Alan Geoffrey Roach

Studies on RX 781094: a selective, potent and specific antagonist of α2‐adrenoceptors

1 The selectivity and specificity of RX 781094 [2‐(2‐(1,4 benzodioxanyl))2‐imidazoline HCl] for α‐adrenoceptors have been examined in peripheral tissues. 2 In isolated tissue experiments RX 781094 was a competitive antagonist at prejunctional α2‐adrenoceptors situated on the sympathetic nerve terminals of the rat (pA2 = 8.56) and mouse (pA2 = 7.93) vas deferens and on the parasympathetic nerve terminals of the guinea‐pig ileum (pA2 = 8.55). 3 Although RX 781094 was also a competitive antagonist at the postjunctional α1‐adrenoceptors of the rat anococcygeus muscle (pA2 = 6.10) its affinity for these receptors was markedly less than that displayed for prejunctional sites. From pA2 values obtained in the rat vas deferens and anococcygeus muscle the calculated α2/α1‐adrenoceptor selectivity ratio for RX 781094 was 288. 4 The rank order of α2/α1‐adrenoceptor selectivities for the antagonists studied was RX 781094 > RS 21361 > yohimbine > piperoxan > phentolamine > WB 4101 > prazosin. 5 RX 781094 had extremely low affinity for β‐adrenoceptors, histamine receptors, cholinoceptors, 5‐hydroxytryptamine and opiate receptors in vitro. 6 In pithed rats, intravenous administration of RX 781094 antagonized the prejunctional α2‐adrenoceptor agonist effects of clonidine and guanabenz on electrically‐induced contractions of the vas deferens and anococcygeus muscle respectively. 7 In the vas deferens the rank order of α2‐adrenoceptor antagonist potencies was RX 781094 > phentolamine > piperoxan > yohimbine > RS 21361 > WB 4101. Only RX 781094, yohimbine and RS 21361 were active against guanabenz in the anococcygeus muscle. 8 In the pithed rat, RX 781094 preferentially antagonized the pressor responses evoked by postjunctional α2‐adrenoceptor activation by UK 14,304 although higher doses also inhibited the effects of phenylephrine and cirazoline at postjunctional α1‐adrenoceptors. 9 RX 781094 had little effect on the cardiovascular responses to 5‐hydroxytryptramine, angiotensin II, histamine, acetylcholine and isoprenaline in pithed rats and rats anaesthetized with pentobarbitone. 10 These results demonstrate that RX 781094 is a potent and selective α2‐adrenoceptor antagonist with a high degree of specificity for these receptors.

Zebrafish: an emerging technology for in vivo pharmacological assessment to identify potential safety liabilities in early drug discovery

The zebrafish is a well‐established model organism used in developmental biology. In the last decade, this technology has been extended to the generation of high‐value knowledge on safety risks of novel drugs. Indeed, the larval zebrafish appear to combine advantages of whole organism phenotypic assays and those (rapid production of results with minimal resource engagement) of in vitro high‐throughput screening techniques. Thus, if appropriately evaluated, it can offer undeniable advantages in drug discovery for identification of target and off‐target effects. Here, we review some applications of zebrafish to identify potential safety liabilities, particularly before lead/candidate selection. For instance, zebrafish cardiovascular system can be used to reveal decreases in heart rate and atrial–ventricular dissociation, which may signal human ether‐a‐go‐go‐related gene (hERG) channel blockade. Another main area of interest is the CNS, where zebrafish behavioural assays have been and are further being developed into screening platforms for assessment of locomotor activity, convulsant and proconvulsant liability, cognitive impairment, drug dependence potential and impaired visual and auditory functions. Zebrafish also offer interesting possibilities for evaluating effects on bone density and gastrointestinal function. Furthermore, available knowledge of the renal system in larval zebrafish can allow identification of potential safety issues of drug candidates on this often neglected area in early development platforms. Although additional validation is certainly needed, the zebrafish is emerging as a versatile in vivo animal model to identify off‐target effects that need investigation and further clarification early in the drug discovery process to reduce the current, high degree of attrition in development.

