Attenuation of PGF2alpha release in ewes infused with the oxytocin antagonist L-368,899.

We have investigated the effects of systemic administration of the oxytocin antagonist (OTA) L-368,899 on luteolytic PGF(2alpha) release in ewes. In the first study, carried out in four ovariectomized ewes primed with progesterone to induce responsiveness to oxytocin, 3-h i.v. infusions of 3, 10 and 30 microg/kg/min OTA, carried out on days 12, 14, 16 and 18 in a Latin Square design, resulted in a significant attenuation of the oxytocin induced increase in PGFM concentration at all doses (OTA 139+/-8.3% of pre-oxytocin baseline; control 206.8+/-18.7%; P<0.005). In a further study, continuous infusion of cyclic ewes (n=6) with 10 microg/kg/min OTA from day 13 to day 17 of the cycle resulted in a reduction in both the frequency (OTA 1.0+/-0.4/ewe; control 2.2+/-0.2/ewe; P<0.05) and amplitude (OTA 31.8+/-11.0 pg/ml; control 68.8+/-10.4 pg/ml; P<0.05) of endogenous PGFM episodes compared to control ewes (n=5) measured during daily 8-h sampling windows on days 14-17. This reduction in PGFM concentrations was accompanied by a modest extension in the day of luteolysis (progesterone <0.5 ng/ml) to day 17.5+/-0.4 in the OTA treated group compared with day 16.4+/-0.5 in the control group (P=0.07). The results demonstrate that treatment with OTA caused a significant reduction in episodes of increased PGFM concentration during the period of luteolysis and may provide an approach by which to reduce early pregnancy failure.

Molecular cloning and pharmacological characterization of the canine B1 and B2 bradykinin receptors.

The dog is a valuable animal model in the study of the physiological role of both the B1 and B2 bradykinin receptors. To more thoroughly characterize the pharmacological properties of the canine kinin receptors we isolated the cDNA sequence encoding the B1 and B2 bradykinin receptor subtypes and overexpressed them in Chinese hamster ovary (CHO) cells. The cDNA sequence of the canine B1 bradykinin receptor encodes a protein comprised of 350 amino acids that is 76% identical to the human B1 bradykinin receptor. The cDNA sequence of the canine B2 bradykinin receptor encodes a protein of 392 amino acids that is 81% identical to the human B2 bradykinin receptor. The amino acid sequence of the canine B1 and B2 receptors are 35% identical. Pharmacological studies of the cloned receptors revealed that the agonist affinity of the dog B1 receptor is similar to the rodent B1 receptors, and differs from the human form in that there is no preference for the presence of the N-terminal Lys residue of [des-Arg10]Lys-bradykinin. Significantly, the B1 receptor antagonist [des-Arg9,Leu8]BK behaves as partial agonist on the cloned dog B1 receptor. The dog B2 receptor exhibits the 'classical' pharmacological properties of this receptor subtype.

In vitro studies on L-771,688 (SNAP 6383), a new potent and selective alpha1A-adrenoceptor antagonist.

L-771,688 (SNAP 6383, methyl(4S)-4-(3, 4-difluorophenyl)-6-[(methyloxy)methyl]-2-oxo-3-[(¿3-[4-(2-pyridin yl)-1-piperidinyl]propyl¿amino)carbonyl]-1,2,3, 4-tetrahydro-5-pyrimidine carboxylate) had high affinity (Ki less than or = 1 nM) for [3H]prazosin binding to cloned human, rat and dog alpha1A-adrenoceptors and high selectivity (>500-fold) over alpha1B and alpha1D-adrenoceptors. [3H]Prazosin / (+/-)-beta-[125I]-4-hydroxy-phenyl)-ethyl-aminomethylteralone ([125I]HEAT) binding studies in human and animal tissues known to contain alpha1A and non-alpha1A-adrenoceptors further demonstrated the potency and alpha1A-subtype selectivity of L-771,688. [3H]L-771,688 binding studies at the cloned human alpha1A-adrenoceptors and in rat tissues indicated that specific [3H]L-771,688 binding was saturable and of high affinity (Kd=43-90 pM) and represented binding to the pharmacologically relevant alpha1A-adrenoceptors. L-771,688 antagonized norepinephrine-induced inositol-phosphate responses in cloned human alpha1A-adrenoceptors, as well as phenylephrine or A-61603 (N-[5-4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7, 8-terahydro-naphthlen-1-yl] methanesulfonamide hydrobromide) induced contraction in isolated rat, dog and human prostate, human and monkey bladder neck and rat caudal artery with apparent Kb values of 0.02-0.28 nM. In contrast, the contraction of rat aorta induced by norepinephrine was resistant to L-771,688. These data indicate that L-771,688 is a highly selective alpha1A-adrenoceptor antagonist.

