John R. Fozard

Cardiovascular response to 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) in the rat: site of action and pharmacological analysis.

Summary: The cardiovascular response to 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), a selective putative 5-HT1A receptor agonist, has been investigated in the rat. Comparisons were made with clonidine, a centrally acting hypotensive agent with negligible affinity for 5-HT receptors. In conscious, spontaneously hypertensive (SH) rats, 8-OH-DPAT caused dose-related and sustained falls in blood pressure and heart rate that were unaffected by depletion of brain 5-HT by p-chlorophenylalanine. 8-OH-DPAT caused hypotension and bradycardia in anesthetized normotensive rats. In pithed rats, 8-OH-DPAT neither lowered blood pressure nor affected the cardiovascular response to spinal sympathetic stimulation or to phenylephrine. The response to 8-OH-DPAT was blocked selectively by intracisternal injection of 8-methoxy-2-(N-2-chloroethyl-N-n propyl) amino tetralin (8-MeO-CIEPAT), a putative irreversible 5-HT1A receptor antagonist, and was abolished in animals whose central monoamine transmitter stores were depleted selectively by combined treatment with dl-α-monofluoromethyl-dopa and dopamine. The cardiovascular response to 8-OH-DPAT was inhibited selectively by metergoline, methiothepin, and 8-MeO-CIEPAT; it was nonselectively inhibited by (±)-pindolol, (±)-cyanopindolol, buspirone, yohimbine, idazoxan, and WY 26392; and was unaffected by prazosin and cis-flupenthixol. These results establish that the cardiovascular response to 8-OH-DPAT in the rat is centrally mediated and point to the putative 5-HT1A receptor as the key site involved. An indirect link involving a catecholaminergic mechanism is suggested by the fact that α2-adrenoceptor antagonists are also inhibitory despite 8-OH-DPAT having no direct agonist effects at α2-adrenoceptors per se.

Regional vasodilation is a prominent feature of the haemodynamic response to endothelin in anaesthetized, spontaneously hypertensive rats

Endothelin is an endothelium-derived, 21-res- idue peptide which has powerful vasoconstrictor effects on a variety of isolated blood vessels and, at low doses, raises blood pressure in the auto- nomically blocked rat (Yanagisawa et al., 1988). In the present report, we demonstrate that re- gional vasodilator effects are a prominent feature of the haemodynamic response to the i.v. injection of endothelin in the anaesthetized spontaneously hypertensive (SH) rat treated with a ganglion blocking agent. Male SH rats (353 + 25 g; n-7) were anaesthetized with Inactin ® 120 mg/kg i.p. A tracheotomy was performed and a catheter in- serted into the right carotid artery for mean arterial pressure (MAP) measurement and a catheter placed in the jugular vein for drug administration. Pulsed Doppler flowprobes (Haywood et al., 1981) were placed around the left carotid artery and, through a midline abdominal incision around the left renal artery, the superior mesenteric artery and the lower abdominal aorta (equated to hindquarter blood flow). The incision was closed and, following a one hour stabilisation period, the rats were ganglion-blocked with mecamylamine (0.25 mg/kg i.v.). Mean resting values (+ S.E.M., n = 7) for MAP (and heart rate: HR) before and after ganglion blockade were 141 + 17 mmHg (302 + 25 b/min) and 84 ± 8 mmHg (251 _+ 15 b/min) respectively. The effectiveness of the ganglion

In Vitro and in Vivo Pharmacological Characterization of 5-[(R)-2-(5,6-Diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (Indacaterol), a Novel Inhaled β2 Adrenoceptor Agonist with a 24-h Duration of Action

