A randomized crossover study to evaluate Ro 115-1240, a selective alpha1A/1L-adrenoceptor partial agonist in women with stress urinary incontinence.

OBJECTIVES: To investigate the potential therapeutic benefits of the selective alpha1A/1l-adrenoceptor partial agonist Ro 115-1240 in women with mild-to-moderate stress urinary incontinence (SUI). PATIENTS AND METHODS: Thirty-seven women with mild-to-moderate SUI were enrolled in a randomized, placebo-controlled crossover study. Patients received 1.5 mg Ro 115-1240 twice daily or matching placebo for 2 or 4 weeks. Voiding diaries were used to record the number of SUI episodes, urge incontinence episodes and pads used. Sitting blood pressures and heart rate were recorded at each visit. RESULTS: Ro 115-1240 was associated with a significantly lower mean weekly number of SUI episodes than placebo (8.4 vs 6.0; P= 0.0079), a 28% relative improvement over placebo. There was also a significantly lower mean number of pads used and wet pads changed/week with Ro 115-1240 than with placebo (P = 0.0055 and 0.0066, respectively). The most frequently reported treatment-emergent adverse events were scalp tingling, headache, chills, piloerection, and pruritus. Generally these events were transient and mild to moderate. There was a slightly lower mean sitting heart rate with Ro 115-1240 than with placebo, but no difference in mean systolic or diastolic blood pressure between treatments. CONCLUSIONS: This study suggests that selective alpha1A/1l-adrenoceptor partial agonists have the potential to improve the symptoms of SUI with little or no cardiovascular effect. These results are encouraging and a randomized controlled trial of Ro 115-1240 in a larger population with SUI is warranted to substantiate these findings.

Pharmacological characteristics of Ro 115-1240, a selective alpha1A/1L-adrenoceptor partial agonist: a potential therapy for stress urinary incontinence.

OBJECTIVE: To describe the preclinical pharmacology of Ro 115-1240, a peripherally acting selective alpha1A/1L-adrenoceptor (AR) partial agonist, compared with the alpha1A/1L-AR full agonist amidephrine, as AR agonists have some utility in the treatment of stress urinary incontinence (SUI) but are limited by undesirable cardiovascular and central nervous system side-effects. RESULTS: In radioligand-binding studies Ro 115-1240 had greater affinity for alpha1A than for alpha1B and alpha1D subtypes. The potency and intrinsic activity of amidephrine and Ro 115-1240 relative to noradrenaline were determined in native and cell-based assays using human recombinant alpha1-ARs; they acted as selective alpha1A/1L-AR full and partial agonists, respectively. In anaesthetized micropigs and rabbits, amidephrine and Ro 115-1240 produced non-selective, dose-dependent increases in intraurethral and arterial blood pressures but the magnitude of the pressure increases evoked by Ro 115-1240 were about a third of those with amidephrine. In conscious micropigs both agents produced dose-dependent increases in urethral tension. Again, the magnitude of the urethral response to Ro 115-1240 was about a third of that with amidephrine. More importantly, only amidephrine produced dose-dependent increases in blood pressure and decreases in heart rate. Ro 115-1240 produced a maximum increase in urethral tension with no effect on blood pressure or heart rate. CONCLUSION: These results show that by combining selectivity for the alpha1A/1L-AR subtype with a reduction in intrinsic agonist efficacy, Ro 115-1240 has reduced haemodynamic effects while retaining to some degree the contractile effects on urethral smooth muscle. These studies indicate that Ro 115-1240 may be useful as a novel treatment for SUI.

Synthesis, pharmacology and pharmacokinetics of 3-(4-aryl-piperazin-1-ylalkyl)-uracils as uroselective alpha1A-antagonists.

Predominance in the urethra and prostate of the alpha(1A)-adrenoceptor subtype, which is believed to be the receptor mediating noradrenaline induced smooth muscle contraction in these tissues, led to the preparation of alpha(1A)-selective antagonists to be tested as uroselective compounds for the treatment of benign prostatic hyperplasia. Thus, a number of selective alpha(1A)-adrenoceptor antagonists were synthesized and assayed in vitro for potency and selectivity. Dog pharmacokinetic parameters of 12 (RO700004) and its metabolite 40 (RO1104253) were established. The relative selectivity of intravenously administered 12, 40 and standard prazosin to inhibit hypogastric nerve stimulation-induced increases in intraurethral prostatic pressure versus phenylephrine-induced increases in diastolic blood pressure in anesthetized dogs was 76, 71 and 0.6, respectively.