Selective alpha1a adrenergic receptor antagonists based on 4-aryl-3,4-dihydropyridine-2-ones.

A series of alpha1a receptor antagonists derived from a 4-aryl-3,4-dihydropyridine-2-one heterocycle is disclosed. Potency in the low nanomolar to picomolar range along with high selectivity was obtained. In vivo efficacy in a prostate contraction model in rats was observed with a few derivatives.

Pharmacological properties of J-104132 (L-753,037), a potent, orally active, mixed ETA/ETB endothelin receptor antagonist.

J-104132 [(+)-(5S,6R, 7R)-2-butyl-7-[2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3, 4-methylenedioxyphenyl)cyclopenteno[1,2-b]pyridine-6-carboxylic; also referred to as L-753,037] is a potent, selective inhibitor of ETA and ETB endothelin (ET) receptors (e.g., Ki: cloned human ETA = 0.034 nM; cloned human ETB = 0.104 nM). In both ligand-binding and isolated tissue preparation protocols, the inhibition of ET receptors with J-104132 is reversible and competitive. In vitro, J-104132 is a potent antagonist of ET-1-induced accumulation of [3H]inositol phosphates in Chinese hamster ovary cells stably expressing cloned human ETA receptors (IC50 = 0.059 nM), ET-1-induced contractions in rabbit iliac artery (pA2 = 9.70) and of BQ-3020-induced contractions in pulmonary artery (pA2 = 10.14). J-104132 is selective for ET receptors because it had no effect on contractions elicited by norepinephrine or KCl in the vascular preparations. The in vivo potency of J-104132 was assessed using challenges with exogenous ET-1. In conscious mice, 5 nmol/kg i.v. ET-1 causes death. Pretreatment with J-104132 prevents the lethal response to ET-1 when administered i.v. (ED50 = 0.045 mg/kg) or p.o. in fed animals (ED50 = 0.35 mg/kg). In conscious, normotensive rats, pressor responses to 0.5 nmol/kg i.v. ET-1 are inhibited by J-104132 after i.v. (0.1 mg/kg) or p.o. (1 mg/kg) administration. In anesthetized dogs, ET-1 was administered directly into the renal artery or brachial artery to generate dose-response (blood flow) curves, and the inhibitory potency of J-104132 (i.v. infusion) was quantified. J-104132 produced greater than 10-fold shifts in the ET-1 dose-response curves at 0.03 mg/kg/h (renal) and 0.3 mg/kg/h (brachial). Oral bioavailability of J-104132 in rats was approximately 40%. These studies indicate that J-104132 is a selective, potent, orally active antagonist of both ETA and ETB receptors and is an excellent pharmacological tool to explore the therapeutic use of a mixed ETA/ETB receptor antagonist.

Nonpeptide oxytocin antagonists: analogs of L-371,257 with improved potency.

Structure-activity studies on the oxytocin antagonist 1 (L-371,257; Ki = 9.3 nM) have led to the identification of a related series of compounds containing an ortho-trifluoroethoxyphenylacetyl core which are orally bioavailable and have significantly improved potency in vitro and in vivo, e.g., compound 8 (L-374,943; Ki = 1.4 nM).

Development of orally active oxytocin antagonists: studies on 1-(1-[4-[1-(2-methyl-1-oxidopyridin-3-ylmethyl)piperidin-4-yloxy]-2- methoxybenzoyl]piperidin-4-yl)-1,4-dihydrobenz[d][1,3]oxazin-2-one (L-372,662) and related pyridines.

