Discovery & development of selective M3 antagonists for clinical use.

The treatment of airway obstructive disease may be improved by antimuscarinic agents which selectively block M1 and M3 receptors but do not inhibit prejunctional cholinergic autoreceptors which limit release of acetylcholine. Revatropate is a novel antimuscarinic agent which shows some 50-fold selectivity for M1 and M3 receptors in guinea pig trachea and rabbit vas deferens over the M2 subtype in atria. This selectivity profile was seen in vivo in anaesthetised guinea pigs and conscious dogs where bronchodilator activity was produced in the absence of any effect on heart rate. Revatropate, in contrast to the non-selective agent ipratropium, did not potentiate bronchoconstrictor responses induced by vagal nerve stimulation, indicating that inhibitory autoreceptors were still functional. Early clinical studies in COAD patients showed that inhaled revatropate was an effective bronchodilator which was well tolerated. Darifenacin differs from revatropate by showing selectivity for M3 receptors relative to both M2 and M1 subtypes. [3H] darifenacin had 5-fold higher affinity for the human m3 relative to m1 receptors while there was significantly reduced binding to m2, m4 and m5 receptors. The degree of selectivity in functional tissue preparations was even greater, with darifenacin showing 100-fold selectivity for the ileum M3 receptors over M2 receptors in atria and 30-fold over M1 receptors in rabbit vas deferens. Darifenacin was able to differentiate between M3 receptors in different tissues; although darifenacin was equipotent with atropine in the ileum and bladder, it was some 10-fold and 6-fold less potent at inhibiting muscarinic responses in the trachea and submandibular salivary gland respectively, relative to atropine. Studies in anaesthetised dogs confirmed this selectivity profile. Thus darifenacin inhibited responses of the gut and bladder to cholinergic stimulation without affecting heart rate. Salivary gland responses were inhibited at doses some 6-10 fold higher than those required to inhibit gut and bladder responses. Clinical studies are ongoing in urge incontinence and functional bowel disease which may confirm this selectivity profile.

Pharmacological profile of UK-74,505, a novel and selective PAF antagonist with potent and prolonged oral activity.

UK-74505, a novel 1,4-dihydropyridine PAF antagonist, exhibited highly selective, time-dependent inhibition of PAF-induced aggregation of rabbit washed platelets (IC50 = 26.3 +/- 0.88 and 1.12 +/- 0.04 nM after 0.25 and 60 min preincubation, respectively), which became irreversible within 15 min, whereas inhibition by WEB-2086 was both independent of preincubation time (IC50 = 145.7 +/- 24.7 nM) and competitive (KI = 27.5 +/- 7.7 nM; Schild slope = 0.98 +/- 0.04). The selective inhibition of specific [3H]PAF binding by UK-74,505 exhibited a slower onset, the IC50 obtained without preincubation (14.7 +/- 2.6 nM) decreasing 2-fold at 45 min. UK-74,505 was 450-fold weaker as an antagonist of [3H]nitrendipine binding to bovine brain membranes and KCl-induced contraction of rat aorta. UK-74,505 was 10-30-fold more potent than WEB-2086 in vivo as an inhibitor of PAF-induced hypotension in rats (ED50 = 35 +/- 5.8 micrograms/kg, i.v.), cutaneous vascular permeability in guinea pigs (ED50 = 0.37 +/- 0.08 mg/kg, p.o.) and lethality in mice, with oral ED50 values of 0.26 +/- 0.03 and 1.33 +/- 0.19 mg/kg at 2 and 8 h, respectively. These data demonstrate that UK-74,505 is a potent, selective, long-acting irreversible PAF antagonist.

UK-74,505, a novel and selective PAF antagonist, exhibits potent and long lasting activity in vivo.

UK-74,505, a potent and selective PAF antagonist in vitro, has been shown to be extremely active given orally or intravenously to various species. In anaesthetised guinea pigs UK-74,505 at 30 & 100 micrograms/kg i.v. inhibited bronchoconstrictor responses to aerosolised PAF without affecting blood pressure or heart rate. The increased cutaneous vascular permeability to intradermal PAF in rats was inhibited by UK-74,505 with an ED50 of 280 +/- 5 micrograms/kg p.o. In conscious dogs, complete inhibition of PAF-induced whole blood aggregation ex-vivo occurred for at least 14 hours after an oral dose of 75 micrograms/kg. It is concluded that UK-74,505 is an effective, orally active PAF antagonist of long duration with the potential for once daily dosing.

A study of the muscarinic receptor subtype mediating mucus secretion in the cat trachea in vitro.