Evaluation of oral ro70-0004/003, an alpha1A-adrenoceptor antagonist, in the treatment of male erectile dysfunction.

Alpha-adrenoceptor antagonists have been used for the treatment of male erectile dysfunction (MED). Ro70-0004/003 (Ro70-0004) is a selective and orally active alpha1A-adrenoceptor antagonist. The objective of this study was to: (1) pharmacologically elucidate the alpha1-adrenoceptor subtype mediating norepinephrine-induced contraction of human isolated corpus cavernosal tissue and (2) conduct a clinical proof-of-concept study with Ro70-0004 to test the hypothesis that selective alpha1A-adrenoceptor blockade would improve erectile function in patients with MED. In vitro organ bath studies were conducted with strips of human isolated corpus cavernosal tissue obtained from patients undergoing penile prosthesis implantation. Prazosin, cyclazosin, RS-100329 and Ro70-0004/003 antagonized norepinephrine-induced contractile responses with affinity estimates (pK(B) or pA2) of 8.4, 7.3, 9.2 and 8.8, respectively, consistent with the singular involvement of alpha1A-adrenoceptor subtype. A clinical study (single center, observer-blind, randomized, placebo-controlled, extended period Latin-Square crossover design) was conducted in 24 male patients (mean age 44 y) with MED of no established organic cause to evaluate the efficacy of a 5-mg oral dose of Ro70-0004. The primary efficacy endpoint was the duration of rigidity > 60% at the base of the penis measured between 0.5 and 2.5 h post-dose. Rigidity was assessed by penile plethysmography using the RigiScan Plus device during visual sexual stimulation. The safety and efficacy of Ro70-0004 was also assessed. A 50-mg dose of sildenafil was included as a positive control. For the primary efficacy endpoint, the mean duration of erection was 9.69 min following administration of placebo, 8.28 min following Ro70-0004, and 22.64 min following sildenafil. Only the difference between sildenafil and placebo reached statistical significance (P < 0.05). A similar pattern was observed when measuring a duration of rigidity > 80% at the base of the penis (secondary endpoint). Ro70-0004 was safe and generally well tolerated (only two out of 20 patients reported at least one adverse event). The highly selective alpha1A-adrenoceptor antagonist, Ro70-0004, given at a single dose of 5 mg, did not improve erectile function when compared to placebo.

In vitro alpha1-adrenoceptor pharmacology of Ro 70-0004 and RS-100329, novel alpha1A-adrenoceptor selective antagonists.

It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the alpha1A-adrenoceptor-mediated contraction of lower urinary tract tissues may, via a selective relief of outlet obstruction, lead to an improvement in symptoms. The present study describes the alpha1-adrenoceptor (alpha1-AR) subtype selectivities of two novel alpha1-AR antagonists, Ro 70-0004 (aka RS-100975) and a structurally-related compound RS-100329, and compares them with those of prazosin and tamsulosin. Radioligand binding and second-messenger studies in intact CHO-K1 cells expressing human cloned alpha1A-, alpha1B- and alpha1D-AR showed nanomolar affinity and significant alpha1A-AR subtype selectivity for both Ro 70-0004 (pKi 8.9: 60 and 50 fold selectivity) and RS-100329 (pKi 9.6: 126 and 50 fold selectivity) over the alpha1B- and alpha1D-AR subtypes respectively. In contrast, prazosin and tamsulosin showed little subtype selectivity. Noradrenaline-induced contractions of human lower urinary tract (LUT) tissues or rabbit bladder neck were competitively antagonized by Ro 70-0004 (pA2 8.8 and 8.9), RS-100329 (pA2 9.2 and 9.2), tamsulosin (pA2 10.4 and 9.8) and prazosin (pA2 8.7 and 8.3 respectively). Affinity estimates for tamsulosin and prazosin in antagonizing alpha1-AR-mediated contractions of human renal artery (HRA) and rat aorta (RA) were similar to those observed in LUT tissues, whereas Ro 70-0004 and RS-100329 were approximately 100 fold less potent (pA2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA respectively). The alpha1A-AR subtype selectivity of Ro 70-0004 and RS-100329, demonstrated in both cloned and native systems, should allow for an evaluation of the clinical utility of a 'uroselective' agent for the treatment of symptoms associated with benign prostatic hyperplasia.

Cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild-type and D79N alpha2A-adrenoceptor transgenic mice.

1. We investigated the cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild-type and D79N alpha2A-adrenoceptor mice. The in vitro pharmacology of these agonists was determined at recombinant (human) alpha2-adrenoceptors and at endogenous (dog) alpha2A-adrenoceptors. 2. In wild-type mice, rilmenidine, moxonidine (100, 300 and 1000 microg kg(-1), i.v.) and clonidine (30, 100 and 300 microg kg(-1), i.v.) dose-dependently decreased blood pressure and heart rate. 3. In D79N alpha2A-adrenoceptor mice, responses to rilmenidine and moxonidine did not differ from vehicle control. Clonidine-induced hypotension was absent, but dose-dependent hypertension and bradycardia were observed. 4. In wild-type mice, responses to moxonidine (1 mg kg(-1), i.v.) were antagonized by the non-selective, non-imidazoline alpha2-adrenoceptor antagonist, RS-79948-197 (1 mg kg(-1), i.v.). 5. Affinity estimates (pKi) at human alpha2A-, alpha2B- and alpha2C-adrenoceptors, respectively, were: rilmenidine (5.80, 5.76 and 5.33), moxonidine (5.37, <5 and <5) and clonidine (7.21, 7.16 and 6.87). In a [35S]-GTPgammaS incorporation assay, moxonidine and clonidine were alpha2A-adrenoceptor agonists (pEC50/intrinsic activity relative to noradrenaline): moxonidine (5.74/0.85) and clonidine (7.57/0.32). 6. In dog saphenous vein, concentration-dependent contractions were observed (pEC50/intrinsic activity relative to noradrenaline): rilmenidine (5.83/0.70), moxonidine (6.48/0.98) and clonidine (7.22/0.83). Agonist-independent affinities were obtained with RS-79948-197. 7. Thus, expression of alpha2A-adrenoceptors is a prerequisite for the cardiovascular effects of moxonidine and rilmenidine in conscious mice. There was no evidence of I1-imidazoline receptor-mediated effects. The ability of these compounds to act as alpha2A-adrenoceptor agonists in vitro supports this conclusion.

Alpha1L-adrenoceptor mediation of smooth muscle contraction in rabbit bladder neck: a model for lower urinary tract tissues of man.

1. The alpha1-adrenoceptor population mediating contractile responses to noradrenaline (NA) in smooth muscles of the bladder neck from rabbit (RBN) has been characterized by use of quantitative receptor pharmacology. 2. Experiments with several 'key' alpha1-adrenoceptor antagonists of varying subtype selectivities (RS-17053, BMY 7378, indoramin, 5-methylurapidil, prazosin, REC 15/2739, SNAP 5089, terazosin, WB 4101, tamsulosin, (+)-cyclazosin and RS-100329) were conducted. Schild regression analyses yielded affinity (mean pKb) estimates of 7.1, 6.2, 8.6, 8.6, 8.4, 9.3, 7.0, 7.4, 8.9, 10.0, 7.1 and 9.3, respectively, although deviations from unit Schild regression slope question the robustness of data for RS-17053 and SNAP 5089. 3. The nature of antagonism by these agents and the profile of affinity determinations generated together suggest that a single alpha1-adrenoceptor subtype mediates contractile responses of RBN to NA. Additional studies with phenylephrine indicated also an agonist-independence of this profile. Pharmacologically, this profile was reminiscent of that described as 'alpha1L'-adrenoceptor, which has been shown to mediate contractions of several tissues including lower urinary tract (LUT) tissues of man. Furthermore, a similarity was noticed between the 'alpha1L'-adrenoceptor described here in RBN and the rabbit and human cloned alpha1a-adrenoceptor (based on data from both whole cell radioligand binding at 37 degrees C and [3H]-inositol phosphates accumulation assays), characterizations of which have been published elsewhere. 4. In conclusion, the RBN appears to provide a predictive pharmacological assay for the study of NA-induced smooth muscle contraction in LUT tissues of man.

