Pyridyl-2,5-diketopiperazines as potent, selective, and orally bioavailable oxytocin antagonists: synthesis, pharmacokinetics, and in vivo potency.

A six-stage stereoselective synthesis of indanyl-7-(3'-pyridyl)-(3R,6R,7R)-2,5-diketopiperazines oxytocin antagonists from indene is described. SAR studies involving mono- and disubstitution in the 3'-pyridyl ring and variation of the 3-isobutyl group gave potent compounds (pK(i) > 9.0) with good aqueous solubility. Evaluation of the pharmacokinetic profile in the rat, dog, and cynomolgus monkey of those derivatives with low cynomolgus monkey and human intrinsic clearance gave 2',6'-dimethyl-3'-pyridyl R-sec-butyl morpholine amide Epelsiban (69), a highly potent oxytocin antagonist (pK(i) = 9.9) with >31000-fold selectivity over all three human vasopressin receptors hV1aR, hV2R, and hV1bR, with no significant P450 inhibition. Epelsiban has low levels of intrinsic clearance against the microsomes of four species, good bioavailability (55%) and comparable potency to atosiban in the rat, but is 100-fold more potent than the latter in vitro and was negative in the genotoxicity screens with a satisfactory oral safety profile in female rats.

Discovery of novel aminothiadiazole amides as selective EP(3) receptor antagonists.

This Letter discloses a series of 2-aminothiadiazole amides as selective EP(3) receptor antagonists. SAR optimization resulted in compounds with excellent functional activity in vitro. In addition, efforts to optimize DMPK properties in the rat are discussed. These efforts have resulted in the identification of potent, selective EP(3) receptor antagonists with excellent DMPK properties suitable for in vivo studies.

Comparison of non-digitalis binding properties of digoxin-specific Fabs using direct binding methods.

Digibind and DigiFab are commercial formulations of polyclonal, ovine, digoxin-specific Fabs in clinical use for treatment of digoxin intoxication. Of interest for extending its use to other clinical indications, Digibind has also been reported to neutralize the effect of endogenous digoxin-like molecules, including ouabain, that are linked to clinical disorders ranging from preeclampsia to congestive heart failure. Although Digibind and DigiFab are equivalent in their digoxin-binding activity, the antigens used to produce these Fabs are different. We therefore explored, using native (3)H-digoxin and (3)H-ouabain in four different types of solution-phase binding methods, whether they might exhibit different profiles with respect to ouabain and other digoxin-like factors. Consistent with previous results, both Fab preparations bound digoxin with the same affinities and capacities. However, (3)H-ouabain was found to bind with high affinity only to Fab sub-populations present in both products. Interestingly, this sub-population was twice as large for Digibind compared to DigiFab. Competition experiments also showed differences in specificity within Fab sub-populations. Therefore, the equivalence in digoxin-binding activity of the two Fab preparations does not extend to ouabain-binding capacity and Fab specificity, with implications for clinical differentiation between the preparations in treatment of disorders related to control of non-digoxin cardenolides. The existence of a small but perhaps clinically relevant sub-population of antibodies was detected using specific radioligands. This sub-population could not have been detected nor quantified using standard cross-reactivity in an ELISA assay.

The discovery of GSK221149A: a potent and selective oxytocin antagonist.

Optimisation of a series of oxazole diketopiperazines has led to the discovery of a very potent and selective oxytocin antagonist GSK221149A. GSK221149A has been shown to inhibit oxytocin-induced uterine contractions in the anaesthetised rat.

Use of a novel and highly selective oxytocin receptor antagonist to characterize uterine contractions in the rat.

Spontaneous and induced uterine contractions in the rat were found to be inhibited by a novel and selective oxytocin receptor antagonist GSK221149A (3R,6R)-3-Indan-2-yl-1-[(1R)-1-(2-methyl-1,3-oxazol-4-yl)-2-morpholin-4-yl-2-oxoethyl]-6-[(1S)-1-methylpropyl]-2,5-piperazinedione. GSK221149A displayed nanomolar affinity (K(i) = 0.65 nM) for human recombinant oxytocin receptors with >1,400-fold selectivity over human V1a, V1b, and V2 receptors. GSK221149A had similar affinity (K(i) = 4.1 nM) and selectivity for native oxytocin receptors from rat and produced a functional, competitive block of oxytocin-induced contractions in isolated rat myometrial strips with a pA(2) value of 8.18. Intravenous administration of GSK221149A produced a dose-dependent decrease in oxytocin-induced uterine contractions in anesthetized rats with an ID(50) = 0.27 +/- 0.60 mg/kg (corresponding plasma concentrations were 88 ng/ml). Oral administration of GSK221149A (5 mg/kg) was effective in inhibiting oxytocin-induced uterine contractions after single and multiple (4-day) dosing. Spontaneous uterine contractions in late-term pregnant rats (19-21 days gestation) were significantly reduced by intravenous administration of 0.3 mg/kg of GSK221149A. These results provide further evidence that selective oxytocin receptor antagonism may offer an effective treatment for preterm labor.

