David R. Blue

In vitroα1‐adrenoceptor pharmacology of Ro 70–0004 and RS‐100329, novel α1A‐adrenoceptor selective antagonists

It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the α1A‐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 α1‐adrenoceptor (α1‐AR) subtype selectivities of two novel α1‐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 α1A‐, α1B‐ and α1D‐AR showed nanomolar affinity and significant α1A‐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 α1B‐ and α1D‐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 α1‐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 α1A‐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 α2A‐adrenoceptor transgenic mice

We investigated the cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild‐type and D79N α2A‐adrenoceptor mice. The in vitro pharmacology of these agonists was determined at recombinant (human) α2‐adrenoceptors and at endogenous (dog) α2A‐adrenoceptors. In wild‐type mice, rilmenidine, moxonidine (100, 300 and 1000 μg kg−1, i.v.) and clonidine (30, 100 and 300 μg kg−1, i.v.) dose‐dependently decreased blood pressure and heart rate. In D79N α2A‐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. In wild‐type mice, responses to moxonidine (1 mg kg−1, i.v.) were antagonized by the non‐selective, non‐imidazoline α2‐adrenoceptor antagonist, RS‐79948‐197 (1 mg kg−1, i.v.). Affinity estimates (pKi) at human α2A‐, α2B‐ and α2C‐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]‐GTPγS incorporation assay, moxonidine and clonidine were α2A‐adrenoceptor agonists (pEC50/intrinsic activity relative to noradrenaline): moxonidine (5.74/0.85) and clonidine (7.57/0.32). 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. Thus, expression of α2A‐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 α2A‐adrenoceptor agonists in vitro supports this conclusion.

Functional evidence equating the pharmacologically‐defined α1A‐ and cloned α1C‐adrenoceptor: studies in the isolated perfused kidney of rat

1 The present study characterizes and classifies α1‐adrenoceptor‐mediated vasoconstriction in the isolated perfused kidney of rat using quantitative receptor pharmacology and compares the results to radioligand binding studies (made in clonedαl‐adrenoceptor subtypes, nativeα1A‐adrenoceptors in submaxillary gland of rat, andα1A‐adrenoceptors in several other tissues of rat). 2 Concentration‐effect curves to noradrenaline in the presence of 5‐methyl‐urapidil were biphasic, indicating αl‐adrenoceptor heterogeneity. Theαl‐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 thisα1‐adrenoceptor subtype or subtypes was abolished by nitrendipine (1 μm), thereby allowing characterization of the second, high affinity site for 5‐methyl‐urapidil. 3 The following antagonists interacted competitively with noradrenaline at theα?l‐adrenoceptor for which 5‐methyl‐urapidil exhibits high affinity (pKB value): WB 4101 (10.3)>prazosin (9.5) ∼ HV 723 (9.3) ∼ 5‐methyl‐urapidil (9.2)> phentolamine (8.6)> spiperone (pA2 = 8.1) ∼ toxymetazoline (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 withα1‐adrenoceptors. Dehydroniguldipine acted as a competitive antagonist (pKB = 9.0). Thus, the results with antagonists define theα1‐adrenoceptor as anα1A‐adrenoceptor. 4 An agonist ‘fingerprint’ was constructed in the presence of nitrendipine to define further theα1A‐adrenoceptor. The following order and relativity of agonist potency was obtained: cirazoline (1) ∼ adrenaline (2)> noradrenaline (5)> phenylephrine (23) ∼ amidephrine (31)> methoxamine (71)>> isoprenaline (1456) ∼ 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 bovineαlC‐adrenoceptor (R2 = 0.85), nativeαlA‐adrenoceptors in submaxillary gland of rat (R2 = 0.79), andα1A‐adrenoceptors from several other tissues of rat (values taken from the literature, R2 = 0.89). 6 The present study demonstrates that theα1A‐adrenoceptor is the predominant α‐adrenoceptor subtype mediating vasoconstrictor responses to exogenously administered noradrenaline in the isolated perfused kidney of rat. More importantly, α1A‐adrenoceptors mediating vasoconstrictor responses to noradrenaline exhibited a pharmacological equivalency to the cloned bovineα1C‐adrenoceptor. Thus, definitive functional pharmacological data are provided for equating the two receptors and support results derived recently from molecular and radioligand binding studies.

α1L-Adrenoceptor mediation of smooth muscle contraction in rabbit bladder neck: a model for lower urinary tract tissues of man

The α1‐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. Experiments with several ‘key’ α1‐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. The nature of antagonism by these agents and the profile of affinity determinations generated together suggest that a single α1‐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 ‘α1L’ ‐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 ‘α1L’ ‐adrenoceptor described here in RBN and the rabbit and human cloned α1a‐adrenoceptor (based on data from both whole cell radioligand binding at 37°C and [3H]‐inositol phosphates accumulation assays), characterizations of which have been published elsewhere. 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.

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

To investigate the potential therapeutic benefits of the selective α1A/1l‐adrenoceptor partial agonist Ro 115–1240 in women with mild‐to‐moderate stress urinary incontinence (SUI).

Evidence for a noradrenergic innervation to α1A‐adrenoceptors in rat kidney

1 Experiments were undertaken to characterize the α1‐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 μm) but were resistant to inhibition by chloroethylclonidine (100 μm). 3 5‐Methyl‐urapidil (30 nm), chloroethylclonidine (100 μm) and nitrendipine (1 μm) did not inhibit the neuronal release of tritium from nerves loaded with [3H]‐noradrenaline. 4 The results suggest that renovascular α1A‐adrenoceptors receive a noradrenergic innervation and that the innervated receptors are coupled to dihydropyridine‐sensitive calcium channels.

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

To describe the preclinical pharmacology of Ro 115–1240, a peripherally acting selective α1A/1L‐adrenoceptor (AR) partial agonist, compared with the α1A/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.

Synthesis, pharmacology and pharmacokinetics of 3-(4-Aryl-piperazin-1-ylalkyl)-uracils as uroselective α1A-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.

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.