N-arylpiperazinyl-N'-propylamino derivatives of heteroaryl amides as functional uroselective alpha 1-adrenoceptor antagonists.

Novel arylpiperazines were identified as alpha 1-adrenoceptor (AR) subtype-selective antagonists by functional in vitro screening. 3-[4-(ortho-Substituted phenyl)piperazin-1-yl]propylamines were derivatized with N,N-dimethyl anthranilamides, nicotinamides, as well as carboxamides of quinoline, 1,8-naphthyridine, pyrazolo[3,4-b]pyridine, isoxazolo[3,4-b]pyridine, imidazo[4,5-b]pyridine, and pyrazolo[1,5-a]pyrimidines. Strips of rabbit bladder neck were employed as a predictive assay for antagonism in the human lower tract. Rings of rat aorta were used as a "negative screen" for the test antagonists. Binding to alpha 1-ARs was relatively sensitive to size and electronic features of the arylpiperazine portion of the antagonists and permissive to these features on the heteroaryl carboxamide side. These structure-affinity findings were exploited to produce nicotinamides (e.g. 13ii and 25x) and pyrazolo[3,4-b]pyridines (e.g. 37f and 37y) ligands with nanomolar affinity at the alpha 1-AR subtype prevalent in the human lower urinary tract(pA2 values: 8.8, 10.7, 9.3, and 9.9, respectively) and displaying 2-3 orders of magnitude selectivity over the alpha 1D-AR.

RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha, alpha-dimethyl-1H-indole-3-ethanamine hydrochloride), a selective alpha 1A-adrenoceptor antagonist, displays low affinity for functional alpha 1-adrenoceptors in human prostate: implications for adrenoceptor classification.

Norepinephrine (NE) contracts smooth muscle cells within the human lower urinary tract (LUT) (bladder neck, prostate, and urethra). Receptor distribution and pharmacological evidence have implicated activation of alpha 1A-adrenoceptors. We disclose the pharmacological properties of the novel, selective alpha 1A-adrenoceptor antagonist N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro- alpha,alpha-dimethyl-1H-indole-3-ethanamine hydrochloride (RS-17053) and examine critically the pharmacological identity of the alpha 1-adrenoceptor mediating contractions to NE in human LUT tissues. In several tissues from rat and cloned adrenoceptors, RS-17053 displayed high affinity for the alpha 1A-adrenoceptor (pKi and pA2 estimates of 9.1-9.9) and a 30-100-fold selectivity over the alpha 1B- and the alpha 1D-adrenoceptor subtypes (pK1 and pA2 estimates of 7.7-7.8). However, in isolated smooth muscle preparations from human LUT tissues, RS-17053 antagonized responses to NE only at high concentrations. Estimates of affinity (pA2) at alpha 1-adrenoceptors mediating NE-induced contractions were 7.5 in prostatic periurethral longitudinal smooth muscle (compared with 8.6 for prazosin), 6.9 in anterior fibromuscular stroma (prazosin, 8.9), and 7.1 in bladder neck (prazosin, 8.5). These findings indicate that contractile responses to NE in human LUT tissues are mediated by a receptor displaying pharmacological properties that are clearly different from those of the defined alpha 1A-adrenoceptor and raise the possibility that multiple forms of the alpha 1A-adrenoceptor may exist in human LUT that are discriminated by RS-17053. In this regard, the affinity estimates obtained with RS-17053 and other alpha 1-adrenoceptor antagonists in human LUT tissues are identical to those described for the putative alpha 1L-adrenoceptor.

Functional evidence equating the pharmacologically-defined alpha 1A- and cloned alpha 1C-adrenoceptor: studies in the isolated perfused kidney of rat.