Here, we describe the preclinical pharmacological profile of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled β2 adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human β2 adrenoceptor (Emax = 73 ± 1% of the maximal effect of isoprenaline; pEC50 = 8.06 ± 0.02), whereas salmeterol displays only partial efficacy (38 ± 1%). The functional selectivity profile of indacaterol over β1 human adrenoceptors is similar to that of formoterol, whereas its β3 adrenoceptor selectivity profile is similar to that of formoterol and salbutamol. In isolated superfused guinea pig trachea, indacaterol has a fast onset of action (30 ± 4 min) similar to formoterol and salbutamol, and a long duration of action (529 ± 99 min) comparable with salmeterol. In the conscious guinea pig, when given intratracheally as a dry powder, indacaterol inhibits 5-hydroxytryptamine-induced bronchoconstriction for at least 24 h, whereas salmeterol, formoterol, and salbutamol have durations of action of 12, 4, and 2 h, respectively. When given via nebulization to anesthetized rhesus monkeys, all of the compounds dose-dependently inhibit methacholine-induced bronchoconstriction, although indacaterol produces the most prolonged bronchoprotective effect and induces the lowest increase in heart rate for a similar degree of antibronchoconstrictor activity. In conclusion, the preclinical profile of indacaterol suggests that this compound has a superior duration of action compatible with once-daily dosing in human, together with a fast onset of action and an improved cardiovascular safety profile over marketed inhaled β2 adrenoceptor agonists.

Subtypes of the 5-HT receptor mediating the behavioural responses to 5-methoxy-N,N-dimethyltryptamine in the rat

The 5-HT receptor subtypes involved in the mediation of reciprocal forepaw treading and the flat body posture induced by the central 5-HT receptor agonist, 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), were examined in intact rats and in rats depleted of monoamines with reserpine. Forepaw treading in non-reserpinised rats was antagonised by the 5-HT2 receptor antagonist, ketanserin, only at doses in excess of those required for occupation of a large proportion of 5-HT2 receptors in brain, and at which there was significant inhibition of stereotyped sniffing induced by the dopamine receptor agonist, apomorphine. Since forepaw treading induced by 5-MeODMT was also blocked in intact rats by haloperidol, blockade of the behaviour by ketanserin may more accurately reflect antagonism at dopamine receptors than at 5-HT2 receptors. In reserpinised rats, i.e. with minimised contributions from other monoamine systems, neither forepaw treading nor the flat body posture were significantly altered by ketanserin, haloperidol or the beta 1- and beta 2-selective adrenoceptor antagonists, betaxolol and ICI 118.551, making a key role for 5-HT2 receptors, dopamine receptors and beta-adrenoceptors unlikely. In contrast, forepaw treading in both reserpinised and non-reserpinised rats was antagonised stereoselectively by pindolol and by spiperone, which interact with 5-HT1 and 5-HT1A recognition sites. The results are consistent with the hypothesis that forepaw treading induced by 5-MeODMT arises by activation of the putative 5-HT1A receptor. Antagonism of the flat body posture by pindolol could be demonstrated only in non-reserpinised rats and the mechanism of induction of this behaviour remains to be established.

Blockade of neuronal tryptamine receptors by metoclopramide

Metoclopramide (0.13, 0.51, 2.0 and 8.1 X 10(-6) M) caused parallel, rightward, shifts in the dose response curves to 5-HT on the isolated rabbit heart. A significant straight line relationship was found between log (5-HT dr--1) and log [metoclopramide] (molar) with a slope of 1.08 +/- 0.13 and giving a pA2 value of 7.20. Metoclopramide did not significantly alter responses to noradrenaline and was 575 times less effective as an inhibitor of DMPP than of 5-HT. The results indicate that metoclopramide is a potent, surmountable and selective antagonist of tryptamine receptors on rabbit cardiac sympathetic nerves.

Evidence that the putative 5-HT1A receptor agonists, 8-OH-DPAT and ipsapirone, have a central hypotensive action that differs from that of clonidine in anaesthetised cats.

