Identification of FDA approved drugs and their synergistic combinations as potent and selective regulators of uterine contractility for therapeutic control of preterm labor

Abstract

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...