The pharmacology of prazosin, a novel antihypertensive agent

During the past few years a large amount of pharmacological and physiological evidence has been obtained in favor of two distinct types of α-adrenoceptors. As a working hypothesis, it is feasible to assume that both α1- and α2-adrenoceptors are abundant on the vascular effector site, whereas the α-adrenoceptors (the blockade of which increases norepinephrine release) predominate at the level of peripheral sympathetic nerve endings. Prazosin is a novel, selective antagonist of α1-adrenoceptors and can be considered an important advancement both pharmacologically and therapeutically since this compound in contrast to classical α-adrenoceptor blocking agents, is effective for the treatment of high blood pressure. Prazosin lacks direct myorelaxant properties and, unlike many vasodilators, in doses lowering blood pressure it does not produce undesirable increases in heart rate and plasma renin activity. Prazosin has proved to be a very useful pharmacological tool since it has permitted us the furtherance of our knowledge with respect to the subclassification of receptors mediating the effects produced by α-adrenoceptor agonists, particularly clonidine. Pharmacokinetic and metabolic studies on prazosin given orally indicate that in animals and in man this compound has a low bioavailability, short half life and undergoes extensive biotransformation. The most common clinical use of prazosin is as an antihypertensive agent and is often given in association with established blood pressure lowering drugs. Recently, it was shown to be useful in the treatment of congestive heart failure, but for this application tolerance has been described. Generally, patients treated chronically with prazosin suffer only minor unwanted effects. This is in contrast to past experience with traditional α-adrenoceptor antagonist. The most serious side effect of prazosin is known as the “first dose phenomenon” which can sometimes lead to syncope. However, it can be avoided if prazosin therapy is initiated with minimally effective doses and individually tailored to obtain the desired antihypertensive effect. Presently, the interesting clinical profile of prazosin is attributed to its novel property of being a selective antagonist of postsynaptic α1-adrenoceptors. Howeverm this is probably an over simplification since some therapeutic observations are not entirely consistent with results which would have been expected for a selective α1-adrenoceptor. For example, prazosin, like the classical antagonists, would be expected to produce sexual dysfunction but, in fact, does not to any significant degree. Future studies with new chemical structures sharing the pharmacological profile of prazosin will clarify the real role of the selectivity towards α1-adrenoceptors in the therapeutic success of prazosin.

Zebrafish based assays for the assessment of cardiac, visual and gut function — potential safety screens for early drug discovery

INTRODUCTIONnSafety pharmacology is integral to the non-clinical safety assessment of new chemical entities prior to first administration to humans. The zebrafish is a well established model organism that has been shown to be relevant to the study of human diseases. The potential role of zebrafish in safety pharmacology was evaluated using reference compounds in three models assessing cardiac, visual and intestinal function.nnnMETHODSnCompound toxicity was first established in zebrafish to determine the non toxic concentration of a blinded set of 16 compounds. In the cardiac assay, zebrafish larvae at 3 days post fertilisation (d.p.f.) were exposed to compounds for 3 h before measurement of the atrial and ventricular rates. To investigate visual function, the optomotor response was assessed in 8 d.p.f. larvae following a 5 day compound exposure. In the intestinal function assay, the number of gut contractions was measured in 7 d.p.f. larvae after a 1 h compound exposure. Finally, compound uptake was determined for 9 of the 16 compounds to measure the concentration of compound absorbed by the zebrafish larvae.nnnRESULTSnSeven compounds out of nine produced an expected effect that was statistically significant in the cardiac and visual functions assays. In the gut contraction assay, six out of ten compounds showed a statistically significant effect that was also the expected result whilst two displayed anticipated but non-significant effects. The compound uptake method was used to determine larval tissue concentrations and allowed the identification of false negatives when compound was poorly absorbed into the zebrafish.nnnDISCUSSIONnOverall, results generated in three zebrafish larvae assays demonstrated a good correlation between the effects of compounds in zebrafish and the data available from other in vivo models or known clinical adverse effects. These results suggest that for the cardiac, intestinal and visual function, zebrafish assays have the potential to predict adverse drug effects and supports their possible role in early safety assessment of novel compounds.

