Design of novel bicyclic analogues derived from a potent oxytocin antagonist.

Eleven new analogues were synthesized by modification of the potent oxytocin antagonist (OTA) [(S)Pmp(1), D-Trp(2), Pen(6), Arg(8)]-Oxytocin, or PA (parent antagonist), in which (S)Pmp = beta,beta-(3-thiapentamethylene)-beta-mercapto-propionic acid. By internal acylation of Lys, Orn, L-1,4-diaminobutyric acid (Dab), L-1,3-diaminopropionic acid (Dap) at position 4 with the C-terminal Gly of the peptide tail, we prepared cyclo-(4-9)-[Lys(4), Gly(9)]-PA (pA(2) = 8.77 +/- 0.

Analogues of oxytocin antagonists bearing a ureido group in the amino acid side chain at position 4 or 5.

Substitution of the side chain carboxamido group at position 4 in the potent oxytocin antagonist (OTA) [ThiaPmp(1), D-Trp(2), Cys(6), Arg(8)]-OT, PA, in which ThiaPmp = beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, led to [Orn(Car)(4)]-PA, ([Cit(4)]-PA), which had uterotonic antagonistic activity equal to that of PA. The same modification at position 5, leading to [Cit(5)]-PA, resulted in antagonistic potency more than 10 times lower than that of PA. This paper also describes the same substitutions introduced in the highly potent OTA [Pen(6)]-PA (antioxytocic in vitro pA(2) = 8.

In vivo activity of the potent oxytocin antagonist on uterine activity in the rat.

Oxytocin antagonist (OTA), TT-235, was developed by our group and shown to inhibit either spontaneous or oxytocin-induced uterine contractions in primates. The purpose of the present study was to confirm the duration of TT-235 to block oxytocin-induced uterine contractions in estrous rats. In Experiment 1, the time-response of the three OTAs on uterine contractility was examined.

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.

Analogues of a potent oxytocin antagonist with truncated C-terminus or shorter amino acid side chain of the basic amino acid at position 8.

Twelve analogues were synthesized, their structure derived from modifications of [(S)Pmp1, D-Trp2, Pen6, Arg8]oxytocin, PA, in which (S)Pmp = beta,beta-(3-thiapentamethylene-beta-mercaptopropionic acid). PA is a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 8.86) and in the baboon.

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.

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.

Antagonists of oxytocin featuring replacement with modified beta-mercaptopropionic acids at position 1.

Twenty analogues were synthesized of [Pmp1, D-Trp2, Arg8]oxytocin, PA, (Pmp = beta,beta-pentamethylene-beta-mercaptopropionic acid), a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 7.77) and in the baboon. Systematic substitution of Pmp1 was made with beta-mercaptopropionic acids featuring replacement of the 4-methylene group of the cyclohexyl ring of Pmp with isosteric O, S, NH or with C=O.

The effect of oxytocin antagonist on uterus in response to exogenous oxytocin.

This study was performed to determine the action mode of oxytocin antagonist. In Study 1, the duration of in vivo action of oxytocin antagonist I (AI) was examined. After infusing AI, oxytocin was given and repeated every hour for 5 hr.

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 bioassays.

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 oxytocin returned to normal (1-8 days) the OCT was repeated with one of the remaining, untested OTAs during the 130-160 day period.

Antiovulatory antagonists of LHRH related to antide.

We report 104 analogues of the potent antiovulatory antagonist of LHRH, N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Nic)-D-Lys(Nic)-Leu-Ilys-Pro-D-Ala- NH2, Antide. We replaced the Nic group in Antide with other acyl substituents to modulate size, hydrophilicity or basicity of the molecule, we also replaced the Lys residues with shorter basic amino acids, and made cyclic 5/6 analogues as well as position 5 or 6 dimers. We substituted Ilys8 with other alkyl groups and acyl derivatives.

Comparison of binding affinity of oxytocin antagonists to human and rat uterine oxytocin receptors and their correlation to the rat uterine oxytocic bioassay.

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.

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.

