Piboserod (SB 207266), a selective 5-HT4 receptor antagonist, reduces serotonin potentiation of neurally-mediated contractile responses of human detrusor muscle.

The aim of this study is to evaluate the potency of piboserod (SB 207266), a selective 5-HT(4) receptor antagonist, at inhibiting the 5-HT(4)-mediated potentiating effect of serotonin (5-HT) on the neurally-mediated contractile responses of human detrusor strips to electrical field stimulations (EFS). Strips of human detrusor muscle were mounted in Krebs-HEPES buffer under a resting tension of 500 mg and EFS (20 Hz, 1 ms duration at 300 mA for 5 s) was applied continuously at 1 min intervals. After stabilization of the EFS-induced contractions, concentration-response curves to 5-HT (0.1 nM-100 microM) were constructed in the absence or presence of 1 or 100 nM of piboserod. The experiments were performed in the presence of methysergide (1 microM) and ondansetron (3 microM) to block 5HT(1)/5HT(2) and 5-HT(3) receptors, respectively. 5-HT potentiated the contractile responses to EFS of human bladder strips in a concentration-dependent manner, with a maximum mean of 60.0+/-19.9% of the basal EFS-evoked contractions. Piboserod did not modify the basal contractions but concentration-dependently antagonized the ability of 5-HT to enhance bladder strip contractions to EFS. In presence of 1 and 100 nM of piboserod, the maximal 5-HT-induced potentiations were reduced to 45.0+/-7.9 and 38.7+/-8.7%, respectively. A mean apparent antagonist dissociation constant value (K(B)) of 0.56+/-0.09 nM was determined. These data show the ability of piboserod to antagonize with high potency the enhancing properties of 5-HT on neurally-mediated contractions of isolated human bladder strips. Therefore, the 5-HT(4) receptor might represent an attractive pharmacological target for the treatment of overactive bladder.

Rabbit alpha2-adrenoceptors: both platelets and adipocytes have alpha2A-pharmacology.

The recombinant alpha2-adrenoceptors, designated as alpha2a and alpha2d, have highly similar amino acid sequences, but distinct pharmacological properties. It has been suggested that these two receptor subtypes are species orthologs, since the alpha2-adrenoceptors of a given species have pharmacological characteristics corresponding to either the alpha2a- (human, pig) or alpha2d- (rat, mouse, guinea pig, cow) adrenoceptor. Radioligand binding assays in rabbit adipocyte suggest alpha2D-adrenoceptor pharmacology. However, functional studies examining prejunctional alpha2-adrenoceptors in several tissues pharmacologically define the receptor of the rabbit as an alpha2A-adrenoceptor rather than an alpha2D-adrenoceptor. We characterized the alpha2-adrenoceptor of rabbit adipocyte and platelet, comparing the ability of norepinephrine and 13 adrenoceptor antagonists to inhibit the binding of [3H]RX821002 with the affinity of these drugs for the human alpha2a-adrenoceptor or the rat alpha2d-adrenoceptor. Pharmacological characteristics of the adipocyte and platelet receptor were very similar, with an excellent correlation between pK(i) values (r2 = 0.95, slope of regression = 1.01). Drug affinities for both platelet and adipocyte receptors correlated better with the alpha2a-adrenoceptor (r2 = 0.68-0.77) than with the alpha2d-adrenoceptor (r2 = 0.37-0.38). Despite the relatively low affinity of the rabbit adipocyte alpha2-adrenoceptor for yohimbine and rauwolscine, this receptor, as well as the platelet receptor, have alpha2A-adrenoceptor pharmacology. Subtle differences in the alpha2-adrenoceptor binding characteristics of these native rabbit tissues compared with the recombinant human alpha2a-adrenoceptor may result either from minor differences in the sequence of human and rabbit alpha2a-adrenoceptors or from differences in the environment to which native and recombinant receptors are exposed.

Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

Activity of N-(phenethyl)phenylethanolamines at beta(1) and beta(2) adrenoceptors: structural modifications can result in selectivity for either subtype.