1. The present study characterizes and classifies alpha 1-adrenoceptor-mediated vasoconstriction in the isolated perfused kidney of rat using quantitative receptor pharmacology and compares the results to radioligand binding studies (made in cloned alpha 1-adrenoceptor subtypes, native alpha 1A-adrenoceptors in submaxillary gland of rat, and alpha 1A-adrenoceptors in several other tissues of rat). 2. Concentration-effect curves to noradrenaline in the presence of 5-methyl-urapidil were biphasic, indicating alpha 1-adrenoceptor heterogeneity. The alpha 1-adrenoceptor subtype mediating the first phase (low affinity for 5-methyl-urapidil) could not be 'isolated' for detailed pharmacological characterization but was defined by a sensitivity to inhibition by chloroethylclonidine and an inability of methoxamine to activate the site. Additionally, vasoconstriction mediated by this alpha 1-adrenoceptor subtype or subtypes was abolished by nitrendipine (1 microM), thereby allowing characterization of the second, high affinity site for 5-methyl-urapidil. 3. The following antagonists interacted competitively with noradrenaline at the alpha 1-adrenoceptor for which 5-methyl-urapidil exhibits high affinity (pKB value): WB 4101 (10.3) > prazosin (9.5) approximately HV 723 (9.3) approximately 5-methyl-urapidil (9.2) > phenotolamine (8.6) > spiperone (pA2 = 8.1) approximately oxymetazoline (7.9). In contrast, insurmountable antagonism was seen with S(+)- and R(-)-niguldipine, the S(+)-isomer being approximately 30 fold more potent than the R(-)-isomer. Receptor protection experiments indicated that S(+)-niguldipine interacted directly with alpha 1-adrenoceptors. Dehydroniguldipine acted as a competitive antagonist (pKB = 9.0). Thus, the results with antagonists define the alpha 1-adrenoceptor as an alpha 1A-adrenoceptor. 4. An agonist 'fingerprint' was constructed in the presence of nitrendipine to define further the alpha 1A-adrenoceptor. The following order and relativity of agonist potency was obtained: cirazoline (1) approximately adrenaline (2) > noradrenaline (5) > phenylephrine (23) approximately amidephrine (31) > methoxamine (71) >> isoprenaline (1456) approximately dopamine (2210). 5. A high correlative association was shown between the affinity of antagonists obtained functionally in the isolated perfused kidney of rat and pKi values obtained from binding experiments with the cloned bovine alpha 1C-adrenoceptor (R2 = 0.85), native alpha 1A-adrenoceptors in submaxillary gland of rat (R2 = 0.79), and alpha 1A-adrenoceptors from several other tissues of rat (values taken from the literature, R2 = 0.89). 6. The present study demonstrates that the alpha 1A-adrenoceptor is the predominant alpha 1-adrenoceptor subtype mediating vasoconstrictor responses to exogenously administered noradrenaline in the isolated perfused kidney of rat. More importantly, alpha 1A-adrenoceptors mediating vasoconstrictor responses to noradrenaline exhibited a pharmacological equivalency to the cloned bovine alpha 1 c-adrenoceptor. Thus,definitive functional pharmacological data are provided for equating the two receptors and support results derived recently from molecular and radioligand binding studies.

Characterization of the alpha-1 adrenoceptor subtype mediating [3H]-arachidonic acid release and calcium mobilization in Madin-Darby canine kidney cells.

The authors characterized the alpha-1 adrenoceptor subtype located on Madin-Darby canine kidney (MDCK) cells by measuring norepinephrine-mediated [3H]-arachidonic acid release and Ca++ mobilization in fura 2-loaded cells. In both assays, prazosin and chloroethylclonidine acted as unsurmountable antagonists, whereas 5-methyl-urapidil acted as a competitive antagonist with pA2 estimates of 7.3 (arachidonic acid assay) and 7.7 (Ca++ assay). Competitive antagonism toward arachidonic acid release was also obtained with the following alpha-1 adrenoceptor antagonists (pA2): (+)-niguldipine (7.6), 2-(2,6-dimethoxy-phenoxy-yethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB 4101; 8.3), phentolamine (7.6) and oxymetazoline (6.4). Arachidonic acid release by norepinephrine was abolished in the absence of extracellular Ca++ and was antagonized by 1-[beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) but was insensitive to antagonism by L-type Ca++ channel antagonists and La . Norepinephrine-mediated increases in intracellular Ca++ consisted of two distinct phases: a transient phase followed by a sustained plateau. The transient phase was resistant to EGTA, whereas the plateau phase was abolished by EGTA. Potassium chloride did not evoke a response in either assay. Northern blot analysis demonstrated that MDCK cells express alpha-1B adrenoceptor messenger RNA. It was concluded that typical alpha-1B adrenoceptors mediate responses to norepinephrine in MDCK cells and that these receptors couple to both intracellular Ca++ release and Ca++ influx by a voltage-independent mechanism. This influx pathway is insensitive to L-type Ca++ channel antagonists but is antagonized by the receptor-operated Ca++ channel antagonist SK&F 96365.

