficacy with nifedipine. Mundulone was found to affect the viability of myometrial cells while MA demonstrated a selectivity index of >10 (Ratio of IC50 in cell viability assay to IC50 in Ca2+‐mobilization assay). Both mundulone and MA showed concentration dependent inhibition of uterine contractions, without affecting fetal DA vasoreactivity at the tissue level. Conclusion Based on differences in uterine‐selectivity and tocolytic efficacy between mundulone and MA, this natural product could benefit from medicinal chemistry efforts to study stru...