Mucus secretion from the cat trachea simulated by muscarinic receptor agonists has been studied by monitoring both the weight and acid glycoconjugate content of samples taken from an in vitro preparation. The nature of the receptor has been probed using a number of competitive muscarinic receptor antagonists by estimating their affinities from the degree to which the response could be blocked. Antagonist affinities have also been compared with those obtained in tracheal smooth muscle and atria from the guinea-pig. Atropine had similar affinities for all receptors investigated. 4DAMP and AF-DX116 had relatively high (pA2 = 9) and low (pA2 = 6) affinities respectively for the secretory receptor. The pA2 value of 7.5 calculated for pirenzepine suggested that the receptor was not of the M1 subtype. However, the value was higher than that for pirenzepine in both guinea-pig tissues indicating that the receptor may be an 'intermediate' between M1 and M2 subtypes. The lack of antagonists with absolute selectivity for a particular subtype of the muscarinic receptor prohibits a definitive classification.

2,4-Diamino-6,7-dimethoxyquinazolines. 2. 2-(4-Carbamoylpiperidino) derivatives as alpha 1-adrenoceptor antagonists and antihypertensive agents.

A series of 4-amino-2-(4-carbamoylpiperidino)-6,7-dimethoxyquinazolines (2) were synthesized and evaluated for alpha-adrenoceptor affinity and antihypertensive activity. These compounds displayed high binding affinity (Ki, 10(-9)-10(-10) M) and selectivity for alpha 1-adrenoceptors in vitro, and 12, 14, 21-26, and 29 showed similar potency to prazosin. Compounds 26 and 28 were also shown to be competitive antagonists of the postjunctional, vasoconstrictor action of norepinephrine with no significant activity at prejunctional alpha 2-sites. The high binding affinity for series 2 is rationalized in terms of enhanced basicity of the quinazoline nucleus (pKa's: 12, 7.38; 26, 7.53; prazosin, 6.8) and hydrophobic interactions of the carbamoyl substituents. Molecular mechanics calculations and computer-assisted superimposition suggest that the quinazoline 2-substituents in prazosin and 2 occupy the alpha 1-adrenoceptor site in different manners. Antihypertensive activity was evaluated after oral administration (5 mg/kg) to spontaneously hypertensive rats, and 11, 15, 21, 22, and 26 displayed sustained prazosin-like efficacy at the 6-h time point. The high alpha 1-adrenoceptor affinity demonstrated by series 2 in vitro suggests that these marked, and prolonged, falls in blood pressure result from selective blockade of the alpha 1-mediated vasoconstrictor effects of norepinephrine.

Comparison of potency of alpha 2-adrenoceptor antagonists in vitro: evidence for heterogeneity of alpha 2-adrenoceptors.

A comparison has been made of affinity of alpha-adrenoceptor antagonists for alpha 2 binding sites in radioligand binding assays, and functional antagonist activity at pre- and postjunctional alpha 2-adrenoceptors in various in vitro preparations. The antagonists displaced [3H]-rauwolscine from rat brain and rabbit spleen membranes but there were substantial differences in rank order and absolute potency in the two tissues. pA2 values for yohimbine, phentolamine and Wy26703 against the selective alpha 2 agonist UK-14,304 were determined in the rat left atrium, rat and rabbit vas deferens and rabbit saphenous vein preparations. The pA2 values varied substantially between the tissues, differing by two orders of magnitude in the case of Wy26703. Yohimbine was more potent in rabbit preparations while Wy26703 was markedly more potent in all the rat preparations. Yohimbine and Wy26703 were compared in the dog saphenous vein preparation where pre- and postjunctional alpha 2 antagonist activity can be compared in the same tissue. As in the rabbit preparations, yohimbine was more potent than Wy26703 at both sites but the absolute potencies were different. It is concluded that alpha 2-adrenoceptors are a heterogeneous population, different subgroups being more apparent between species rather than between tissue types or location.

The alpha 1-adrenoceptor antagonist profile of doxazosin: preclinical pharmacology.