A series of phenylethanolamines bearing a 2-[1-phenylpropyl] substituent on the nitrogen atom was evaluated in vitro for activity at beta(1)- and beta(2)-adrenoceptors. As previously observed, the presence of 3,4-dihydroxy substitution on phenylethanolamine is required for potent activation of both subtypes, whereas the 3,5-dihydroxy analog showed selectivity for the beta(2)-subtype. Replacement by a carboxyl group of the 4-hydroxyl group on the aralkyl nitrogen substituent produced only a small reduction in beta(1) potency (5-fold), whereas beta(2) potency was reduced by more than 100-fold. Hence this structural class includes agonists having either a beta(1), nonselective beta(1)/beta(2) or beta(2) selectivity profile.

Cloning, expression, and pharmacological characterization of a novel human histamine receptor.

Using a genomics-based reverse pharmacological approach for screening orphan G-protein coupled receptors, we have identified and cloned a novel high-affinity histamine receptor. This receptor, termed AXOR35, is most closely related to the H3 histamine receptor, sharing 37% protein sequence identity. A multiple responsive element/cyclic AMP-responsive element-luciferase reporter assay was used to identify histamine as a ligand for AXOR35. When transfected into human embryonic kidney 293 cells, the AXOR35 receptor showed a strong, dose-dependent calcium mobilization response to histamine and H3 receptor agonists including imetit and immepip. Radioligand binding confirmed that the AXOR35 receptor was a high-affinity histamine receptor. The pharmacology of the AXOR35 receptor was found to closely resemble that of the H3 receptor; the major difference was that (R)-alpha-methylhistamine was a low potency agonist of the AXOR35 receptor. Thioperamide is an antagonist at AXOR 35. Expression of AXOR35 mRNA in human tissues is highest in peripheral blood mononuclear cells and in tissues likely to contain high concentrations of blood cells, such as bone marrow and lung. In situ hybridization analysis of a wide survey of mouse tissues showed that mouse AXOR35 mRNA is selectively expressed in hippocampus. The identification and localization of this new histamine receptor will expand our understanding of the physiological and pathological roles of histamine and may provide additional opportunities for pharmacological modification of these actions.

Role of neurokinin receptors in the behavioral effect of intravesical antigen infusion in guinea pig bladder.

PURPOSE: To characterize a guinea pig behavior model of bladder pain due to intravesical antigen infusion and to determine the role of neurokinin receptor subtypes in mediating this behavior. MATERIALS AND METHODS: The influence of subtype-selective neurokinin receptor antagonists on increased abdominal licking behavior in response to intravesical antigen infusion in guinea pigs immunized with ovalbumin (OA) was determined. RESULTS: Intravesical OA infusion for 30 minutes induced a significantly greater frequency (about 3-fold) of abdominal licking behavior than during either the 30 minutes pre-challenge or post challenge saline infusions. Treatment with IP capsaicin 7 to 10 days before OA challenge abolished the intravesical antigen-induced behavior. IP injection of the NK1 receptor antagonist CP 99994 (10 mg./kg. or 30 mg./kg.), 30 minutes pretreatment, inhibited the increase in the average number of abdominal licks during antigen infusion. The 30 mg./kg., but not the 10 mg./kg. dose increased the percent of animals showing antinociceptive activity (defined as 4 or less abdominal licks during the antigen infusion). The NK2 receptor antagonist SR 48968 reduced the antigen-induced abdominal licking behavior at IP doses of 3 and 10 mg./kg. but was ineffective at 1 mg./kg. The NK3 receptor antagonist SB 235375 (30 mg./kg., IP) did not reduce this behavior. CONCLUSIONS: These results suggest a role for activation of NK1 and NK2, but not NK3 receptors, by tachykinins released from capsaicin-sensitive nerves, in the increased abdominal licking behavior response of guinea pigs to intravesical antigen infusion.

Adrenoceptor subclassification: an approach to improved cardiovascular therapeutics.