Determination of conformational changes in the progesterone receptor using ELISA-like assays.

The conformation of proteins often influences their functional activity. The effect of progesterone receptor ligands on the C-terminal conformation of the progesterone receptor affects the recruitment of transcriptional cofactors. These conformations can be studied by differential sensitivity to proteolytic cleavage or immunoprecipitation with a conformation-specific antibody. This study describes an ELISA-like method using conformation-specific antibodies to the C-terminal or an area adjacent to the DNA binding region. Progesterone receptor ligands are shown to influence how the progesterone receptor interacts with these antibodies in a concentration dependent manner. This method allows for quick determination of the potency of agonists as well as mechanistic studies of antagonism. The conformation inducing activity of several standard agonist and antagonist compounds were compared to their binding affinity and ability to induce alkaline phosphatase in T47D cells. This method is useful for screening compounds for functional activity at the progesterone receptor and demonstrates that J867 induces an antagonist conformation of the progesterone receptor similar to the antagonist RU486.

Mechanism of vasopeptidase inhibitor-induced plasma extravasation: comparison of omapatrilat and the novel neutral endopeptidase 24.11/angiotensin-converting enzyme inhibitor GW796406.

We describe N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1H-pyrazol-1-yl)-L-phenylalanine (GW796406), a vasopeptidase inhibitor (VPI) that possessed approximately 3-fold selectivity for neutral endopeptidase 24.11 (NEP) versus angiotensin-converting enzyme (ACE) in in vitro assays using rat and human enzymes. In the same assays, omapatrilat, the most extensively studied VPI, displayed approximately 3-fold selectivity for ACE. The in vivo ACE and NEP inhibition profile and the liability of the compounds to increase plasma extravasation were compared at two (low and high) therapeutically equivalent intravenous doses in the rat. At the low dose, both agents inhibited ACE activity by approximately 85%. Consistent with their in vitro ACE/NEP selectivity, omapatrilat produced 49% inhibition, whereas GW796406 produced >95% inhibition of NEP. Neither compound increased plasma extravasation. When the low dose was administered to rats pretreated with the NEP inhibitor ecadotril to normalize NEP background to <5% of control, only omapatrilat significantly increased plasma extravasation. At the high dose, omapatrilat and GW796406 produced profound, nonselective inhibition of ACE (>90%) and NEP (>95%), and they significantly increased plasma extravasation. The activity of the agents as inhibitors of dipeptidylpeptidase IV (DPP IV) and aminopeptidase P (APP) was also investigated. Neither compound inhibited DPP IV. Interestingly, omapatrilat, but not GW796406, was a relatively potent inhibitor of APP (IC50 = 260 nM). We investigated whether APP inhibition increased the plasma extravasation liability of GW796406. The low dose of GW796406 administered with apstatin, an APP inhibitor, did not increase plasma extravasation. This finding inferred that APP inhibition is not involved in plasma extravasation in the rat and that APP inhibition does not explain the increased plasma extravasation produced by omapatrilat in NEP-inhibited rats.

Neuromedin U elicits cytokine release in murine Th2-type T cell clone D10.G4.1.

Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for (125)I-labeled NmU (K(D) 364 pM and B(max) 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca(2+) concentration (EC(50) 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.

Characterization of the neutralizing activity of digoxin-specific Fab toward ouabain-like steroids.

Digoxin-specific Fab (Digibind) is a mixture of antidigoxin Fab fragments prepared from sheep sera and is used as a treatment for digoxin poisoning. Digoxin-specific Fab has been shown to neutralize an endogenous Na+/K+ ATPase inhibitor (endogenous digoxin-like Na+/K+ ATPase regulatory factor; EDLF) in rats and humans and to lower blood pressure. Although the exact structure of EDLF is unknown, compounds identical to or structurally related to ouabain, bufalin, and marinobufagenin have been detected in mammalian plasma. In this study, some structural characteristics of EDLF were inferred from the ability of digoxin-specific Fab to neutralize the Na+/K+ ATPase inhibitory activity of several known cardenolides and bufodienolides. Additional structural information was obtained from [3H]ouabain binding and enzyme-linked immunosorbent assay experiments. Digoxin-specific Fab had the ability to interact to some extent with all of the cardenolides and bufodienolides tested. However, digoxin-specific Fab was more than 20-fold more potent in neutralizing ouabain and bufalin than marinobufagenin. The antihypertensive effect of digoxin-specific Fab seen in preeclampsia and animal models of hypertension may therefore be due to a molecule identical to or structurally similar to ouabain or bufalin.

The effect of acute angiotensin-converting enzyme and neutral endopeptidase 24.11 inhibition on plasma extravasation in the rat.