Thoracic preganglionic sympathetic nerve activity, blood pressure, heart rate and femoral arterial conductance were recorded in anaesthetised, paralysed cats. Cumulative dose-response curves were constructed for 8-OH-DPAT, ipsapirone and clonidine. All three drugs caused dose-related falls in blood pressure which were associated with minimal changes in femoral arterial conductance. However, 8-OH-DPAT and ipsapirone differed from clonidine in that their hypotensive action was associated with moderate sympathoinhibition and a profound bradycardia, whereas clonidine caused profound sympathoinhibition and, as it did not increase central vagal tone, only a moderate bradycardia. 8-OH-DPAT also caused sympathoinhibition in bi-vagotomised cats and decreased carotid sinus nerve activity along with blood pressure. As 8-OH-DPAT and ipsapirone bind selectively to central 5-HT1A receptors it is concluded that central stimulation of these receptors causes sympathoinhibition and an increase in vagal tone, whereas stimulation of central alpha 2-adrenoceptors causes only sympathoinhibition. In addition, the present data suggest a peripheral vasodilator mechanism may also contribute to the hypotensive effects of 8-OH-DPAT and ipsapirone in the cat. The nature and relative importance of this remains to be established.

Mediation of the discriminative stimulus properties of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) by the putative 5-HT1A receptor

Male Sprague-Dawley rats were trained to discriminate the putative 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) from saline in a 2-lever operant drug discrimination paradigm. The 8-OH-DPAT cue was found to be highly selective; neither the 5-HT receptor agonists, quipazine, LSD, MK 212 and RU 24969, the 5-HT releasing agent, p-chloroamphetamine, nor the alpha 2-adrenoceptor agonist, clonidine, were able to substitute for 8-OH-DPAT in tests of generalization. In contrast, both buspirone and TVX Q 7821, which like 8-OH-DPAT have high affinity and selectivity for the 5-HT1A recognition site, generalized to the 8-OH-DPAT cue in a dose-dependent manner. The discriminative stimulus properties of 8-OH-DPAT were not antagonized by the 5-HT2 receptor antagonist, ketanserin, or the selective beta 1- and beta 2-adrenoceptor antagonists, betaxolol and ICI 118.551, indicating that neither 5-HT2 receptors, nor beta-adrenoceptors play a significant role in the behaviour. However, the 8-OH-DPAT cue was antagonized stereoselectively by pindolol and alprenolol, which have relatively high affinity and stereoselectivity for 5-HT1, but not 5-HT2, recognition sites. Similarly, the capacity of TVX Q 7821 to generalize to the 8-OH-DPAT cue could be blocked by pindolol. In view of the fact that 8-OH-DPAT has negligible affinity for the 5-HT1B site, the above results are consistent with its discriminative stimulus properties being mediated by the putative 5-HT1A receptor. Moreover, agonist activity at central 5-HT1A receptors may be an important mechanism contributing to the anxiolytic properties of buspirone and TVX Q 7821.

A role for mast cells in adenosine A3 receptor‐mediated hypotension in the rat

1 The adenosine A3 receptor agonist, N6–2‐(4‐aminophenyl)ethyladenosine (APNEA) induces hypotension in the anaesthetized rat. The present experiments were carried out to explore the role of mast cells in the response. 2 Intravenous injection of APNEA (1–30 μg kg−1 to rats in which the A3 receptor‐mediated response had been isolated by pretreatment with 8‐(p‐sulphophenyl) theophylline (8‐SPT)), induced dose‐related falls in blood pressure accompanied at higher doses by small falls in heart rate. Responses to the mast cell degranulating agent, compound 48/80 (10–300 μg kg−1, i.v.) were qualitatively similar to those to APNEA. 3 Pretreatment with sodium cromoglycate (0.25‐20 mg kg−1, i.v.) induced dose‐related, although incomplete, blockade of the hypotensive responses to APNEA. At 20 mg kg−1, sodium cromoglycate also inhibited the cardiovascular response to compound 48/80 but had no effects on those to the selective A1 receptor agonist, N6–cyclopentyladenosine (CPA) or the selective A2A receptor agonist, 2‐[p‐(2‐carboxyethyl)phenylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680). Lodoxamide (0.01–20 mg kg−1) also blocked selectively but incompletely the response to APNEA. 4 The cardiovascular responses to compound 48/80 (10–300 μg kg−1, i.v.) were markedly suppressed in animals which had received repeated doses of the compound by the intraperitoneal route. Similarly APNEA was essentially devoid of cardiovascular activity in such preparations. In contrast, responses to CPA were similar in animals treated repeatedly with compound 48/80 to those obtained in control animals. 5 Plasma and serum histamine concentrations were markedly increased associated with the pronounced hypotensive responses induced by intravenous injections of APNEA (30 or 100 μg kg−1) in the presence of 8‐SPT, or compound 48/80 (300 μg kg−1). 6 Taken together the data implicate the mast cell in a key role in adenosine A3 receptor‐mediated hypotension in the anaesthetized rat.

