The renin-angiotensin system plays a key role in the regulation of blood pressure, fluid and electrolyte balance, and blood volume. In the initial step of the biochemical cascade, the proteolytic enzyme renin catalyzes the breakdown of the high-molecular-weight peptide, angiotensinogen, to angiotensin I. The report of the benzylimidazole 1 sparked our interest in the application of an overlay hypothesis strategy to aid in the design of nonpeptide angiotensin II antagonists. Careful examination of existing AII peptide structure-activity relationship helped refine our overlay hypothesis strategy and focus our synthetic efforts. Thus, a Tyr-Phe overlay hypothesis became the focus of our molecular modeling efforts. According to this overlay hypothesis, the small molecule covered the Tyr aromatic ring, Ile sidechain, and the Phe carboxylate of AII but failed to reach other regions of the octapeptide known to be important for affinity, such as the Arg and His sidechains and Phe aromatic ring.

Burke et al. Current Eye Research; Sep. 5 (9): 665-76 (1986). Mittag. Annals of Ophthalmology 1983; 15(3): 201-202. Renal Effects of Selective Alpha-1 and Alpha-2 Adre noceptor Agonists in Conscious, Normotensive Rats; Miklos Gellai & Robert R. Ruffolo, Jr. Selective Alpha-2. Adrenoceptor Agonists Alter Fluid and Electrolyte Transport in Mammalian Small Intes tine; Joseph D. Fondacaro, David Kolpak & Gerald P. McCafferty. Ocular Effects of a Relatively Selective Alpha-2 Ago nist (UK-14, 304-18) in Cats, Rabbits and Monkeys; J. A. Burke and D. E. Potter. Alpha-2 Adrenergic Modulation of Norepinephrine Secretion in the Perfused Rabbit Iris-Ciliary Body; James E. Jumblatt, John G. H. Liu & Ginnie T. North. Ocular Effects of Selective Alpha-Adrenergic Agents: A New Drug Paradox Tom Mittag, Ph.D. Mechanism of Alpha-2-Adrenoceptor Agonist-In duced Diuresis Miklos Gellai & Richard M. Edwards. Clonidine and Some Bridge Analogues; Cardiovascular Effects and Nuclear Magnetic Resonance Data (H/C); Pieter B.M.W.M. Timmermans & Pieter A. van Zwi

A series of 3-urea-1-(phenylmethyl)-pyridones was discovered as novel EP(3) antagonists via high-throughput screening and subsequent optimization. The synthesis, structure-activity relationships, and optimization of the initial hit that resulted in potent and selective EP(3) receptor antagonists such as 11g are described.

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.

The excitatory roles of EP3 receptors at the peripheral afferent nerve innervating the rat urinary bladder have been evaluated by using the selective EP3 antagonist (2E)-3-{1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1H-indol-7-yl}-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide (DG- 041). The bladder rhythmic contraction model and a bladder pain model measuring the visceromotor reflex (VMR) to urinary bladder distension (UBD) have been used to evaluate DG-041 in female rats. In addition, male rats [spontaneously hypertensive rat (SHR), Wistar-Kyoto (WKY), and Sprague-Dawley (SD)] were anesthetized with pentobarbital sodium, and primary afferent fibers in the L-6 dorsal root were isolated for recording the inhibitory response to UBD following intravenous injection of DG-041. Intravenous injection of DG- 041 (10 mg/kg), a peripherally restricted EP3 receptor antagonist, significantly reduced the frequency of bladder rhythmic contraction and inhibited the VMR response to bladder distension. The magnitude of reduction of the VMR response was not different in the different strains of rats ( SD, SHR, and WKY). Furthermore, quantitative characterization of the mechanosensitive properties of bladder afferent nerves in SHR, WKY, and SD rats did not show the SHR to be supersensitive to bladder distension. DG-041 selectively attenuated responses of mechanosensitive afferent nerves to UBD, with strong suppression on the slow-conducting, high-threshold afferent fibers, with equivalent activity in the three strains. We conclude that sensitization of afferent nerve activity was not one of the mechanisms of bladder hypersensitivity in SHR. EP3 receptors are involved in the regulation of bladder micturition and bladder nociception at the peripheral level.

Prostaglandin EP3 receptors in the central nervous system (CNS) may exert an excitatory effect on urinary bladder function via modulation of bladder afferent pathways. We have studied this action, using two EP3 antagonists, (2E)-3-{1-[(2,4-dichlorophenyl) methyl]-5-fluoro-3-methyl- 1H-indol-7-yl}-N-[(4,5-dichloro-2-thienyl) sulfonyl]-2-propenamide (DG041) and (2E)-N-{[5-bromo-2-(methyloxy) phenyl] sulfonyl}-3-[2-(2-naphthalenylmethyl) phenyl]-2-propenamide (CM9). DG041 and CM9 were proven to be selective EP3 antagonists with radioligand binding and functional fluorescent imaging plate reader (FLIPR) assays. Their effects on volume-induced rhythmic bladder contraction and the visceromotor reflex (VMR) response to urinary bladder distension (UBD) were evaluated in female rats after intrathecal or intracerebroventricular administration. Both DG041 and CM9 showed a high affinity for EP3 receptors at subnanomolar concentrations without significant selectivity for any splice variants. At the human EP3C receptor, both inhibited calcium influx produced by the nonselective agonist PGE(2). After intrathecal or intracerebroventricular administration both CM9 and DG041 dose-dependently reduced the frequency, but not the amplitude, of the bladder rhythmic contraction. With intrathecal administration DG041 and CM9 produced a long-lasting and robust inhibition on the VMR response to UBD, whereas with intracerebroventricular injection both compounds elicited only a transient reduction of the VMR response to bladder distension. These data support the concept that EP3 receptors are involved in bladder micturition at supraspinal and spinal centers and in bladder nociception at the spinal cord. A centrally acting EP3 receptor antagonist may be useful in the control of detrusor overactivity and/or pain associated with bladder disorders.

