Arrest of mouse preterm labor until term delivery by combination therapy with atosiban and mundulone, a natural product with tocolytic efficacy.

There is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and mundulone acetate (MA) as inhibitors of in vitro intracellular Ca2+-regulated myometrial contractility. In this study, we probed the tocolytic potential of these compounds using human myometrial samples and a mouse model of preterm birth. In a phenotypic assay, mundulone displayed greater efficacy, while MA showed greater potency and uterine-selectivity in the inhibition of intracellular-Ca2+ mobilization. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted inhibition of myometrial contractions and that neither compounds affected vasoreactivity of ductus arteriosus. A high-throughput combination screen identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these combinations, mundulone+atosiban demonstrated a significant improvement in the in vitro therapeutic index compared to mundulone alone. The ex vivo and in vivo synergism of mundulone+atosiban was substantiated, yielding greater tocolytic efficacy and potency on myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone after mifepristone administration dose-dependently delayed the timing of delivery. Importantly, mundulone+atosiban permitted long-term management of PL, allowing 71% dams to deliver viable pups at term (>day 19, 4-5 days post-mifepristone exposure) without visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the development of mundulone as a single or combination tocolytic for management of PL.

Endothelium-independent vasorelaxant effect of Asphodelus tenuifolius Cav. via inhibition of myosin light chain kinase activity in the porcine coronary artery.

ETHNOPHARMACOLOGICAL RELEVANCE: Asphodelus tenuifolius Cav. (Asphodelaceae), a wild, terrestrial, annual stemless herb, is widely used in traditional medicine for the treatment of hypertension, diabetes, atherosclerosis and circulatory problems. A previous research study from our laboratory revealed that A. tenuifolius has beneficial effects in reducing blood pressure and improves aortic endothelial dysfunction in chronically glucose fed rats. Despite the fact that A. tenuifolius reduces blood pressure and improves endothelial function in vivo, there are no detailed studies about its possible mechanism of action. AIM OF THE STUDY: This study was designed to provide pharmacological basis and mechanism of action for the traditional use of A. tenuifolius in hypertension and circulatory problems. We explored the vasorelaxant effect of A. tenuifolius and its underlying vasorelaxation mechanism in porcine coronary artery rings. MATERIALS AND METHODS: Aqueous methanolic crude extract of A. tenuifolius was prepared by maceration process and then activity guided fractionation was carried out by using different polarity based solvents. Phytochemical studies were carried out using LC-DAD-MS. Segments of porcine distal coronary artery were set up in a wire myograph for isometric force measurements. Extract/fractions of A. tenuifolius seeds were tested for vasodilator activity by measurement of changes in tone after pre-contraction with the thromboxane mimetic U46619 in the presence or absence of inhibitors of intracellular signaling cascades. RESULTS: Crude extract/fractions of A. tenuifolius produced dose dependent endothelium independent vasorelaxant response in coronary rings, whereas, the butanol fraction of A. tenuifolius (BS-AT) produced the largest relaxation response with 100% relaxation at 1 mg/ml, therefore the mechanism of relaxation of this fraction was determined. The relaxation to BS-AT was unaffected by removal of the endothelium, pre-contraction with KCl, or the presence of the non-selective potassium channel blocker tetraethylammonium, indicating that the relaxation was endothelium-independent, and does not involve activation of potassium channels. BS-AT (1 mg/ml) inhibited the contractile response to calcium,the L-type calcium channel activator BAY K8664,and ionomycin, indicating that it inhibits calcium-induced contractions. The relaxation response to BS-AT was attenuated in the absence of extracellular calcium. However, relaxations to BS-AT were also reduced after deletion of calcium from intracellular stores with cyclopiazonic acid. Incubation with 1 mg/ml BS-AT also inhibited phosphorylation of myosin light chains in homogenates of coronary artery. CONCLUSION: The butanol extract of Asphodelus tenuifolius produces a large endothelium-independent relaxation of the porcine coronary artery through inhibition of calcium-induced contractions. The effect appears to be downstream of calcium influx, possibly through inhibition of myosin light chain kinase. This study supports previous studies demonstrating that A. tenuifolius reduces blood pressure. Future studies will aim to determine the active compounds underlying this response.

Hesperetin inhibits rat coronary constriction by inhibiting Ca(2+) influx and enhancing voltage-gated K(+) channel currents of the myocytes.

Hesperetin (HSP, one of the most common flavonoids in Citrus) has been reported to possess many benificial effects and is indicated for many diseases both as a therapeutic drug and as a supplement. Although its vascular effects have been extensively studied, little is known about its effects and the underlying mechanisms on coronary artery. In the present study, the myogenic effects of HSP were studied with a wire myograph in isolated rat coronary artery (RCA). Molecular probe and the patch clamp technique were used to study effects of HSP on intracellular free Ca(2+) concentration, inward Ca(2+) currents through L-type voltage-gated Ca(2+) channels (LVGC) and outward K(+) currents through voltage-gated K(+) channels (KV). HSP (0.01-0.1mM) concentration-dependently depressed concentration-contraction curves of both KCl and thromboxane receptor agonist 9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U46619), and relaxed RCA precontracted by the both vasoconstrictors. The vasospasmolytic effect was more potent in KCl- than in U46619-induced contraction. The vasorelaxation was attenuated by 4-aminopyridine, a specific KV inhibitor, but not affected by NG-nitro-L-arginine methylester ester, indomethacin, glibenclamide, iberiotoxin, BaCl2 or endothelium denudation. At the same concentrations, HSP inhibited extracellular Ca(2+) influx-induced contraction, reduced intracellular free Ca(2+) concentration, inhibited inward Ca(2+) currents through LVGC and increased outward K(+) currents through KV in the vascular smooth muscle cells (VSMCs) freshly isolated from RCA. Collectively, our results show that HSP is vasospasmolytic in RCA and suggest that the vasospasmolysis is mediated by inhibition of LVGC and enhancement of KV currents in RCA VSMCs.