Systematic substitution of an oxytocin antagonist with D-amino acids: unexpected high antagonistic potency of the D-Cys6-substituted analogue.

We report twelve analogues (1-12) of [Pmp1,D-Trp2,Arg8]oxytocin, PA (parent antagonist), (Pmp = beta,beta-pentamenthylene-beta-mercaptopropionic acid), which is a potent antagonist (pA2 = 7.77) of the uterotonic effect of oxytocin (OT) in rats. The analogues were designed by replacement of each optically active amino acid residue at positions 3-8 in PA with a D-amino acid.

Decreased histamine release by luteinizing hormone-releasing hormone antagonists obtained upon translocation of the cationic amino acid from position 8 to position 7.

We report analogues of N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Pic)-D-Lys(Pic)-Leu-Ilys-Pro-D-Ala- NH2, the parent antagonist (PA), which is a potent antagonist of LHRH. To simplify future radioactive labeling we prepared N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Pic)-D-Lys(Pic)-Leu-Arg-Pro-D-Ala-NH2 (4), [Arg8]PA, which had good activity in the antiovulatory assay (AOA). Other analogues were designed at first by substituting with Arg at positions 5, 6, 7, 9, and 10, and Trp or Leu at position 8.

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.

Multiple carriers for dipeptide transport: carrier-mediated transport of glycyl-L-proline in renal BBMV.

To determine whether multiple carriers are responsible for luminal uptake of glycyl-L-proline (Gly-Pro) in the renal proximal tubule, transport of Gly-[3H]Pro was measured in brush-border membrane vesicles (BBMV). A Line-weaver-Burk analysis of Michaelis-Menten kinetics revealed the presence of two carriers: a lower affinity, higher capacity carrier (Km = 1.3 x 10(-2) M; Vmax = 4.

Inhibition of oxytocin-induced uterine contractions by an oxytocin antagonist in the pregnant baboon.

Uterine contractions were induced with oxytocin in anesthetized pregnant baboons (Papio anubis) at three stages of pregnancy (days 140, 156, and 169; normal gestation length, 184 days). After the contractile activity was greater than two to three contractions every 10 minutes, beta-mercapto-beta, beta-cyclopentamethylenepropionic acid1-[D-Trp2,Phe3,Ile4,Arg8]-oxytocin, a novel oxytocin antagonist produced in our laboratories, was given simultaneously with the oxytocin for 90 minutes. Contractile force (frequency x mean amplitude) was determined for 30 minutes before and for three 30-minute intervals after the oxytocin antagonist was administered.

Some pharmacological properties of cyclic and linear analogs obtained by substituting each residue of an oxytocin antagonist with D-tryptophan.

We synthesized 10 analogs (1-10) derived from the sequence of [Pmp1,D-Trp2,Arg8]oxytocin, (parent antagonist or PA), (Pmp = beta,beta-pentamethylene-beta-mercaptopropionic acid) which is a potent antagonist (pA2 = 7.77) of the uterotonic effect of oxytocin (OT) in rats, as determined in our uterotonic assay. Eight of the following analogs were designed by replacement of each residue in the PA sequence, other than the residue at position 2, with D-tryptophan: Ac-D-Trp-D-Trp-Ile-Gln-Asn-Val-Pro- Arg-Gly-NH2, (1); [Pmp1,D-Trp(For)2,Arg8] OT, (2); [Pmp1,D-Trp2,D-Trp3,Arg8] OT, (3); [Pmp1,D-Trp2,D-Trp4,Arg8] OT, (4); [Pmp1,D-Trp2,D-Trp5,Arg8] OT, (5); Aaa-D-Trp-Ile-Gln-Asn-D-Trp-Pro-Arg- Gly-NH2, (6); [Pmp1,D-Trp2,D-Trp7,Arg8] OT, (7); [Pmp1,D-Trp2,D-Trp8] OT, (8); [Pmp1,D-Trp2,Arg8,D-Trp9] OT, (9); [Pmp1,D-Trp2,Arg8,D-Trp(For)9] OT, (10).