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.

Nonsteroidal anti-inflammatory cyclooxygenase inhibitors that function to reduce prostaglandin E2 (PGE2) production have been widely reported as effective agents in models of urinary bladder overactivity. We therefore investigated a potential role for the PGE2 receptor, EP3, in urinary bladder function by performing conscious, freely moving cystometry on EP3 receptor knockout (KO) mice. EP3 KO mice demonstrated an enhanced bladder capacity compared with wild-type (WT) mice ( approximately 185% of WT) under control conditions, based on larger voided and infused bladder volumes. Infusion of the EP3 receptor agonist GR63799X into the bladder of WT mice reduced the bladder capacity. This was ineffective in EP3 KO mice that demonstrated a time-dependent increase in bladder capacity with GR63799X, an effect similar to that observed with vehicle in both genotypes. In addition, infusion of PGE2 into WT mice induced bladder overactivity, an effect that was significantly blunted in the EP3 KO mice. The data reported here provide the first evidence supporting a functional role for EP3 receptors in normal urinary bladder function and implicate EP3 as a contributor to bladder overactivity during pathological conditions of enhanced PGE2 production, as reported previously in overactive bladder patients.

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.

Non-selective cyclooxygenase (COX) inhibitors exert effects on lower urinary tract function in several species. The exact contributions of COX-1 and COX-2 isozymes have not been studied much. The present studies investigated the effects of non- and selective COX inhibitors on bladder irritation in the cat.Chloralose-anaesthetised female cats were catheterised through the bladder dome for cystometric evaluation of bladder responses to intravesical infusion of saline or acetic acid. Bladder capacity, voiding efficiency, threshold pressure, and reflex-evoked bladder contraction amplitude and duration were measured. The cat COX selectivity of the doses of inhibitors examined was determined using an in vitro whole-blood assay and analysis of plasma levels. Pretreatment with indomethacin or ketoprofen (non-selective COX inhibitors; 0.3 mg kg(-1) i.v.) inhibited acetic acid-evoked irritation (characterised by a decrease in bladder capacity in vehicle pretreated animals). FR-122047 (selective COX-1 inhibitor), NS-398 and nimesulide (selective COX-2 inhibitors; 1 and 3 mg kg(-1) i.v.) had no effects on bladder irritation. Analysis of plasma levels of the doses examined and determination of COX-1 and COX-2 inhibition in cat whole blood confirmed the reported selectivity of these compounds in this species. The present studies suggest that dual COX inhibition is required to attenuate acetic acid-evoked bladder irritation in the cat.

Urotensin-II (U-II), a ligand for the G-protein-coupled receptor UT, has been characterized as the most potent mammalian vasoconstrictor identified to date. Although circulating levels of U-II are altered in lower species (e.g., fish) upon exposure to hypo-osmotic stress, little is known about the actions of this cyclic undecapeptide within the kidney, an organ that plays a pivotal role in the control of cardiovascular homeostasis, influencing both cardiac preload (plasma volume) and after load (peripheral resistance). The present study reports the identification of specific, high affinity [125I]hU-II binding sites in Sprague-Dawley rat kidney outer medulla by autoradiography and also through membrane radioligand binding (Kd 1.9 +/- 0.9 nM and Bmax 408 +/- 47 amol mm(-2) and Kd 1.4 +/- 0.3 nM and Bmax 51.3 +/- 7.8 fmol mg(-1) protein, respectively). Differences were observed in the binding characteristics within rat strains. Compared to the Sprague-Dawley, Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rat kidney outer medulla displayed low density < 20 fmol mg(-1) protein and low affinity (> 1 microM) [125I]hU-II binding sites. Thus, the relative contribution of specific U-II binding sites to the physiological actions of U-II in the control of cardiorenal homeostasis is worthy of further investigation.

Hyperphosphatemia directly or indirectly contributes to the progression of chronic renal disease and is an important factor in the development of secondary hyperparathyroidism and uremic bone disease. New therapeutic advances include the development of calcium-free intestinal phosphate binders, calcimimetics to control parathyroid hormone secretion and non-calcemic vitamin D analogs.

Previous studies have shown that basolateral ATP inhibits vasopressin action in the renal collecting tubule. Although there is evidence for an apical P2Y2 receptor in this tubule segment, it is not known whether apical ATP has similar effects. In the rat inner medullary collecting duct basolateral, but not apical, ATP (0.1-100 microM) reversibly inhibited vasopressin-induced increases in water permeability with an IC50 of 1.09 microM. Basolateral UTP, but not ADP, alpha,beta-methylene-ATP or 2-methylthio-ATP also inhibited vasopressin action. It is concluded that basolateral but not apical P2Y2 receptors inhibit vasopressin action in the collecting duct.

reductase inhibitor 172, 193 Alkylating agents 129

The function of the kidney has been a focus of study since time immemorial. Considerable work is still being conducted to augment better understanding of the dominant role of the kidney in the regulation of both blood pressure and the internal millieu. Much work remains to be performed to comprehend fully the processes and mechanisms involved in both renal function and dysfunction. This chapter aims to provide some general information on one particular aspect of kidney function, the renal circulation. It describes some of the different methods used to study the renal circulation and provides a synopsis of some factors which affect blood flow through the kidney. Where appropriate, information on the pathophysiological role these factors may have in renal disease is also provided.