Human Vascular Smooth Muscle Cells Express Receptors for CC Chemokines

Arteriosclerotic lesions are characterized by the accumulation of T lymphocytes and monocytes and the proliferation of intimal smooth muscle cells. Expression of the chemokine monocyte chemoattractant protein-1 (MCP- 1) has been observed in arteriosclerotic plaques and has been proposed to mediate the transendothelial migration of mononuclear cells. More recently, MCP-1 has been proposed to affect the proliferation and migration of vascular smooth muscle cells (VSMCs). We have used reverse transcription-polymerase chain reaction (RT-PCR) to investigate chemokine mRNA expression in human arteriosclerotic lesions obtained from surgical biopsy of diseased vascular tissue and show, in addition to MCP-1, expression of the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) at higher levels than in normal aortic tissue. We have also used RT-PCR to characterize the expression of known chemokine receptors by primary human VSMCs. Messenger RNA for the MIP-1alpha/RANTES receptor, CCR-1, and the MCP-1/MCP-3 receptor, CCR-2, was expressed by unstimulated VSMCs grown under serum-free culture conditions for 24 hours. The receptors CCR-3, CCR-4, CCR-5, CXCR-1, and CXCR-2 were not expressed by VSMCs. The presence of functionally coupled receptors for MIP-1alpha on VSMCs was demonstrated by specific binding of biotinylated MIP-1alpha and increases in intracellular Ca2+ levels after exposure to this chemokine. Taken together, these results suggest that chemokines are likely to be involved in arteriosclerosis and may play a role in modulating the function of VSMCs in vivo.

Non-Associative Learning in Larval Zebrafish

Habituation, where a response is reduced when exposed to a continuous stimulus is one of the simplest forms of non-associative learning and has been shown in a number of organisms from sea slugs to rodents. However, very little has been reported in the zebrafish, a model that is gaining popularity for high-throughput compound screens. Furthermore, since most of the studies involving learning and memory in zebrafish have been conducted in adults, we sought to determine if zebrafish larvae could display non-associative learning and whether it could be modulated by compounds identified in previous rodent studies. We demonstrated that zebrafish larvae (7 days post fertilization) exhibit iterative reduction in a startle response to a series of acoustic stimuli. Furthermore, this reduction satisfied criteria for habituation: spontaneous recovery, more rapid reductions in startle to shorter intertrial intervals and dishabituation. We then investigated the pathways mediating this behavior using established compounds in learning and memory. Administration of rolipram (PDE4 inhibitor), donepezil (acetylcholinesterase inhibitor), and memantine (N-methyl-D-aspartic acid (NMDA) receptor antagonist) all increased the acoustic startle response and decreased habituation in the larvae, similar to previous rodent studies. Further studies demonstrated that NMDA blocked the memantine response and the effect of donepezil was blocked by mecamylamine but not atropine suggesting that the donepezil response was mediated by nicotinic rather than muscarinic receptors. Zebrafish larvae possess numerous advantages for medium to high-throughput screening; the model described herein therefore offers the potential to screen for additional compounds for further study on cognition function.