The antihypertensive efficacy of the new alpha 1-adrenoceptor antagonist doxazosin is described, and its selectivity for alpha 1-adrenoceptors is reported from both in vivo and in vitro studies. Groups of beagle dogs with chronic perinephritic hypertension were given doxazosin orally, and systolic blood pressure was recorded indirectly from an exteriorized carotid loop. Dogs given doxazosin 0.5 mg kg-1 daily for 10 days showed consistent daily falls in systolic pressure in addition to a progressive reduction in daily pre-dose pressures. A clear indication of antihypertensive action in excess of 24 h post dose was evident. Heart rate changes were minimal. In pentobarbitone anaesthetized dogs pretreated with desimipramine, doxazosin 10-500 micrograms kg-1 i.v. reduced responses of the nictitating membrane to electrical stimulation of the vagosympathetic-depressor nerve trunk (an alpha 1-adrenoceptor response) but had no effect on the chronotropic response of the heart to electrical stimulation of the ansa subclavia. In contrast, the prejunctional alpha 2-adrenoceptor antagonist activity of yohimbine 10-100 micrograms kg-1 i.v. was manifest as a marked dose-related increase in both the heart rate and nictitating membrane responses. The lack of effect of doxazosin on postjunctional alpha 2-adrenoceptors in vivo was demonstrated in the anaesthetized cat. Doxazosin at 50 and 100 micrograms kg-1 i.v. inhibited pressor responses to injected phenylephrine (an alpha 1-adrenoceptor agonist) but had no effect on pressor responses to either alpha-methylnoradrenaline (an alpha 2-adrenoceptor agonist) or angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of activity of alpha-adrenoceptor agonists and antagonists in dog and rabbit saphenous vein.

The alpha adrenoceptors present on the saphenous vein of the dog and rabbit were characterised in vitro using the selective alpha 1 antagonist prazosin and the alpha 2 antagonists rauwolscine and yohimbine. In the dog saphenous vein, prazosin and rauwolscine competitively antagonised contractile responses to phenylephrine and UK-14,304 respectively. Noradrenaline was competitively blocked by prazosin only. In contrast, phenylephrine and methoxamine-induced responses in the rabbit saphenous vein were insensitive to prazosin and corynanthine. Rauwolscine competitively blocked responses to UK-14,304, noradrenaline and phenylephrine and pA2 values were similar against each agonist. Noradrenaline and UK-14,304 were equipotent agonists on the rabbit saphenous vein while in the dog vein noradrenaline has a lower potency than UK-14,304. These results suggest that the dog saphenous vein has a mixed population of alpha adrenoceptors, while the alpha adrenoceptors on the rabbit vein are homogeneous, with characteristics of the alpha 2 subtype.

Lack of differential inhibition by nifedipine of pressor responses induced by alpha 1- and alpha 2-adrenoceptor agonists and by angiotensin II in anaesthetized cats.

The effect of nifedipine on pressor dose-response curves to phenylephrine, alpha-methylnoradrenaline and angiotensin II was determined in anaesthetized cats pretreated with propranolol and atropine. The selectivity of phenylephrine and alpha-methylnoradrenaline for postjunctional alpha 1- and alpha 2-adrenoceptors respectively was demonstrated by using the selective alpha 1-adrenoceptor antagonist doxazosin and the relatively selective alpha 2-adrenoceptor antagonist rauwolscine. Nifedipine infused intravenously produced a degree of inhibition of rises in diastolic blood pressure similar to that induced by all three agonists. It is concluded that alpha 1- and alpha 2-adrenoceptor activation induced by phenylethanolamine derivatives is equally dependent on extracellular calcium for vascular smooth muscle contraction. Antagonism of sympathetically mediated or angiotensin-induced vasoconstriction could contribute to the vasodilator effects of nifedipine.

Metabolism of arachidonic acid and its endoperoxide (PGH2) to myotropic products in guinea-pig and rabbit isolated lungs.

1 Conversion of arachidonic acid (AA) and its endoperoxide (PGH(2)) to myotropic metabolites has been studied in isolated Krebs-perfused lungs of guinea-pig and rabbit. A continuous differential bioassay technique was used to measure myotropic metabolites in the lung perfusate.2 AA was metabolized in guinea-pig lungs to thromboxane A(2) (TxA(2)), prostacyclin (PGI(2)) and small amounts of a prostaglandin E(2) (PGE(2))-like substance. The amounts of products were dose-related over the AA range used (1 to 10 mug). PGH(2) was detected only after AA (10 mug).3 Rabbit lungs converted AA (2.5 to 10 mug) to the same products in similar relative proportions but the amounts were 15 to 25% of those produced by guinea-pig lungs.4 Indomethacin (10 nM) preferentially inhibited metabolism of AA to prostaglandins in guinea-pig lungs but preferentially inhibited metabolism to TxA(2) in rabbit lungs. Higher concentrations (50 nM) greatly reduced conversion to all the myotropic metabolites in lungs from both species.5 Imidazole (50 muM) selectively inhibited conversion of AA to TxA(2) and increased conversion to PGI(2) in rabbit lungs. A similar effect was produced in guinea-pig lungs but with much higher concentrations of imidazole (2.5 to 5 mM).6 PGH(2) (800 ng) was converted in guinea-pig lung to TxA(2) (100 ng) and very small amounts of PGI(2) (10 to 16 ng) but only unchanged PGH(2) and some PGE(2) were present in the lung perfusate after injection of PGH(2) in rabbit lung.7 It is concluded that guinea-pig and rabbit lung differ in their ability to metabolize AA to myotropic substances and also in their response and sensitivity to drugs which interfere with prostaglandin and TxA(2) synthesis. The lungs do not appear to have an important role in converting PGH(2) to PGI(2).