The subdivision of alpha adrenoceptors into the alpha 1 and alpha 2 classes was the impetus for the design of the selective alpha 1-adrenoceptor antagonists, which remain useful antihypertensives. alpha 2-Adrenoceptor agonists also have application as antihypertensive drugs, based on their ability to reduce sympathetic outflow. Likewise, subdivision of the beta adrenoceptors has lead to the development of selective beta 1-adrenoceptor antagonists as antihypertensive and selective beta 2 agonists as bronchodilators. In the past decade, both the alpha 1 and alpha 2 adrenoceptors have been further subdivided, each into three subclasses. In addition, there is strong functional evidence to suggest the presence of additional adrenoceptor subtypes, such as the "alpha 1L" adrenoceptor and "beta 4" adrenoceptor. alpha 1A (or alpha 1L)-Adrenoceptor antagonists have been evaluated for benign prostatic hyperplasia (BPH), and selective alpha 1A agonists for stress incontinence. Gene knockout experiments in mice suggest an important role for the alpha 1B adrenoceptor in the control of vascular tone. Hence, selective alpha 1B antagonists may offer a new approach toward hypertension. Although targeting of specific adrenoceptors can be used to optimize the therapeutic profile of a drug, there are also cases where blockade of multiple adrenoceptors is desirable, as with the alpha/beta-adrenoceptor antagonist carvedilol in congestive heart failure. It is possible that combination of affinities for selected adrenoceptor subtypes within a single molecule may be desirable for certain applications.

Drugs targeting adrenergic receptors: does interaction with a specific subtype confer therapeutic advantage?

Many important drugs act via the activation or blockade of adrenergic receptors. Although research has been ongoing in this area for over fifty years, the continual subdivision of the major adrenoceptor classes has provided new opportunities for drug discovery. This review focuses on the recent developments directed towards a few of the many potential targets now available. While there is a rationale to suggest that limiting pharmacological activity to one particular subtype may improve the therapeutic profile for some indications, it is also possible for a drug to have beneficial effects resulting from interactions with multiple adrenergic receptor subtypes.

Characterization of postjunctional alpha-adrenoceptors in the pithed mouse.

The adrenoceptor subtypes responsible for the pressor response to alpha1- and alpha2-adrenoceptor agonists have not yet been established, although gene knockout experiments in the mouse have provided evidence for a role of the alpha1B- and alpha2B-adrenoceptor. We have evaluated the blood pressure response to selective activation of postjunctional alpha1- and alpha2-adrenoceptors in the pithed mouse. The pressor response to phenylephrine was sensitive to blockade by terazosin, a selective alpha1-adrenoceptor antagonist, but insensitive to rauwolscine, an antagonist at alpha2-adrenoceptors. Phentolamine, a nonselective alpha-adrenoceptor antagonist, blocked the response to either phenylephrine or the selective alpha2-adrenoceptor agonist B-HT 933, whereas rauwolscine blocked only B-HT 933. A dose of terazosin effective against phenylephrine had no effect on B-HT 933; however, the B-HT 933 response was antagonized when the terazosin dose was increased tenfold. A high dose of doxazosin, an alpha1-adrenoceptor antagonist having no affinity for the alpha2B adrenoceptor, blocked the response to phenylephrine but not B-HT 933. Comparison of the potencies of these antagonists against the pressor response to phenylephrine with their affinities for recombinant alpha1-adrenoceptor subtypes suggests that this response is mediated by either alpha1B- or alpha1D-adrenoceptors. The alpha2B-adrenoceptor subtype is likely to take part in the response to B-HT 933. The ability of certain quinazoline alpha1-adrenoceptor antagonists to block the alpha2B adrenoceptor may contribute to their activity as antihypertensive agents.

Adrenoceptor pharmacology: urogenital applications.