Chemokine production by human vascular smooth muscle cells: modulation by IL-13

1 The production of chemokines by vascular smooth muscle cells (SMC) is implicated in the pathogenesis of atherosclerosis, although the factors regulating chemokine production by these cells are incompletely characterized. 2 We describe the differential stimulation of interleukin‐(IL)‐8, monocyte chemoattractant protein (MCP)‐1 and regulated on activation normal T‐cell expressed and secreted (RANTES) synthesis following treatment of human vascular SMC with IL‐1α or tumour necrosis factor α (TNFα). Under basal conditions, cultured SMC release very low amounts of IL‐8, MCP‐1 and RANTES as assessed by specific ELISA. Concentration‐response studies with IL‐1α or TNFα revealed that each stimulus induced a similar amount of MCP‐1. In contrast approximately three fold more IL‐8 was induced by IL‐1α than by TNFα whereas significant RANTES production was induced only by TNFα. These findings point to a divergence in the regulation of synthesis of the different chemokines in response to IL‐1α or TNFα stimulation. 3 The T‐cell derived cytokines IL‐10 and IL‐13 were also found to have differential effects on chemokine production by SMC. IL‐13, but not IL‐10, significantly enhanced IL‐8 and MCP‐1 release in response to IL‐1α or TNFα. This increase in chemokine release appeared to be accounted for by increased mRNA expression. 4 These findings provide support for the concept that smooth muscle cells can have an active role in a local immune response via the production of chemokines which can be selectively modulated by T‐cell derived cytokines.

α2‐Adrenoceptor agonists induce mydriasis in the rat by an action within the central nervous system

1 The effects of intravenous administration of the selective α2‐adrenoceptor agonists clonidine, UK 14,304 and guanoxabenz on rat pupil diameter were investigated. 2 In rats anaesthetized with pentobarbitone, each agonist produced a marked dose‐related increase in pupil diameter; the rank order of potency was: clonidine > UK 14,304 > guanoxabenz. 3 Pretreatment with the selective α2‐adrenoceptor antagonist, RX 781094 (0.5 mg/kg, i.v.), produced a parallel 30–40 fold shift to the right of the dose‐pupil dilator response curves for the three agonists. Yohimbine (1.5 mg/kg, i.v.) produced about a 10 fold rightward shift of the dose‐response curve for guanoxabenz. In contrast, the α1‐selective antagonist, prazosin (0.5 mg/kg, i.v.), failed to affect the dose‐response relation for guanoxabenz. 4 Several antagonists of varying selectivities towards α1‐ and α2‐adrenoceptors were tested for their ability to reverse the maximal mydriasis induced by guanoxabenz (0.3 mg/kg, i.v.). The rank order of potency of the antagonists producing a 50% reversal of this effect was: RX 781094 > yohimbine > piperoxan = rauwolscine > mianserin > RS 21361. Neither corynanthine nor prazosin reversed the guanoxabenz‐induced mydriasis. 5 Topical application of RX 781094 (0.1 to 3% w/v solutions) onto one eye produced a slow reversal of guanoxabenz‐induced mydriasis; the time course and degree of reversal were virtually the same in both eyes. 6 Intracerebroventricular administration of RX 781094 (1.25–15 μg total dose) caused a rapid dose‐related reversal of the maximal mydriasis induced by guanoxabenz (0.3 mg/kg, i.v.). 7 Guanoxabenz (0.3 and 1.0 mg/kg, i.v.) did not produce any dilation of the physostigmine‐constricted undamaged pupil of the pithed rat. Intravenous adrenaline was found to produce a small mydriatic effect, while atropine completely antagonized the effects of physostigmine in this preparation. 8 These results indicate that α2‐adrenoceptor agonists induce mydriasis in the rat through a central α2‐adrenoceptor mechanism. However, the site of action within the central nervous system remains to be determined.

Studies on the mechanism of the vasodilator effects of prazosin in dogs and rabbits