The previously reported oxytocin antagonist L-371,257 (2) has been modified at its acetylpiperidine terminus to incorporate various pyridine N-oxide groups. This modification has led to the identification of compounds with improved pharmacokinetics and excellent oral bioavailability. The pyridine N-oxide series is exemplified by L-372,662 (30), which possessed good potency in vitro (Ki = 4.1 nM, cloned human oxytocin receptor) and in vivo (intravenous AD50 = 0.71 mg/kg in the rat), excellent oral bioavailability (90% in the rat, 96% in the dog), good aqueous solubility (>8.5 mg/mL at pH 5.2) which should facilitate formulation for iv administration, and excellent selectivity against the human arginine vasopressin receptors. Incorporation of a 5-fluoro substituent on the central benzoyl ring of this class of oxytocin antagonists enhanced in vitro and in vivo potency but was detrimental to the pharmacokinetic profiles of these compounds. Although lipophilic substitution around the pyridine ring of compound 30 gave higher affinity in vitro, such substituents were a metabolic liability and caused shortfalls in vivo. Two approaches to prevent this metabolism, addition of a cyclic constraint and incorporation of trifluoromethyl groups, were examined. The former approach was ineffective because of metabolic hydroxylation on the constrained ring system, whereas the latter showed improvement in plasma pharmacokinetics in some cases.

Cloning and expression of the rhesus monkey oxytocin receptor.

The oxytocin receptor belongs to the family of G-protein-coupled receptors (GPCRs) characterized by seven transmembrane spanning domains and mediates numerous neurotransmitter and hormonal functions. The cloning of this receptor was initiated to validate the use of the rhesus monkey (Macaca mulatta) as a viable animal model for therapeutic development of oxytocin receptor antagonists by ruling out potential species variations that are sometimes present among GPCRs. The rhesus monkey oxytocin receptor was cloned by the polymerase chain reaction (PCR) and expressed transiently in 293/EBNA cells. The cDNA encodes a protein of 389 amino acids and is highly homologous to that from other species, especially the human receptor which exhibits 97% identity to the rhesus protein. The cloned receptor shows a very similar pharmacological profile to the human oxytocin receptor for a variety of agonists and antagonists from various structural classes. These results substantiate the validity of the rhesus monkey as a useful model for the evaluation of human therapeutics.

Nonpeptide oxytocin antagonists: potent, orally bioavailable analogs of L-371,257 containing a 1-R-(pyridyl)ethyl ether terminus.

Structure-activity studies on the oxytocin antagonist 1 (L-371,257) have identified a new series of high affinity, receptor-selective OT antagonists in which the N-acetyl-4-piperidinyl ether terminus in 1 has been replaced with a 1-(aryl)ethoxy group.

Progress in the development of oxytocin antagonists for use in preterm labor.

There is currently a need for new therapeutic agents for treating preterm labor which could offer improved safety and efficacy beyond what has been achieved with the widely employed beta-mimetics. In this regard, the longstanding hypothesis of oxytocin receptor blockade as representing a potentially more selective method of tocolysis has continued to gain support from results obtained in clinical studies with the peptide oxytocin antagonist, atosiban. Our laboratory has focussed on the identification of non-peptide oxytocin antagonists with properties suitable for both oral and intravenous administration. We have previously described the development of potent, camphor-based oxytocin antagonists, including L-368,899 which entered phase I human studies. More recently we have pursued a new structural class of oxytocin antagonists based on the 1-(N-benzoylpiperidin-4-yl)-4H-3,1-benzoxazin-2(1H)-one template. L-372,662 is a new member of this structural class and in our preclinical assays possesses an attractive overall profile from the standpoint of human oxytocin receptor affinity (Ki = 4.9 nM), human oxytocin vs. vasopressin receptor selectivity (> 500-fold), potency as an antagonist of oxytocin-induced uterine contractions in late gestation pregnant rhesus monkeys (AD50 = 36 micrograms/kg), oral bioavailability (F = 90% in dogs), and aqueous solubility (10 mg/mL).