The effect of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) inhibition on microvascular plasma leakage (extravasation) was evaluated in a rat model. Progressive inhibition of ACE using captopril caused increased extravasation when lung ACE was inhibited by >55%. In contrast, the selective inhibition of renal NEP by >90% using ecadotril did not increase extravasation. In NEP-inhibited rats, extravasation produced by the ACE inhibitors captopril and lisinopril was markedly enhanced. The dual ACE and NEP inhibitor omapatrilat, at oral doses of 0.03, 0.1, and 0.3 mg/kg, selectively inhibited lung ACE by 19, 61, and 76%, respectively, and did not cause significant extravasation. Doses of 1 and 10 mg/kg omapatrilat, which produced >90% inhibition of ACE and also inhibited renal NEP by 54 and 78%, respectively, significantly increased extravasation. In this model, bradykinin and substance P produced extravasation that could be abolished by the bradykinin 2 (B2) receptor antagonist Hoe 140 (icatibant) or the neurokinin1 (NK1) antagonist CP99994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine], respectively. Bradykinin induced extravasation was also partially ( approximately 40%) inhibited by CP99994, indicating that a portion of the response involves B2 receptor-mediated release of substance P. In conclusion, this study is the first to relate the degree of ACE and/or NEP inhibition to extravasation liability in the rat model. Our data clearly demonstrate that ACE inhibitor-induced plasma extravasation is enhanced by concomitant inhibition of NEP. In addition, this study provides further evidence for the role for B2 and NK1 receptors in mediating plasma extravasation in the rat.

Effects of angiotensins II and IV on blood pressure, renal function, and PAI-1 expression in the heart and kidney of the rat.

The role of angiotensin II (AII) and angiotensin IV (AIV) as inducers of PAI-1 expression during hypertension was studied in vivo. A 2-week infusion of AII (300 ng/kg/min) via an osmotic pump increased systolic blood pressure (171 +/- 2 vs. 138 +/- 6 mm Hg), urinary protein excretion (32 +/- 6 vs. 14 +/- 2 mg/day), and renal (2.2 +/- 0.5 vs. 1.0 +/- 0.1) and cardiac (1.8 +/- 0.3 vs. 1.0 +/- 0.1) gene expression of plasminogen activator inhibitor 1 (PAI-1). AIV infusion did not affect any of the above with the exception of PAI-1 gene expression which was increased in the left ventricles (1.7 +/- 0.3 vs. 1.0 +/- 0.1). AII-infused rats displayed a decreased creatinine clearance (538 +/- 75 vs. 898 +/- 96 ml/min) and hypertrophic left ventricles (0.275 +/- 0.006 vs. 0.220 +/- 0.011 g/100 g). Our results demonstrate that AII but not AIV infusion is associated with increased renal PAI-1 gene expression.

The angiotensin type 1 receptor antagonist, eprosartan, attenuates the progression of renal disease in spontaneously hypertensive stroke-prone rats with accelerated hypertension.

The effects of the angiotensin type 1 (AT(1)) receptor antagonist, eprosartan, were studied in a model of severe, chronic hypertension. Treatment of male spontaneously hypertensive stroke prone rats (SHR-SP) fed a high-fat, high-salt diet with eprosartan (60 mg/kg/day i.p.) for 12 weeks resulted in a lowering of blood pressure (250 +/- 9 versus 284 +/- 8 mm Hg), renal expression of transforming growth factor-beta mRNA (1.5 +/- 0.2 versus 5.4 +/- 1.4) and the matrix components: plasminogen activator inhibitor-1 (5.2 +/- 1.4 versus 31.4 +/- 10.7), fibronectin (2.2 +/- 0.6 versus 8.2 +/- 2.2), collagen I-alpha 1 (5.6 +/- 2.0 versus 23.8 +/- 7.3), and collagen III (2.7 +/- 0.9 versus 7.6 +/- 2.1). Data were corrected for rpL32 mRNA expression and expressed relative to Wistar Kyoto (WKY) rats [=1.0]. Expression of fibronectin protein was also lowered by eprosartan (0.8 +/- 0.1 versus 1.9 +/- 0.5), relative to WKY rats. Eprosartan provided significant renoprotection to SHR-SP rats as measured by decreased proteinuria (22 +/- 2 versus 127 +/- 13 mg/day) and histological evidence of active renal damage (5 +/- 2 versus 195 +/- 6) and renal fibrosis (5.9 +/- 0.7 versus 16.4 +/- 1.9) in vehicle- versus eprosartan-treated rats, respectively. Our results demonstrated that AT(1) receptor blockade with eprosartan can reduce blood pressure and preserve renal structure and function in this model of severe, chronic hypertension. These effects were accompanied by a decreased renal expression of transforming growth factor-beta1, plasminogen activator inhibitor-1, and several other extracellular matrix proteins compared with vehicle-treated SHR-SP.