Although the selective alpha1-adrenoceptor antagonists were initially developed as antihypertensive drugs, and they are still utilized for this indication, the alpha1-adrenoceptor blockers are now used extensively for the symptomatic treatment of benign prostatic hyperplasia (BPH). As a result, a number of new drugs in this class have been specifically developed for use in BPH. The utility of alpha1-adrenoceptor antagonists in BPH derives from the observation, made several decades ago, that the irreversible, alpha1- adrenoceptor selective antagonist phenoxybenzamine, blocked the contractile activity of norepinephrine in isolated strips of rat or human prostate. Following the further subclassification of alpha1-adrenoceptors into the alpha1A-, alpha1B- and alpha1D-adrenoceptor subtypes, the relationship between subtype selectivity and efficacy in BPH has been investigated in the hope of developing more selective drugs for the treatment of this disorder. Molecular characterization of the adrenoceptor population in human prostate clearly shows the alpha1A-adrenoceptor subtype to predominate, and highly selective alpha1A-adrenoceptor antagonists have been identified and investigated in BPH. However, controversy remains as to whether prostatic smooth muscle contraction is mediated by the alpha1A-adrenoceptor, or by another novel alpha1-adrenoceptor subtype (not corresponding to any of the three known recombinant alpha1-adrenoceptors), or both. alpha1-Adrenoceptor agonists have been used clinically for the treatment of stress incontinence, acting to increase urethral tone by contracting urethral smooth muscle. Research efforts are ongoing to identify agents of this class having a selective action on urethral versus vascular smooth muscle, in order to produce a greater effect on the urethra without producing dose-limiting increases in blood pressure. Local administration of vascular smooth muscle relaxants, either alone or in combination, has been used for the treatment of erectile dysfunction. An alpha1-adrenoceptor antagonist is often used as one comportent in such mixtures, which act to relax trabecular smooth muscle. The recent demonstration that a systemically administered drug can produce a sufficiently selective action on cavernosal smooth muscle to allow efficacy without producing limiting systemic side effects has renewed interest in the possibility of systemic administration of alpha1-adrenoceptor antagonists for this indication.

Effects of alpha1-adrenoceptor antagonists on agonist and tilt-induced changes in blood pressure: relationships to uroselectivity.

We evaluated the uroselectivity of a series of alpha1-adrenoceptor antagonists by comparing their potency against phenylephrine-induced increases in urethral perfusion pressure and diastolic blood pressure in the anesthetized rabbit and pithed rat. In the rabbit, Rec 15/2739 (N-[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-2-phenyl -4H-1-benzopyran-8-carboxamide) as well as analogs with a chlorine substituent on the methoxyphenyl ring (Rec 15/2869) or this substituent combined with the replacement of the phenyl substituent on the pyran ring by cyclohexyl (Rec 15/3011) were 2-6-fold more potent against the urethral vs. vascular response to phenylephrine. Rec 15/2841 (N-[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-2-cyc lohexy-4H-1-benzopyran-8-carboxamide) was only 1.5-fold more potent against the urethral response. SL 89.0591 and tamsulosin also showed selectivity for the urethral response (2-2.5-fold), while the quinazolines produced equipotent blockade of urethral and vascular responses (selectivity ratio = 0.9-1.1). The urethral selectivities of Rec 15/2739 and its derivatives were confirmed by evaluation of the response to tilt in sedated, hypovolemic rabbits. Phenylephrine challenge assays did not show any of the antagonists, with the exception of terazosin at 300 microg kg(-1), to be uroselective in the rat (selectivity ratios = 0.2-1.5); potentiation of tilt-induced hypotension in the anesthetized rat showed substantial differences from the rabbit, with Rec 15/2739, but not Rec 15/3011 and Rec 15/2841 showing orthostatic effects equivalent to that observed for prazosin. Hence, Rec 15/2739 was uroselective in the rabbit, but not in the rat, while two of its close structural analogs were highly uroselective in both species. An assay for orthostatic activity in the conscious rat yielded different results, showing prazosin and terazosin, but not Rec 15/2739, to cause a reversal of the pressor response to tilt. Hence, the apparent uroselectivity of an alpha1-adrenoceptor antagonist is both species- and assay-dependent.