Pharmacology of L-744,453, a novel nonpeptidyl endothelin antagonist.

L-744,453 ((+/-)3-[4-(1-carboxy-1-(3,4-methylenedioxyphenyl)methoxy)-3,5-diprop ylphenyl methyl]-3H-imidazo[4,5-c]pyridine) is an endothelin (ET) receptor antagonist from a new structural class, the dipropyl-alpha-phenoxyphenylacetic acid derivatives. L-744,453 competitively and reversibly inhibits [125I]-ET-1 binding to Chinese Hamster Ovary cells expressing cloned human ET receptors (K(i)s: hET(A)=4.3 nM; hET(B)=232 nM), and is selective for endothelin receptors compared to other peptide receptors. It is an antagonist of ET-1 stimulated phosphatidyl inositol hydrolysis in rat uterine slices (IC50=220 nM) and exhibits no agonist activity. This compound also inhibits ET-1 stimulated contraction of rat aortic rings with a K(b) value of 50 nM. L-744,453 protects against ET-1 induced lethality in mice after i.v. (AD50=13 mg/kg i.v.) or oral administration. This compound also antagonizes ET-1 induced increases in diastolic blood pressure in conscious normotensive rats (AD50=0.67 mg/kg i.v.) and anesthetized ferrets (AD50=1.6 mg/kg i.v.). L-744,453 is a potent, selective, orally active endothelin antagonist which may be useful in elucidating the role of endothelin in normal and pathophysiological states.

Pharmacology of L-754,142, a highly potent, orally active, nonpeptidyl endothelin antagonist.

L-754,142, (-)-N-(4-iso-propylbenzenesulfonyl)-alpha-(4-carboxyl-2-n-propy lphenoxy)-3,4- methylenedioxyphenylacetamide, is a potent nonpeptidyl endothelin antagonist (e.g., Ki: cloned human ETA = 0.062 nM: cloned human ETB = 2.25 nM), with high specificity for endothelin receptors. In vitro, L-754,142 is a potent antagonist of ET-1-induced phosphatidyl inositol hydrolysis in Chinese hamster ovary cells expressing cloned human endothelin receptors (IC50: hETA = 0.35 nM; hETB = 26 nM) and of ET-1 induced contractions in rabbit iliac artery rings (pA2 = 7.74) and rat aortic rings (pA2 = 8.7). In vivo, L-754,142 is a potent and specific antagonist of exogenously administered ET-1 or big ET-1, L-754,142 fully protects against ET-1-induced lethality in mice (AD50 = 0.26 mg/kg i.v.). The pressor response to big ET-1 in the anesthetized ferret is blocked by this compound with an ED50 value of 0.019 mg/kg i.v. L-754,142 also blocks the pressor response to big ET-1 in the conscious rat with ED50 values of 0.30 mg/kg i.v. and 0.56 mg/kg p.o. The duration of action of L-754,142 in this rat model is more than 12 hr after an oral dose of 3 mg/kg. In summary, L-754,142 is a potent, orally active ET antagonist with a long duration of action in several in vivo models.

The role of drug metabolism in drug discovery: a case study in the selection of an oxytocin receptor antagonist for development.

Drug discovery is a process involving multiple disciplines and interests. During the research phase of drug discovery, usually a large number of compounds are evaluated for biological activity and toxicological potential in animal species. Various types of problems with respect to pharmacodynamics, pharmacokinetics, and toxicity are commonly encountered at this stage. Drug metabolism, as a discipline participating in a drug discovery team, can play an important role in identifying factors underlying the problems, facilitate the optimal selection of compounds for further development, provide information on metabolites for possible improvement in drug design, and contribute to the identification of the appropriate animal species for subsequent toxicity testing. During the process of evaluating oxytocin receptor antagonists for further development for treatment of preterm labor, in vivo and in vitro drug metabolism studies conducted in rats, dogs, and monkeys contributed to the selection of L-368,899 as the development candidate on the basis of pharmacokinetic and metabolism observations. The presence of active N-demethylated metabolites of two other equipotent compounds in rats and dogs was found to be the major factor responsible for the discrepancy between oral bioavailability and efficacies observed for these 2 compounds. For L-368,899, a compound that demonstrated 20-40% oral bioavailability in rats, dogs, and chimpanzees, extensive first-pass metabolism rather than absorption was determined as the major factor responsible for the poor bioavailability (< 1%) in rhesus monkeys. In vitro metabolism studies with hepatic microsomes from rats, dogs, monkeys, and humans substantiated the conclusion that the rate of hepatic metabolism of L-368,899 in monkeys is faster than in the other species.