Release of smooth muscle-contracting substances from isolated perfused lungs.

Infusion of tryptamine (1-4 mug/ml) through the pulmonary circulation of rat isolated lung perfused with Krebs solution caused release of a mixture of spasmogens contracting isolated smooth muscle preparations. One component of this mixture had biological activity comparable to E-type prostaglandins. Other components included a slow reacting substance comparable to SRS-A and a rabbit aorta-contracting substance comparable to RCS. Infusions of 5-hydroxytryptamine, acetylcholine and histamine (0.5-2 mug/ml) also caused release. Release induced by the tryptamines but not that by acetylcholine and histamine was prevented by methysergide whereas acetylcholine-induced release was prevented by hyoscine which did not affect tryptamine-induced release. The tryptamines and histamine released spasmogens from dog isolated lungs but only histamine was effective in guinea-pig lungs. We conclude that amine-induced release from isolated lungs is a fairly general phenomenon and that it may represent an endocrine function of lung.

The bradykininase activities of extracts of dog lung.

1. Homogenates of dog lungs freed from blood inactivate bradykinin. The bradykininase activity is present in soluble and particulate subcellular fractions.2. The fraction with the highest relative specific activity is that sedimenting between 8,700 g and 78,000 g. This fraction has been studied in more detail.3. There is evidence for two bradykinin inactivating enzymes in this fraction with apparent K(m) values of 7.5 muM and 120 muM.4. The bradykininase activity is not dependent on the concentration of chloride ion.5. The bradykininase activity is inhibited by EDTA, 2:3-dimercaptopropanol, nickel ions and some of the peptides from the venom of Bothrops jararaca and of Agkistrodon halys blomhoffii but not by 2-mercaptoethanol or N-ethylamaleimide.6. Angiotensin II in either 15 or 170 mM chloride ion is not an inhibitor of bradykininase activity but angiotensin I at either chloride concentration is an inhibitor. It is proposed that chloride is not necessary for binding of angiotensin I to converting enzyme but is necessary to ensure the correct orientation of substrate for hydrolysis to proceed.

The removal of noradrenaline in the pulmonary circulation of rat isolated lungs.

1. Removal of noradrenaline by isolated lungs of the rat, perfused via the pulmonary artery with Krebs bicarbonate solution has been studied.2. A constant removal (40.2%) was observed over a concentration range of 2-50 ng noradrenaline/ml (12-300 nM). At 100 ng/ml (600 nM), the removal is significantly reduced to 33.5%.3. The removal of noradrenaline was inhibited by cocaine (1 muM), but not by normetanephrine (5 muM), metaraminol (10 muM), phenoxybenzamine (10 muM) and 5-hydroxytryptamine (110 and 560 nM).4. We conclude that the removal of noradrenaline in the lungs does not involve an uptake process comparable with those previously described for this amine. The uptake process for noradrenaline in the lung is similar to that for 5-hydroxytryptamine and may be unique to this tissue.

The inactivation of bradykinin in the pulmonary circulation of isolated lungs.

1. The isolated lungs of guinea-pigs, rats and dogs, perfused with Krebs bicarbonate solution via the pulmonary artery, inactivated 95, 98 and 99% respectively of the bradykinin infused through them.2. This inactivation process was not inhibited by 2-mercaptoethanol (1 mM) or EDTA (500 muM).3. The inactivation was inhibited by 2:3-dimercaptopropanol (100 muM), N-ethylmaleimide (100 muM), and several of the bradykinin potentiating peptides from Bothrops jararaca, in both natural and synthetic forms.4. The effect of these compounds, 2-mercaptoethanol, EDTA, 2:3-dimercaptopropanol, N-ethylmaleimide and Bothrops peptides on the pulmonary bradykininase activity is the same as their effect on pulmonary angiotensin I converting enzyme.