Molecular basis for the stereoselective interactions of catecholamines with alpha-adrenoceptors.

The catecholamines were found to inhibit the binding of the alpha 2-adrenoceptor agonist, [3H]-clonidine, to the recombinant wild type alpha 2a-adrenoceptor (Table 1) with potencies that are consistent with their functional activity in alpha 2-adrenoceptor test systems [6,7]. Mutation of Ser165 to alanine had no significant effect (less than 2-fold) on the affinity of any of the catecholamines for the alpha 2a-adrenoceptor, and in particular, the ratios of affinities between the corresponding (-)- and (+)-enantiomers of the catecholamines were not altered by the point mutation at Ser165. These findings indicate clearly that Ser165, in contrast to predictions made by molecular modeling, plays little if any role in the binding of the catecholamines in general, and cannot be involved in the attachment of the beta-hydroxyl group to the alpha 2a-adrenoceptor. Mutation of either Ser90 on transmembrane helix II or Ser419 on transmembrane helix VII to alanine produced dramatic and selective reductions in the affinity of the (-)-enantiomers of the catecholamines for the alpha 2a-adrenoceptor, with no changes occurring in affinities of the (+)-enantiomers. Thus, the affinities of (-)-norepinephrine and (-)-epinephrine for the Ser90 and Ser419 mutants of the alpha 2a-adrenoceptor were 35-75 fold lower than their affinities for the wild type receptor (Table 1), suggesting that Ser90 and/or Ser419 are involved in the attachment of the beta-hydroxyl groups of the catecholamines to the receptor. Similarly, the affinity of (+/-)-6-fluoronorepinephrine was reduced by 100-fold for the Ser90 mutant receptor (Table 1). Importantly, the affinities of the (+)-enantiomers of the catecholamines, as well as dopamine and epinine, which are the corresponding analogs of norepinephrine and epinephrine which lack the beta-hydroxyl group, were not affected by mutation of Ser90 or Ser419 to alanine (Table 1). Asn293 in transmembrane helix VI has also been proposed to be involved in the interaction of the beta-hydroxyl group of isoproterenol with the beta 2-adrenoceptor [4]. The alpha 2a-adrenoceptor contains three hydroxyl bearing amino acids at a position corresponding to this site (Thr393-Tyr394-Thr395). These amino acids could theoretically form a hydrogen bond with the beta-hydroxyl group of a catecholamine, and therefore could serve as a potential point of attachment. Simultaneous mutation of all three of these amino acids to Ala-Phe-Ala reduced the affinity of the (-)-enantiomers of the catecholamines by 12-20 fold, which is somewhat less than what was observed for mutation of either Ser90 or Ser419 (Table 1). However, in contrast to mutation of Ser90 or Ser419, which had no effect on the affinity of the (+)-enantiomers, mutation of the three residues in transmembrane helix VI did significantly reduce the affinities of the (+)-enantiomers of the catecholamines by approximately 5- to 9-fold, indicating that mutations at these points of the receptor are not selective for the (-)-enantiomers, and are therefore not likely to be involved in the attachment of the beta-hydroxyl group of the catecholamines.

Comparison of metoprolol and carvedilol pharmacology and cardioprotection in rabbit ischemia and reperfusion model.