Characterization of the human oxytocin receptor stably expressed in 293 human embryonic kidney cells.

The human oxytocin (OT) receptor was stably expressed in 293 embryonic kidney cells (293/OTR), characterized pharmacologically and compared to human uterine myometrial receptors. The cloned receptor is expressed at a reasonably high density (0.82 fmole/microgram protein) and exhibits high affinity for [3H]OT (Kd = 0.32nM), similar to the value found in human myometrial tissue. The rank-order of potency for various antagonist and agonist ligands from several structural classes is also similar between the cloned and native receptor, as seen in a comparison of their inhibitory constants for [3H]OT binding. Agonist affinity at the cloned OT receptor is decreased by guanine nucleotide analogs, demonstrating functional G-protein-coupling. The OT receptor in 293 cells, like in human myometrium, is also coupled to the inositol phosphate pathway. In 293/OTR cells, OT stimulates inositol phosphate accumulation with an EC50 = 4.1 nM, an effect blocked by a potent and selective OT antagonist, L-366,948. Additionally, the cloned receptor in 293 cells desensitizes to high concentrations of OT, similar to the desensitization in myometrial tissue and also described for several other G-protein-coupled receptors. These results illustrate the utility of the 293 cell line for expressing human OT receptors in an environment quite comparable to the native myometrial tissue.

Progress in the development of oxytocin antagonists for use in preterm labor.

From a targeted screening effort and medicinal chemistry program, L-368,899 was selected as the first orally-active oxytocin (OT) antagonist to enter clinical trials. In animal studies, L-368,899 was shown to be a potent and selective OT antagonist and was orally bioavailable in rats, dogs and chimpanzees. L-368,899 was further shown to be a potent OT antagonist in pregnant rhesus and to inhibit spontaneous nocturnal uterine contractions. In Phase I human studies, L-368,899 was generally well-tolerated given intravenously and showed significant plasma levels after oral administration. In addition, L-368,899 blocked OT-stimulated uterine activity in postpartum women with a potency similar to that in the pregnant rhesus monkey. More recently, another structural series has been pursued, represented by L-371,257 [1-(1-(4-(N-acetyl-4-piperidinyloxy)-2-methoxybenzoyl)pip eridin-4-yl)- 1,2-dihydro-4(H)-3,1-benzoxazin-2-one]. L-371,257 exhibits high affinity (Ki, 4.6 nM) for human uterine OT receptors with high selectivity vs. human vasopressin receptors. In rat tissues in vitro, L-371,257 is a potent and competitive OT antagonist (pA2, 8.4) and, in vivo, blocks OT-stimulated uterine activity given both i.v. and intraduodenally. L-371,257 highlights the promise of this novel structural class.

Conformationally constrained o-tolylpiperazine camphorsulfonamide oxytocin antagonists. Structural modifications that provide high receptor affinity and suggest a bioactive conformation.

A series of new o-tolylpiperazine camphorsulfonamide OT antagonists is described. Analogs containing conformationally constrained 1-acylamino-2-propyl substituents at the camphor C2 endo position exhibit high affinity for OT and AVP-V1a receptors or high affinity and selectivity for OT receptors, depending on functionalities present in the acyl group. Determination of the preferred conformation of potency-enhancing 1-acylamino-2-propyl substituents using molecular mechanics energy calculations and X-ray crystallography, along with topological similarities to a conformationally constrained cyclic hexapeptide OT antagonist, suggests a receptor-bound conformation for this series of non-peptide OT antagonists.