Improvement in potency of an oxytocin antagonist after systematic substitutions with L-tryptophan.

We report twelve analogues of [Pmp1,D-Trp2,Arg8]oxytocin, ANTAG (Pmp = beta, beta-pentamethylene-beta-mercaptopropionic acid), which is a potent antagonist (pA2 = 7.77) of the uterotonic effect of oxytocin (OT) in rats, as measured in a uterotonic assay. Nine of the following analogues were designed by replacement of each of the nine residues in ANTAG with an L-tryptophan residue: [Ac-Trp1,D-Trp2,Val6,Arg8]OT, [Pmp1,Trp2,Arg8]OT, [Pmp1,D-Trp2,Trp3,Arg8]OT, [Pmp1,D-Trp2,Trp4,Arg8]OT, [Pmp1,D-Trp2,Trp5,Arg8]OT, [Aaa1,D-Trp2,Trp6,Arg8]OT, [Aaa1,D-Trp2,Val6,Arg8]OT, [Pmp1,D-Trp2,Ica7,Arg8]OT, [Pmp1,D-Trp2,Trp7,Arg8]OT, [Pmp1,D-Trp2,Trp8]OT, [Pmp1,D-Trp2,Arg8,Trp9]OT (11), [Pmp1,D-Trp2,Arg8,Trp(For)9]OT (12).

Design of potent oxytocin antagonists featuring D-tryptophan at position 2.

We prepared nine analogues (1-9) of MCPA-D-Phe-Phe-Ile-Asn-Cys-Pro-Arg-Gly-NH2, [MCPA1, D-Phe2, Phe3, Ile4, Arg8]oxytocin (MCPA = beta-mercapto-beta,beta-pentamethylenepropionic acid), a potent antagonist of the rat uterotonic action of oxytocin (OT). We replaced D-Phe with D-Trp and made [MCPA1,D-Trp2,Phe3,Ile4,Arg8]OT (1), which had OT pA2 of 7.51, somewhat higher than that of the D-Phe2 antagonist which has OT pA2 = 7.

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.

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.

Inhibition of catalase and epoxide hydrolase by the renal cystogen 2-amino-4,5-diphenylthiazole and its metabolites.

Subchronic feeding of 2-amino-4,5-diphenylthiazole (DPT) to rats results in the development of renal cysts and has been used as a model system to study polycystic kidney disease. Because previous studies revealed changes in renal enzymes following DPT administration, a possible direct effect of DPT and its phenolic metabolites on catalase and a related enzyme, epoxide hydrolase, was examined. Experiments with three in vitro systems (suspensions of rabbit renal tubules, rat kidney homogenates, and commercially obtained bovine liver catalase) revealed direct inhibition of catalase activity by the diphenolic metabolite (diOH- DPT: 2-amino-4,5di(4'-hydroxyphenyl)-thiazole), the known renal cystogen nordihydroquaiaretic acid (NDGA) 2-amino-4(4'-hydroxyphenyl),5-phenyl-thiazole (4OH-DPT), and the known catalase inhibitor 3-amino-1,2,4-triazole; DPT did not inhibit catalase activity.

The N- and C-terminal boundaries of myelin basic protein determinants required for encephalitogenic and proliferative responses of Lewis rat T cells.

Highly purified synthetic peptides representing portions of the 68-86 sequence of guinea pig (GP) myelin basic protein (GPMBP) were used to define the N- and C-termini of encephalitogenic determinants that cause experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Each peptide was tested for: (a) induction of EAE, (b)in vitro potentiation of EAE transfer activity by GPMBP-sensitized lymph node cells (LNC), (c) in vitro proliferation of GPMBP-sensitized LNC, and (d) in vitro proliferation of a GPMBP-reactive line of EAE-inducing T cells. In these bioassays, the general rank order of potency was: GPMBP greater than or equal to GP68-86 greater than or equal to GP72-86 greater than [G84]GP68-86 greater than or equal to GP68-84 much greater than GP75-85 greater than or equal to GP75-84 = virtually no activity.