George Flouret

Forward shift in the initiation of the nocturnal estradiol surge in the pregnant baboon: Is this the genesis of labor?

Daily (9 AM and 6 PM) blood samples were obtained from the inferior vena cava during the last trimester of pregnancy in a tethered baboon model. In addition, three 24-hour (hourly blood sampling) studies were performed at days 143 to 147, 158 to 162, and 172 to 177 of pregnancy. Dramatic 24-hour rhythms in progesterone and estradiol were detected, with both steroids surging nocturnally. Early in the third trimester the estradiol surge followed the progesterone surge. However, approximately 10 to 12 days before delivery, the initiation of the nocturnal estradiol surge shifted forward, thus preceding the progesterone surge. This forward shift in the estradiol surge created a daily (3 to 5 hours) window of elevated estradiol-to-progesterone ratio and appears to coincide with the initiation of nocturnal uterine contractions. The nocturnal uterine contractions can be inhibited by an oxytocin antagonist. We hypothesize that this forward shift in the initiation of the estradiol surge induces nocturnal uterine contractions by oxytocin release and/or increase in uterine oxytocin receptors and generates molecular messages that are the genesis for labor and delivery in the baboon.

Inhibition of spontaneous uterine contractions during the last trimester in pregnant baboons by an oxytocin antagonist

The effect of a potent oxytocin antagonist, produced in our laboratories, on spontaneous uterine contractions in the pregnant baboon was examined. Three types of uterine contractions were studied: immediately after operation, during the nocturnal period, and near or at labor. Bolus intravenous injections of oxytocin antagonist were given and uterine activity was examined +/- 1 hour after the injection. The oxytocin antagonist caused a precipitate decrease (approximately 70%) in contractile force (mean amplitude x frequency) in the first 15 minutes after injection (p less than 0.05); this force diminished to approximately 90% at the end of 1 hour for both nocturnal and labor contractions. In contrast, uterine contractions immediately after operation were diminished by only 60% within 60 minutes after the oxytocin antagonist. These results indicate that the oxytocin antagonist is a potent inhibitor and suggest that oxytocin is a primary regulator of spontaneous nocturnal and labor uterine contractions in the pregnant baboon.

A new tocolytic agent: development of an oxytocin antagonist for inhibiting uterine contractions.

A potent oxytocin antagonist has been developed and tested on both the rat and human uterus. In the rat the oxytocin antagonist: (1) inhibited in vitro and in vivo uterine contractions in the nonpregnant animal in response to exogenous oxytocin, (2) inhibited milk letdown, and (3) disrupted the progress of labor. In addition, the oxytocin antagonist inhibited the in vitro contractile response to exogenous oxytocin of human myometrial tissue obtained by cesarean section at term. The results of these studies suggest that the oxytocin antagonist can be used to study the role of oxytocin in labor and has the potential of inhibiting preterm labor in humans.

Adsorption chromatography of cyclic nucleotides on silica gel and alumina thin-layer sheets

Abstract Several solvent combinations of graded polarity have been developed, capable of separating cAMP or cGMP, not only from related nucleotides but from bases and nucleosides on alumina and silica gel thin-layer sheets (tls). The solvent system chloroform (C), methanol (M), water (W) (40:20:3) gave effective separations of cAMP on silica gel TLS. Identical results were obtained when this adsorption chromatography method was compared with the ion-exchange chromatography method involving PEI for its effectiveness in the separation of [α- 32 P]cAMP formed from [α- 32 P]ATP by a preparation of bovine sperm cells. In addition, this solvent system effectively separates cAMP from inosine, hypoxanthine, xanthine, and uric acid which may be useful in determinations of cAMP arising from 3 H- or 14 C-prelabeled ATP. Effective separation of cGMP on silica gel tls was accomplished with C:M:W (30:30:5). On alumina tls, cAMP and cGMP were separated from related compounds with M:W combinations; the solvent M:W (50:50) gave the lowest blanks (0.02%) for cGMP and it may prove useful in cGMP determinations.