In pentobarbital (35.0 mg/kg) anaesthetised dogs, bolus injections of prazosin into the femoral artery (3.0--300.0 microgram) provoked a dose-related fall in the vascular resistance of the innervated hind limb. In contrast to papaverine, prazosin failed to produce the same effect in dogs under spinal anaesthesia even when the intrinsic femoral vascular tone was increased with vasopressin. However, vasodilator effects of prazosin were again observed when the tone of the limb was elevated by either stimulating the sympathetic lumbar chain or by infusing alpha-adrenoceptor agonists. A significant reduction of both aortic blood pressure and pressor response to bilateral carotid artery occlusion was noted in a group of normotensive dogs anaesthetised 12 h after the last dose of prazosin given twice daily at 0.5 mg/kg, p.o., for 3 day period. This short-term treatment modified neither the resting heart rate nor the positive chronotropic effect induced by either intravenous noradrenaline or electrical stimulation of pre- and post-ganglionic nerve fibres of the right stellate ganglion. However, it prevented the larger increase in heart rate in response to bilateral carotid occlusion in placebo-treated dogs after section of the vagi. A decrease in baseline sympathetic tone of the perfused hind limb as well as vasoconstrictor effects produced by i.a. injections of several alpha-adrenoceptor agonists and electrical stimulation of the lumbar sympathetic chain was observed in prazosin-treated animals. The dose--pressor response profiles to these alpha-adrenoceptor stimulants after prazosin were not parallel to those obtained in the control group. The vasoconstrictor response to angiotensin II was not changed by prazosin. In rabbit aortic strips, prazosin (0.1--3.0 micrometer) produced competitive antagonism of the contractile responses induced by cirazoline, noradrenaline and phenylephrine. In contrast to papaverine, prazosin in concentrations up to 100.0 micrometer neither relaxed the aortic strips contracted by potassium ions nor modified the concentration-response curve to calcium ions. These studies indicate that blood pressure lowering effects of prazosin given acutely or for three days can be accounted for by a clear-cut functional impairment of vascular postsynaptic alpha-adrenoceptors. No evidence for a direct myorelaxant property of prazosin could be obtained in these studies.

EFFECTS OF CLONIDINE, PRAZOSIN AND PHENTOLAMINE ON HEART RATE AND CORONARY SINUS CATECHOLAMINE CONCENTRATION DURING CARDIOACCELERATOR NERVE STIMULATION IN SPINAL DOGS

1 In spinal dogs, continuous electrical stimulation of the cardioaccelerator nerve produced a transient rise in aortic blood pressure and a sustained increase in both heart rate and coronary sinus blood flow. The latter effects were accompanied by a significant elevation in the coronary sinus plasma noradrenaline concentration without significant changes in the levels of dopamine and adrenaline. The concentrations of the three catecholamines in thoracic aorta plasma were not significantly changed by cardioaccelerator nerve stimulation. 2 Clonidine (20 μg/kg, i.v.), given during cardioaccelerator nerve stimulation, increased both mean aortic blood pressure and coronary sinus blood flow and decreased heart rate and coronary sinus venous plasma noradrenaline overflow. 3 Phentolamine (0.3 mg/kg, i.v.) completely antagonized these effects of clonidine. Prazosin (0.3 mg/kg, i.v.) inhibited by only 43 and 38% the respective reductions in heart rate and noradrenaline overflow elicited by clonidine. 4 On termination of cardioaccelerator stimulation (about 10 min after either prazosin or phentolamine), heart rate and coronary sinus noradrenaline overflow returned to control prestimulation levels. 5 Phentolamine or prazosin, administered alone during stimulation of the cardioaccelerator nerve, increased heart rate and noradrenaline overflow into the coronary sinus plasma. However, intravenous phentolamine and prazosin, in contrast to desipramine (0.3 mg/kg, i.v.) or tyramine (1.0 mg, i.a.), failed to change the tachycardia resulting from the local administration of noradrenaline into the sinus node artery (i.a.). 6 These results show that in spinal dogs the clonidine‐induced reduction in heart rate (elevated by electrical stimulation of the cardioaccelerator nerve) is accompanied by a fall in the quantity of noradrenaline overflowing into the coronary sinus plasma. The latter effect is presumably the result of an action of clonidine on cardiac presynaptic α‐adrenoceptors, the activation of which is followed by a reduction in the release of noradrenaline per nerve impulse. Phentolamine and prazosin are both antagonists of cardiac presynaptic α‐adrenoceptors in spinal dogs, as suggested by their action against clonidine and by their positive chronotropic effect when administered during stimulation of the cardioaccelerator nerve.