Novel Drug Discovery Pathways in the Myometrium for the Treatment of Preterm Labor

Abstract

Introduction Approximately 10% of all US births occur preterm (<37 weeks). Half of preterm births are the result of spontaneous preterm labor (sPTL), which is idiopathic in nature. We are developing novel therapeutics that build upon our previous findings that nitric oxide‐mediated relaxation of the myometrium is independent of cGMP accumulation and occurs via protein S‐nitrosations (S‐NO). An important mediator of protein S‐nitrosation is the availability of S‐nitrosoglutathione (GSNO), an endogenous NO donor. Nitric oxide synthase (NOS) and GSNO reductase (GSNOR) alter GSNO availability in smooth muscle. We have previously determined that GSNOR is upregulated in preterm laboring myometrium, and we hypothesize that increasing endogenous NO availability will restore the quiescent phenotype in sPTL tissue. Here we describe that GSNOR inhibition (SPL‐334, N6022) and β3AR activation (mirabegron, nebivolol), both of which increase endogenous NO levels in the cell, exhibit tocolytic effects on human myometrium. These findings are significant because currently available tocolytics are ineffective at delaying birth beyond 48‐hours, and they are innovative as they constitute a novel approach to preventing preterm birth. Methods Strips of (~0.5x15mM) were attached to a force transducer and isometrically stretched in an organ bath containing Krebs buffer. Tissues were maintained at 37°C and gently bubbled with balanced oxygen. Tissues were then challenged with KCl (60mM replacing NaCl) and oxytocin (8nM), followed by washout, then treated with either mirabegron (100μM), nebivolol (100μM), N6022 (100μm) or SPL‐334 (10μM). Data were analyzed with LabChart (version 8.1.13). Western blots were run on a 10–15% polyacrylamide gel with 30μg of total protein and using a student’s t‐test (sig. p<0.05). Results Both GSNOR inhibition (SPL‐334 or N6022), and β3AR activation (mirabegron or nebivolol), exhibit negative inotropic effect on human myometrium. SPL334 decreased ‘area under the curve’ by 21.6% (p<0.001) and reduced the number of ‘contractions per unit time’ by 20.3%. Similarly, mirabegron decreased peak force by 18.2% (p=0.0113). The combined administration of N6022 and nebivolol had a synergistic effect that all but abolished contractile force (92% decrease in peak force), as compared to their individual effects (N6022 40.8%, nebivolol 86.7% decrease in peak force). Of note, β3ARs are expressed in both endothelium and uterine smooth muscle, and we found that both β3AR and eNOS are downregulated in PTL (n=6 p=0.0317, p=0.0012) as compared to other pregnancy states (n=12 each: TL, TNL, PTNL), indicating a potential role for therapies that target these pathways. Conclusion The importance of protein S‐nitrosation in the myometrium, combined with the known dysregulation of NO metabolism in sPTL tissue, have led us to seek novel therapeutics (tocolytics) that will mitigate preterm labor by enhancing endogenous NO availability (and SNOs) in the myometrium. SPL‐334 and N6022 increase NO through inhibition of GSNOR, and mirabegro...