Characterization of MDL 73005EF as a 5‐HT1A selective ligand and its effects in animal models of anxiety: comparison with buspirone, 8‐OH‐DPAT and diazepam

1 With radioligand binding techniques, MDL 73005 EF (8‐[2‐(2,3‐dihydro‐1,4‐benzodioxin‐2‐yl‐methylamino)ethyl]‐8‐azaspiro[4,5]decane‐7,9‐dione methyl sulphonate) shows high affinity (pIC50 8.6) and selectivity (>100 fold compared to other monoamine and benzodiazepine receptor sites) for the 5‐hydroxytryptamine (5‐HT)1A recognition site; it was both more potent and more selective than buspirone in this respect. 2 In rats pretreated with reserpine, 8‐hydroxy‐2‐(di‐n‐propyl‐amino) tetralin (8‐OH‐DPAT) induced forepaw treading and flat body posture; in the same model, MDL 73005EF and buspirone showed minimal agonist activity and at high doses MDL 73005EF inhibited responses to 8‐OH‐DPAT. 3 In rats trained to discriminate 8‐OH‐DPAT from saline in a drug discrimination paradigm, both MDL 73005EF and buspirone generalized dose‐dependently and completely to the 8‐OH‐DPAT cue. 4 To define the anxiolytic potential of MDL 73005EF, it was examined in the elevated plus‐maze test and in the water‐lick conflict test in comparison with diazepam and buspirone. In both tests MDL 73005EF induced effects similar to those seen following diazepam. Buspirone had similar effects to both MDL 73005EF and diazepam in the water‐lick conflict test but opposite effects in the elevated plus‐maze. 8‐OH‐DPAT also had opposite effects in the elevated plus‐maze test to MDL 73005EF and diazepam. 5 The anti‐conflict effects of MDL 73005EF were reversed by low doses of the 5‐HT1A receptor agonist, 8‐OH‐DPAT; those of buspirone were neither antagonised nor mimicked by 8‐OH‐DPAT. 6 These results suggest that an interaction with 5‐HT1A receptors is the basis of the anxiolytic‐like activity of MDL 73005EF. However, its mechanism of action is clearly different from that of buspirone, possibly reflecting a greater selectivity for the 5‐HT1A receptors located presynaptically on central 5‐hydroxytryptaminergic neurones.

Pulmonary edema induced by allergen challenge in the rat: noninvasive assessment by magnetic resonance imaging.

The course of pulmonary edema formation after an intratracheal (i.t.) instillation of ovalbumin was followed noninvasively by magnetic resonance imaging ( MRI ) in actively sensitized Brown Norway (BN) rats. Changes in edema volume assessed by MRI mimicked the results from the analysis of the number and activation of inflammatory cells recovered from the broncho‐alveolar lavage (BAL) fluid. Rats treated with budesonide did not develop edema following challenge with ovalbumin, and these animals showed a significant decrease in BAL fluid inflammatory cell numbers and eosinophil peroxidase and myeloperoxidase activities. Thus, following lung edema formation by MRI provides a reliable means of assessing pulmonary inflammation after allergen challenge. Unlike BAL fluid analysis, which requires killing animals at each time point, this method is noninvasive. MRI could be of importance for the noninvasive profiling of anti‐inflammatory drugs in animal models of asthma and in the clinic. Magn Reson Med 45:88–95, 2001.