Comparison of Binding Affinity of Oxytocin Antagonists to Human and Rat Uterine Oxytocin Receptors and their Correlation to the Rat Uterine Oxytocic Bioassay1

Abstract

One of the primary methods used to screen the development of oxytocin antagonists (OTAs) is the rat oxytocic bioassay. The purpose of this study was to determine whether the rat oxytocic bioassay is a good predictor of binding affinity to human and rat uterine oxytocin receptors (OTr). The binding affinities of five OTAs to human and rat uterine OTr were determined and correlated with pA2 values derived from the rat uterine oxytocic bioassay. Human uterine myometrial tissue was obtained from patients at the time of cesarean section. Rat uterine tissue for the OTr assay was taken at Day 21 of pregnancy. OTr assays were accomplished by isolating uterine cell membranes and performing saturation analysis with cold OTAs and tritium-labeled oxytocin. The association constants (Ka; 10(+8)/M) were calculated by nonlinear curve-fitting techniques. The Ka for the five OTAs (Mpa1-OT, Antag I, L366948, Antag II, and Antag III), as estimated from the human OTr assay, were 0.55, 0.60, 2.27, 1.91, and 47.20, respectively. Ka estimates obtained through use of rat uterine membranes were 0.51, 1.16, 5.89, 2.03, and 24.40, respectively. Correlation of the log10 of the rat oxytocic bioassay results with those of the rat and human OTr assay was 0.94 and 0.98, respectively (p < 0.01). Antag III was approximately 55, 48, and 90 times more potent than Mpa1-OT as determined by the rat bioassay and rat and human uterine OTr assays, respectively. Mpa1-OT is an OTA that is currently undergoing clinical evaluation.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of Oxytocin and Oxytocin Antagonist Metabolism in the Plasma of Pregnant Humans and Baboons

“…It was important to test TT-235 in a primate model because compounds that show antioxytocic properties in one animal species may have different relative activities in other species or even turn out to have oxytocic properties. The results of our previous studies [18,20,21] indicate that the relative activity of TT-235 was much better than that of atosiban in various species and different assay methods used. Based on these studies in the baboon, it is anticipated that TT-235 will have a prolonged activity in pregnant women.…”

“…This assay was a modification of a previously described procedure [18]. The oxytocin receptors used in this assay were obtained from the pregnant rat uterus at delivery.…”

“…Among these, we have developed a very potent compound, [PMP(s) 1 , D-Trp 2 , Pen 6 , Arg 8 ]-oxytocin, referred to as TT-235. TT-235, previously referred to as Antag III, is a potent oxytocin antagonist undergoing development as a potential inhibitor of preterm labor [17][18][19][20][21]. It has significant clinical potentials based on data from bioassay [18], receptor assay affinity [21], and antagonist response interval [21].…”

Development of a Radioreceptor Assay for Measuring an Oxytocin Antagonist in Blood

“…One might be critical of designing the different protocols for the rat and the baboon as was done in our study. Based on the data of receptor-binding affinity, bioassay, and antagonist-response interval [11,13], the in vivo biological activity of TT-235 is expected to be species-specific.…”

“…The oxytocin receptors used for this assay were isolated from the pregnant uterus of rats at delivery as previously described [11]. Following delivery of 3 or more fetuses, uterine tissues were isolated, trimmed of fat and rinsed in ice-cold buffer.…”