Oxytocin antagonist inhibitory effect on the rat and baboon uterus may be overcome by prostaglandins

OBJECTIVE A potent, long-acting oxytocin antagonist produced in our laboratory (ANTAG-III) can inhibit uterine response to oxytocin in the rat and baboon for hours and even days. The purpose of this study was to evaluate uterine response to prostaglandins subsequent to the administration of ANTAG-III. STUDY DESIGN For the rat study one cannula was inserted in the jugular vein, and another cannula to measure uterine activity was inserted in the uterus. In study 1 saline solution or 5 micrograms of ANTAG-III was administered to five rats each, followed by 100 mU of oxytocin at 0.1, 1, and 2 hours. In study 2 six rats each were infused with saline solution of 5 micrograms of ANTAG-III, followed 1 hour later by 5 micrograms of 15-methyl-prostaglandin F2 alpha and uterine activity monitored. After baseline activity returned to normal 100 mU of oxytocin was infused and the uterine response reassessed. For the baboon study ANTAG-III was administered into the aorta of tethered pregnant baboons (n = 2). An oxytocin challenge test was performed starting with 10 mU/min and going up to 400 mU/min. After a significant uterine contractile response was established and activity returned to baseline, a 15-methyl-prostaglandin F2 alpha challenge test was performed. RESULTS During the period in which the response to oxytocin was inhibited the uterine response to 15-methyl-prostaglandin F2 alpha of the estrous rat and pregnant baboon was maintained. CONCLUSIONS The inhibition of the estrous rat and pregnant baboon uterus to oxytocin caused by ANTAG-III may be prolonged. During this period uterine response to prostaglandins is not altered.

Oxytocin receptors coupled to uterine contraction in estrogen-dominated rabbits

The present study investigated whether specific [3H]oxytocin binding sites previously demonstrated in estrogen-dominated rabbit uterus have properties expected of physiologic receptors coupled to uterine contraction. Microsomal membranes from estrogen-dominated rabbit uterus were found to contain high-affinity specific oxytocin binding sites with Kd = 2-3 nM. These sites were predominantly myometrial in locus. Specific oxytocin binding exhibited a pH optimum between 7.5 and 8.0. Mg2+ or Mn2+ was necessary for maximal specific [3H]oxytocin binding; in contrast, Ca2+ at submillimolar concentrations inhibited specific binding. Oxytocin binding sites were not detectable in microsomal membranes isolated from progesterone-dominated rabbit uterus. Relative binding and uterotonic activities of 10 synthetic neurohypophyseal hormone analogues were determined in estrogen-dominated rabbit uterus. A qualitative correlation was observed between binding and uterotonic responses. Angiotensin II and insulin did not compete with [3H]oxytocin for uterine binding sites. It is concluded that the specific high affinity [3H]oxytocin binding sites demonstrated in estrogen-dominated rabbit uterus have the selectivity for neurohypophyseal hormone analogues expected for physiologic receptors coupled to uterine contraction.

Comparison of the in vivo activity of different oxytocin antagonists in the pregnant baboon.

Objective: To ascertain the relative activity of five oxytocin antagonists (OTAs) in vivo in a tethered pregnant baboon model and compare these results to previously reported affinities in human and rat oxytocin receptor assays and median effective dose in rat uterotonic bioassay. Methods: Pregnant tethered baboons between days 130 and 160 of pregnancy were given an oxytocin challenge test 1 minute after infusion of 1 mg of one of five randomly selected OTAs: ANTAG I, ANTAG II, ANTAG III, L366948, and Atosiban. Once the uterine response to exytocin returned to normal (1-8 days) the OCT was repeated with one of the remaining, untested OTAs during the 130-160 day period. Uterine activity, the time until the first significant response, and the dose of oxytocin needed to induce this response were all factored into one expression, the antagonist-response interval (ARI). Results: When expressed as ratio to ANTAG I the relative ARI for the OTAs were 0, .5, 1.0, 2.4 and 59.2 for L366948, Atosiban, ANTAG I, ANTAG II, and ANTAG III, respectively. ANTAG III and L366948 were significantly different from each other and the three other OTAs (P < .05). The log10 ARI for the 4 active OTAs when correlated with the log10 of the human and rat oxytocin receptor affinities and the rat uterotonic bioassay were all highly correlated (r = .99; P < .05). Conclusion: ANTAG III is a potent, long-acting OTA in vivo in the pregnant baboon and has the potential as a tocolytic in humans.

