Targeted downmodulation of OXTR and Cx43 in the myometrium by SERTIs as a novel tocolytic strategy.

Preclinical investigation using human cell cultures, human ex vivo tissue, and mouse models

“Here we investigate the ability of a novel CADA compound to provide tocolysis by downmodulating both OXTR and Cx43 in human and mouse myometrium.”

Primary pregnant human uterine smooth muscle cells, human myometrial biopsies from term pregnancies, and C57BL/6J mice

“Myometrial biopsies were obtained with written informed-consent from women with pregnancies undergoing Cesarean section, as previously described [18].”

Human cell pools from 8 patients, human tissue from 5 patients, and mice groups of 3 to 6 animals

“1° phUSMCs aggregated from 8 term pregnant patients were treated with TK047 at ½ log doses from 1 to 30 for an additional 48 h (n=5 per condition), or received DMSO (volumetric equivalent).”

Cell treatments ranged from 4 to 48 hours, tissue pretreatments from 10 minutes to 24 hours, and mouse injections for 3 days

“Cells were treated with varying doses of TK047 (10–30 µM, or DMSO vehicle control < 1% final volume) for periods between 4 and 48 h, then rinsed 2× in KREBS buffer containing, in mM: NaCl (118), KCl (4.75), CaCl2 (2.5), KH2PO4 (1.2), NaHCO3 (25), MgCl2 (1.2), dextrose (20), adjusted to pH 7.4, with the addition of 0.04% Pluronic® F-127 and 1 mM probenecid (anion transporter inhibitor), followed by 5 µM Calbryte™ 520 AM (Cat. 20650—AAT Bioquest, Sunnyvale, CA) for 1 h.”

TK047 reduced plasma membrane localization of oxytocin receptor and connexin 43 while increasing cytosolic concentrations

“TK047, SG004 and SG007 reduced PM localization of OXTR by − 6.98% ± 2.43 (p = 0.0455), − 9.64% ± 15.60; (p=0.5700) and − 28.99% ± 1.22 (p = < 0.0001) respectively, while with increasing cytosolic concentrations of OXTR by +214.30% ± 30.71; (p = 0.0022), +28.31% ± 46.17 (p = 0.5728), and +37.49% ± 10.64 (p = 0. 0244; Fig. 2B, left panel).”

TK047 decreased oxytocin-induced calcium influx and gap junction channel activity in human uterine smooth muscle cells

“TK047 treated cells experienced a reduction in the ‘area under the curve’ (AUC) by 20.32% ± 6.8 (p = 0.0087) and a reduction in peak height by 26.25% ± 12.1 (p = 0.0316), relative to control cells (Fig. 5C).”

TK047 reduced contractile force in human myometrium and did not affect fetal viability in mice

“Relative to control, in response to OT simulation, the AUC of TK047-treated tissues decreased by 98.17% ± 25.50 (p = 0.0049), while the peak tension decreased by 90.21% ± 32.21 (p = 0.0116).”

Use of nonpregnant mice for in vivo studies, potential species differences between mouse and human, and lack of direct confirmation of Sec61 pathway interaction in tissue

“For this study, the use of nonpregnant mice is a limitation in our 3 day in vivo treatment study (Fig. 10), as myometrial OXTR and Cx43 expression increase approximately 2.1-fold and 6.2-fold, respectively, in late pregnancy [75]; however, both targets are detectable in nonpregnant myometrium, making these animals informative for assessing baseline target modulation for tocolytic potential in pregnant mice.”