Carvedilol, a selective alpha1 and non-selective beta-adrenoceptor antagonist and antioxidant, has been shown to provide significant cardiac protection in animal models of myocardial ischemia. To further explore the mechanisms contributing to carvedilol cardioprotection efficacy, the effects of carvedilol on hemodynamic variables, infarct size and myeloperoxidase activity (an index of neutrophil accumulation) were compared with a beta1-selective adrenoceptor antagonist, metoprolol. Carvedilol (1 mg/kg) or metoprolol (1 mg/kg or 1 mg/kg + 0.5 mg/kg 90 min later) was given intravenously 5 min before reperfusion. In vehicle-treated rabbits, ischemia (60 min) and reperfusion (180 min) resulted in significant increments in left ventricular end diastolic pressure, large infarcts (59+/-2.6% of area-at-risk) and marked increase in myeloperoxidase activity (0.59+/-0.09 U/100 mg tissue). Carvedilol treatment resulted in sustained reduction of pressure-rate-index and significantly smaller infarcts (22.0+/-2.5%, P < 0.01 vs. vehicle) as well as decreased myeloperoxidase activity (0.186+/-0.056 U/100 mg tissue, P < 0.01 vs. vehicle). The highest dose of metoprolol, 1 mg/kg + 0.5 mg/kg, that resulted in pressure-rate-index comparable to that of 1.0 mg/kg carvedilol, failed to reduce myeloperoxidase activity in the ischemic myocardial tissue, and the infarct size (35+/-3.1%) was significantly larger than in carvedilol-treated animals. Taken together, this study suggests that the superior cardioprotection of carvedilol over metoprolol is not a consequence of hemodynamic variances but possibly the result of the additional pharmacological properties of carvedilol such as the antioxidant and anti-neutrophil effects.

Ser165 of transmembrane helix IV is not involved in the interaction of catecholamines with the alpha-2a-adrenoceptor.

Molecular modeling studies have predicted that the beta-hydroxyl group of the catecholamines interacts with the beta 2-adrenoceptor at the serine residue at position 165 (Ser165) located on transmembrane helix IV; however, this has not been confirmed by site-directed mutagenesis. It has been inferred that this site, which is conserved in all of the nine known alpha- and beta-adrenoceptor subtypes, is also involved in the interaction of catecholamines with the alpha 2a-adrenoceptor. To test the hypothesis that the beta-hydroxyl group of the catecholamines interacts with Ser165 of the alpha 2a-adrenoceptor, we prepared a mutant alpha 2a-adrenoceptor where Ser165 was mutated to alanine. Mutation of Ser165 of the alpha 2a-adrenoceptor to alanine had no effect on the affinity of dopamine (which lacks the beta-hydroxyl group) or either enantiomer of norepinephrine or epinephrine (both of which possess the beta-hydroxyl group), indicating that Ser165 is not involved in the interaction of the catecholamines with the alpha 2a-adrenoceptor. We have previously shown that mutation of Ser90, located in transmembrane helix II, to either alanine or cysteine produces a selective reduction in the affinity of the (-)-enantiomers of the catecholamines for the alpha 2a-adrenoceptor, with no effect on the (+)-enantiomers or the corresponding beta-desoxy analogs. This is consistent with the known stereoselectivity involved in the interactions of catecholamines with the alpha 2a-adrenoceptor. The results of the present investigation indicate that Ser165 is not involved in the interaction of catecholamines with the alpha 2a-adrenoceptor. Because all known alpha-adrenoceptor subtypes have a serine residue at a position corresponding to Ser90 of the alpha 2a-adrenoceptor, it would appear that this site represents an important point for attachment of the beta-hydroxyl group of catecholamines.

Pharmacological characterization of the uroselective alpha-1 antagonist Rec 15/2739 (SB 216469): role of the alpha-1L adrenoceptor in tissue selectivity, part I.