Comparison of oxytocin and oxytocin antagonist metabolism in the plasma of pregnant humans and baboons

Oxytocin antagonists may be useful in inhibiting the uterine contractions of preterm labor. One such compound is TT-235 (previously referred to as Antag III). The purpose of this study was to compare the resistance of TT-235 and oxytocin to enzymatic degradation by oxytocinase in the blood of humans and baboons during their 3rd trimester of pregnancy. Blood samples from pregnant women and baboons not in labor were incubated in vitro with known amounts of oxytocin and TT-235. Samples were collected at 0, 15, 30, 45, and 60 min for oxytocin analysis and at 0, 10, 60, and 360 min for TT-235 analysis. Oxytocin was analyzed by radioimmunoassay after extraction, while TT-235 was analyzed by radioreceptor assay. In human blood, oxytocin was readily metabolized with >83% disappearance over the 60-min incubation period. In contrast, TT-235 was stable up to 360 min of incubation. In the baboon, oxytocin did not diminish over the 60-min incubation period. The level of TT-235 was similar to that in human blood without change over 360 min of incubation. This study suggests (1) that in contrast to blood from pregnant humans, blood from pregnant baboons lacks oxytocinase at least in vitro and (2) that TT-235 is resistant to enzymatic degradation by human blood, implying that this oxytocin antagonist may have a prolonged activity in vivo in humans.

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

The purpose of the present study was to develop a radioreceptor assay to monitor the pharmacokinetics of the oxytocin antagonist, TT-235, in the blood of the pregnant rat and baboon. The receptors used for this assay were prepared from the pregnant rat uterus at delivery. The assay using blood from pregnant rats and baboons was performed on filter plates and analyzed for radioactivity in a gamma counter. The assay was sensitive to 10 pg/well with a range from 10 to 1,000 pg. The average recovery was 86%. A new radioreceptor assay was developed for the oxytocin antagonist, TT-235. TT-235 had a much longer half-life than oxytocin in the pregnant rat and baboon. This longer half-life may partially explain the prolonged in vivo tocolytic activity of TT-235 in these animals.

Effect of an oxytocin antagonist on plasma oxytocin levels during nocturnal uterine contractions in the pregnant baboon.

TT-235 is a potent oxytocin (OT) antagonist that blocks the action of OT at the receptor level. Previous studies have shown that pregnant baboons demonstrate nocturnal uterine contractions induced by OT as they near delivery. The purpose of this study was to evaluate the changes in plasma OT levels following uterine contraction blockage with TT-235. A tethered pregnant baboon model in its last trimester of pregnancy was used. Three blocks of arterial blood samples, immediately before, plus 1 h and plus 2 h following an OT antagonist injection, were collected once nocturnal uterine contractions were detected. Each block consisted of a continuous 10 min withdrawal with 10 samples per block (1 ml/min). A TT-235 dosage of 300 μg/kg and saline for control were utilized. Uterine activities were monitored as pressure changes in the amniotic fluid, and the frequency and mean amplitude of contractile activity per 10 min intervals were expressed as contractile force. Plasma OT levels were determined by a radioimmunoassay following plasma extraction with petroleum ether. The contractile force was decreased by 77% (p < 0.05) within 2 h after TT-235 administration while it increased 23% following saline infusion. Plasma OT levels were unchanged following saline infusion while they increased 82% (p < 0.05) 2 h after the administration of TT-235. If a positive feedback existed between uterine contractions and OT release, one would expect plasma OT levels to be decreased with contractile activity following TT-235 infusion. Since this is not the case in the present study, the data suggest that there is either a negative feedback or an independent relationship between nocturnal uterine contractions and OT release.