Evidence for a noradrenergic innervation to alpha 1A-adrenoceptors in rat kidney.

1. Experiments were undertaken to characterize the alpha 1-adrenoceptor subtype mediating vasoconstrictor responses to periarterial noradrenergic nerve stimulation (PNS) in the isolated perfused kidney of the rat. 2. Vasoconstrictor responses to nerve stimulation were inhibited by prazosin (10 nM), 5-methyl-urapidil (30 nM), and nitrendipine (1 microM) but were resistant to inhibition by chloroethylclonidine (100 microM). 3. 5-Methyl-urapidil (30 nM), chloroethylclonidine (100 microM) and nitrendipine (1 microM) did not inhibit the neuronal release of tritium from nerves loaded with [3H]-noradrenaline. 4. The results suggest that renovascular alpha 1A-adrenoceptors receive a noradrenergic innervation and that the innervated receptors are coupled to dihydropyridine-sensitive calcium channels.

Slow off-rate of prazosin accounts for deviations from competitive antagonism in the isolated perfused kidney of rat.

1. The kinetics of the interaction of prazosin with noradrenaline, administered by bolus injection and infusion, was examined in the isolated perfused rat kidney. 2. Prazosin (1-30 nM) competitively antagonized vasoconstrictor responses to infusion of noradrenaline (pA2 = 9.51) whereas antagonism toward bolus injections of noradrenaline deviated from competition. 3. In the presence of prazosin, responses to bolus injection of noradrenaline became biphasic whereas the kinetics of the responses to infusions of noradrenaline were unchanged. 4. In contrast, phentolamine (30-300 nM) competitively antagonized responses to bolus injection of noradrenaline without altering response kinetics and in coperfusion studies with prazosin, prevented prazosin from inducing biphasic responses to bolus injection of noradrenaline. 5. It is concluded that prazosin acts as a pseudoirreversible antagonist toward vasoconstrictor responses to bolus injections noradrenaline (non-steady state conditions) whereas steady state conditions and competitive kinetics are observed with infused noradrenaline.

Antagonist characterization of atypical beta adrenoceptors in guinea pig ileum: blockade by alprenolol and dihydroalprenolol.

The present study was undertaken to further characterize the atypical beta adrenoceptor in guinea pig ileum. Tension was developed in isolated segments of ileum using transmural electrical stimulation of enteric cholingeric nerves. The ability of isoproterenol to relax the ileum, via beta-1 adrenoceptor and atypical beta adrenoceptor agonism, was measured. Propranolol (5 x 10(-6) M) and bromoacetylaprenololmetane blocked beta-1 adrenoceptors but, at the concentrations tested, were without affinity at atypical beta adrenoceptors. (-)-Alprenolol and (-)-dihydroalprenolol, however, acted as competitive antagonists at both sites (pA2 values of 8.2 and 8.81 at beta-1 adrenoceptors and 6.47 and 6.43 at atypical beta adrenoceptors, respectively). (-)-Alprenolol also exerted agonistic activity at the atypical beta adrenoceptor. [3H](-)-Dihydroproalprenolol failed to identify beta-1 adrenoceptors or atypical beta adrenoceptors but, instead, bound to a putative lipophilic site unrelated to ileal adrenoceptors. Before this study, nadolol (pA2 = 4.7) was the only documented antagonist at the atypical beta adrenoceptor in guinea pig ileum. Thus, the present results detail two additional pharmacological probes which exhibit about a 100-fold greater affinity than nadolol for the atypical site.