Alpha adrenoceptor antagonists have been convincingly shown to be beneficial in reducing both subjective and objective indices of urethral obstruction in benign prostatic hyperplasia. Rec 15/2739 (SB 216469) is a novel alpha-1 adrenoceptor (alpha-1 AR) antagonist currently being developed for benign prostatic hyperplasia. When evaluated in radioligand binding assays with expressed animal or human alpha-1 ARs, Rec 15/2739 shows marked to moderate selectivity for the alpha-1a AR subtype. Its affinity for the recombinant alpha-2 AR subtypes or native dopaminergic D2 receptor was about 100-fold lower than that for alpha-1a AR subtype. In canine tissues, Rec 15/2739 was 20-fold more potent as an inhibitor of [3H]prazosin binding to prostate vis-a-vis aorta. Functional studies in isolated rabbit tissues also confirmed the uroselectivity of Rec 15/2739, with substantially higher affinity (Kb = 2-3 nM) being observed in urethra and prostate, compared with ear artery and aorta (Kb = 20-100 nM). The in vitro selectivity observed with Rec 15/2739 was confirmed in vivo in the anesthetized dog, comparing potency against norepinephrine- or hypogastric nerve stimulation-induced urethral contraction with its ability to reduce diastolic blood pressure. In this model, Rec 15/2739 had greater selectivity than any other alpha-1 AR antagonist examined, including terazosin and tamsulosin. Based on the low potency of prazosin and some of its structural analogs in the rabbit and dog lower urinary tract tissues, it appears that norepinephrine contracts these tissues via activation of the alpha-1L AR. Hence this alpha-1 AR subtype, rather than the alpha-1A AR, may mediate the contraction in vivo.

Pharmacological characterization of the uroselective alpha-1 antagonist Rec 15/2739 (SB 216469): role of the alpha-1L adrenoceptor in tissue selectivity, part II.

The aim of the present work was to investigate whether or not the uroselectivity of Rec 15/2739 and several other alpha-1 adrenoceptor (alpha1-AR) antagonists observed in the anesthetized dog could be related to selectivity of these compounds for a particular alpha-1 AR subtype. The binding affinity of the tested compounds for canine prostate alpha-1 ARs and their in vitro functional affinity for the alpha-1 ARs of rabbit urethra and prostate correlated with their functional affinity for the alpha-1L AR subtype, but not with the binding affinity for recombinant animal and human alpha-1a, alpha-1b and alpha-1d AR subtypes. Similar results were obtained when the in vivo potency on urethral pressure was correlated with the affinity for the alpha-1 AR subtypes; also in this case alpha-1L AR gave the best correlation. No correlation was obtained by considering the other alpha-1 AR subtypes. The in vivo hypotensive effects observed in dog after i.v. administration of the considered compounds correlated only with the binding affinity for the animal and human alpha-1d subtype. In conclusion, the results shown in the present paper indicate that the potencies of different alpha-1 antagonists against the contractions induced by norepinephrine on tissues of the lower urinary tract of rabbits and dogs are better correlated with their affinity for the putative alpha-1L subtype than for the alpha-1a subtype. Only the compounds showing selectivity for the alpha-1L subtype versus the alpha-1d subtype proved highly selective in vivo for the lower urinary tract versus the vascular tissues.

Recent advances in the identification of alpha1- and alpha2-adrenoceptor subtypes: therapeutic implications.

The cloning of multiple subtypes of both alpha1- and alpha2-adrenoceptors has renewed interest in the therapeutic application of agents interacting with these receptors. Effort has primarily been directed towards the design of uroselective alpha1-adrenoceptor antagonists for the treatment of benign prostatic hyperplasia (BPH). Evidence is accumulating for the involvement of a novel alpha1-adrenoceptor, designated as alpha1L-adrenoceptor, in alpha1-adrenoceptor-mediated smooth muscle contraction in prostatic and other urogenital tissues. While several antagonists showing a high degree of uroselectivity in animal models have been identified, their clinical superiority over the currently available alpha1-adrenoceptor antagonists has not yet been demonstrated. It is possible that the interaction with alpha1-adrenoceptors, as yet uncharacterised subtypes, at non-prostatic sites contributes to the therapeutic activity of this drug class in BPH. The alpha1-adrenoceptor subtypes involved in the control of vascular tone are currently being evaluated, and the profile of interaction with the various alpha1-adrenoceptor subtypes may play a key role in the efficacy of cardiovascular drugs such as carvedilol. Alpha2-adrenoceptor agonists are now being employed for a variety of therapeutic applications, most involving actions on receptors within the central nervous system (CNS). These agents are useful in the treatment of hypertension, glaucoma, opiate withdrawal and attention deficit hyperactivity disorder (ADHD), and as analgesics and adjuncts to general anaesthesia. While subtype selectivity has not yet been applied to the design of new alpha2-adrenoceptor agonists for these applications, recent gene mutation/knock-out experiments have identified the alpha2-subtypes involved in some of these actions, and optimisation of a therapeutic profile may be possible. Furthermore, the design of agents combining affinities for multiple adrenoceptor subtypes, or the combination of a specific adrenoceptor affinity profile with another pharmacological action, may offer advantages over molecules selective for an individual adrenoceptor subtype.

