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Introduction Currently, there are no FDA‐approved tocolytics for the management of preterm labor (PTL) due to adverse effects and lack of efficacy. Repurposing FDA‐approved drugs offers a better risk‐vs‐benefit compared to traditional drug development. Drug combinations targeting different molecular signaling pathways involved in uterine contractility may provide a synergistic effect, permitting lower doses that reduce off‐target effects or increase therapeutic efficacy. The study objective was to: 1) screen FDA‐approved drugs for their ability to inhibit Ca2+‐mobilization in uterine myometrial (UT‐myo) cells, 2) omit hit‐drugs that display cytotoxicity and lack uterine‐selectivity, 3) identify synergistic combinations of hit‐drugs and 4) evaluate ex vivo tocolytic efficacy and potency of single and combination drugs. Methods A phenotypic high‐throughput Ca2+‐mobilization assay using primary term‐pregnant human myometrial cells was performed to screen the SelleckChem FDA library of 1,180 drugs at 10μM to identify antagonists of oxytocin (OT)‐induced Ca2+‐mobilization. Uterine‐selectivity was determined by comparing the concentration‐response (Emax and IC50) of hit‐antagonists between UT‐myo and vascular smooth muscle cells (VSMCs) – the main off‐target limiting the use of current tocolytics. The high‐throughput assay was adapted for combination screening in 8×8 dose matrices to determine synergy using Combenefit. A WST‐1 assay with myometrial, hepatic and renal cells was used to determine cytotoxicity and safety index (SI, ratio of IC50 values from cell viability and Ca2+ assays). The tocolytic efficacy and potency were examined using human myometrial tissue in an ex vivo isometric contractility assay. Results We identified 27 hit‐antagonists of Ca2+‐mobilization from the screen of 1,180 FDA‐approved drugs. Twelve drugs were identified as uterine selective (≥ 5‐fold potency towards myometrial cells), of which 9 were potent with an IC50 ≤10μM without reported reproductive toxicity. Combining these drugs that target different molecular signaling pathways, with one another and 3 currently utilized tocolytics (atosiban, indomethacin and nifedipine), yielded 66 non‐redundant combinations. We found 25 combinations that synergistically resulted in increased efficacy and 39 combinations displayed increased potency for at least one drug. At present, at least 12 combinations have a favorable SI. Fosaprepitant dimeglumine demonstrated the greatest ex vivo tocolytic efficacy and potency, and to date, we have tested and confirmed tocolytic potency and efficacy of two drug combinations (atosiban and indomethacin; fosaprepitant dimeglumine and atosiban). Conclusion Phenotypic high‐throughput screening of FDA‐approved drugs against UT‐myo and VSMCs identified potent and uterine‐selective modulators of myometrial contractions with potential to be repurposed for the treatment of PTL. Furthermore, the HTS combination screen was an important tool to rapidly and reliably identify synergistic drug combinations with improved potency an...

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...

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