The use of alpha-adrenoceptor antagonists in the pharmacological management of benign prostatic hypertrophy: an overview.

Benign prostatic hypertrophy (BPH) produces symptomatic urethral obstruction in a significant percentage of older men. Since the incidence of BPH is age related, the clinical and economic impact of this disease will continue to progress as average lifespan increases. BPH is associated with growth of both glandular and stromal elements of the prostate gland. Glandular hyperplasia can be partially reversed by withdrawal of androgenic tone with androgen receptor antagonists or steroid-5-alpha-reductase inhibitors. However, the reduction in prostatic size produced by these agents has little effect on the dynamic tone induced by nerve mediated contraction of stromal smooth muscle. This tone is mediated by activation of alpha-adrenoceptors. Therefore the alpha-adrenoceptor antagonists represent a useful pharmacological approach to the treatment of BPH. Studies in isolated strips of human prostate show that either exogenous alpha-adrenoceptor agonists or electrical field stimulation will induce contraction. Studies with selective antagonists such as prazosin show that this response is mediated by the alpha 1-adrenoceptor, even though radioligand binding studies show the presence of alpha 1 and alpha 2 adrenoceptor subtypes in approximately equal density. Following the cloning of multiple alpha 1-adrenoceptors, the contractile response in human prostate has been assigned to the alpha 1A adrenoceptor. However, recent data would suggest a functional role for another subtype, which has not yet been cloned, and designated as alpha 1L based on a relatively low affinity for prazosin. Clinical trials have shown efficacy of a variety of alpha-adrenoceptor antagonists in BPH, including non-selective agents such as phenoxybenzamine, as well as a variety of selective alpha 1-adrenoceptor antagonists, most structurally related to prazosin. The agents most commonly employed at the present time include the prazosin analogs terazosin, doxazosin and alfuzosin, as well as the structurally unrelated indoramin and tamsulosin. The design of new alpha 1-antagonists for BPH has concentrated on agents producing preferential blockage of urogenital vis-á-vis vascular alpha 1-adrenoceptors, based either on selectivity for the alpha 1A-adrenoceptor subtype or on functional uroselectivity in animal models. While these newer agents offer the prospect of reducing the incidence of the cardiovascular side effects associated with current therapy their superiority over nonselective alpha 1-adrenoceptor antagonists remains to be demonstrated in the clinical setting.

"Vinylogs" and "acetylenylogs" of beta-adrenergic agents.

Vinylogous (Groups III and V) and acetylenologous (Group IV) analogs of the classical beta-adrenergic agents--stimulants and blockers--were prepared in order to evaluate the effect of degree of saturation, position of unsaturation and rigidity of the chain linking the aromatic ring and the amino containing functional group on biological activity. Derivatives from Group III, which represent 4-aryl-3-butenyl-2-ol-amine analogs of Group II, retained beta 1-adrenoceptor antagonist activity albeit substantially less potent (50-200-fold) than that possessed by their aryloxy counterparts. Consistent with the SAR for Group II compounds, substitution at position 2 of the aromatic ring yielded the most potent antagonists (5a, 5d, 5g), with KB's ranging from 73-93 nM while 3,4-dichloro substitution (5e) markedly reduced antagonist potency (KB = 2,400 nM). Agonist activity was also noted for 5b and 5d, suggesting that these compounds may be best classified as partial agonists. Representatives from Groups IV and V were inactive as antagonists at the beta 1-adrenoceptor confirming the importance of the spatial relationship between the hydroxyl and the amino nitrogen.