British Journal of Pharmacology

Published by Wiley
Online ISSN: 1476-5381
Print ISSN: 0007-1188
Publications
1. The effects of three analogues of NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA), inhibitors of nitric oxide (NO) synthase, on hydrogen peroxide (H2O2)-induced endothelial cell injury were studied. 2. Endothelial cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH) and 51Cr. 3. Addition of H2O2 (250-1,000 microM) to endothelial cells induced the release of LDH dose-dependently. The release of LDH was reduced by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4)-4 x 10(-3) M), L-NOARG (10(-4)-4 x 10(-3) M) and NG-nitro-L-arginine benzyl ester (L-NABE, 10(-4)-4 x 10(-3) M), inhibitors of NO synthase. 4. L-NOARG analogues also reduced H2O2-induced 51Cr release from endothelial cells, while L-NMMA had no effect. 5. The protective effect of L-NAME was not reversed by addition of L-arginine (L-Arg, 1-10 mM). 6. Both L-NAME and L-NMMA completely inhibited L-Arg metabolism to L-citrulline coupled with NO synthesis. 7. These findings suggest that L-NOARG analogues but not L-NMMA reduced H2O2-induced endothelial cell injury, and that these effects may not be related to inhibition of NO production.
 
1. The effects of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on mean circulatory filling pressure (MCFP), total peripheral resistance (TPR), cardiac output (CO) and resistance to venous return (Rv) were studied in rats. 2. In conscious, unrestrained rats, L-NAME (0.5-16 mg kg-1) dose-dependently increased mean arterial pressure (MAP) but not MCFP, an inverse index of venous compliance, either in the absence or presence of the ganglionic blocker mecamylamine (10 mg kg-1). 3. In pentobarbitone-anaesthetized rats, L-NAME (2, 4, 8 mg kg-1) increased MAP and reduced CO in a dose-related manner but did not change MCFP, TPR (+84, +140 and +192%) as well as Rv (+62, +72, +110%) were dose-dependently increased by L-NAME. 4. Our results show that L-NAME reduces CO by increasing arterial as well as venous resistances. L-NAME does not affect MCFP.
 
1. Some postganglionic sympathetic axons possess P2Y-like P2-purinoceptors which, when activated, decrease the release of noradrenaline. We examined the question of whether such receptors also occur at the noradrenergic axons in the rat brain cortex. Slices of the brain cortex were preincubated with [3H]-noradrenaline, then superfused with medium containing desipramine (1 microM) and stimulated electrically, in most experiments by trains of 4 pulses/100 Hz. 2. The selective adenosine A1-receptor agonist, N6-cyclopentyl-adenosine (CPA; 0.03-3 microM) as well as the non-subtype-selective agonist 5'-N-ethylcarboxamido-adenosine (NECA; 0.3-3 microM) reduced the evoked overflow of tritium, whereas the adenosine A2a-receptor agonist, 2-p-(2-carbonylethyl)-phenethylamino-5'-N-ethylcarboxamido-a denosine (CGS-21680; 0.003-30 microM) and the adenosine A3-receptor agonist N6-2-(4-aminophenyl)ethyl-adenosine (APNEA; 0.03-3 microM) caused no change. Of the nucleotides tested, ATP (30-300 microM), adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S; 30-300 microM), adenosine-5'-O-(2-thiodiphosphate) (ADP beta S; 30-300 microM), P1,P4-di(adenosine-5')-tetraphosphate (Ap4A; 30-300 microM) and the preferential P2Y-purinoceptor agonist, 2-methylthio-ATP (300 microM) decreased the evoked overflow of tritium. The P2X-purinoceptor agonist, alpha,beta-methylene-ATP (3-300 microM) caused no change. 3. The A1-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM) attenuated the effects of the nucleosides CPA (apparent pKB value 9.8) and NECA as well as of the nucleotides ATP (apparent pKB 9.3), ATP gamma S (apparent pKB 9.2) and ADP beta S (apparent pKB 8.7). CGS-21680 and APNEA were ineffective also in the presence of DPCPX. The A2-selective antagonist 1,3-dipropyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KF-17837) reduced the effects of CPA, NECA and ATP gamma S only when given at a concentration of 300 nM but not at 1O nM.4. The P2-purinoceptor antagonists, suramin (300 micro M), reactive blue 2 (30 micro M) and cibacron blue 3GA(30 micro M) did not change the effect of CPA. Suramin and cibacron blue 3GA shifted the concentration response curve of ATP gamma S to the right (apparent pKB values 3.7 and 5.0, respectively). Reactive blue 2 also attenuated the effect of ATPyS, and cibacron blue 3GA attenuated the effect of ATP, but in these cases the agonist concentration-response curves were not shifted to the right. There was no antagonistic effect of suramin against ATP and ADP beta S.5. The results indicate that rat cerebrocortical noradrenergic axons possess, in addition to the knownadenosine Al-receptor, a separate purinoceptor for nucleotides (P2) which, in contrast to the Al-receptor,is blocked by suramin, reactive blue 2 and cibacron blue 3GA. Nucleotides such as ATP and ATP gamma S activate both receptors. Inconsistencies in antagonist effects against nucleotides are probably due to this activation of two receptors. The presynaptic P2-purinoceptor is P2Y-like, as it is in the peripheral sympathetic nervous system.
 
1. The influence of the nitric oxide (NO) biosynthesis inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on the gastric relaxation induced by peripheral vagal stimulation was investigated in the anaesthetized rat. 2. Peripheral vagal stimulation (10 Hz, 10 V, 1 ms for 20 s) induced a reproducible biphasic response: a short-lasting increase followed by a more pronounced decrease in intragastric pressure. This response also occurred in reserpinized animals (5 mg kg-1, i.p., 24 h before the experiment) while atropine (1 mg kg-1, i.v.) abolished the initial increase in intragastric pressure. 3. L-NAME (1-30 mg kg-1, i.v.) induced an increase in arterial blood pressure. L-NAME (1 mg kg-1, i.v.) had no influence on the vagally induced gastric response while L-NAME (10 and 30 mg kg-1 i.v.) significantly changed it: the initial increase in intragastric pressure was enhanced while the decrease in intragastric pressure was reduced or abolished. N(G)-nitro-L-arginine (L-NNA, 10 mg kg-1, i.v.) had the same effect. 4. An i.v. infusion of phenylephrine (10-mu-g kg-1 min-1) inducing a pressor response similar to that produced by L-NAME (30 mg kg-1, i.v.) did not influence the vagal gastric response. Infusion of L-arginine (300 mg kg-1 bolus, then 100 mg kg-1 h-1) starting 30 min beforehand, reduced the pressor effect and prevented the influence of L-NAME (10 mg kg-1, i.v.) on the vagal gastric response. After injection of both atropine (1 mg kg-1, i.v.) and L-NAME (30 mg kg-1, i.v.), the vagally induced decrease in intragastric pressure was similar to that obtained under control conditions. 5. These results are consistent with NO being released and inducing gastric relaxation during peripheral vagal stimulation. In addition to NO, another inhibitory non-adrenergic non-cholinergic neurotransmitter is released.
 
The efflux of 86Rb from rat isolated pancreatic islets preloaded with the isotope and perifused in vitro, has been used to monitor the effects of sulphonylureas on the potassium permeability, Pk, of pancreatic beta-cells. Tolbutamide (5 microM to 5 mM) had a dual effect, causing initially a decrease in 86Rb efflux (the 'on' response) which was rapidly superseded on drug removal by a large phasic increase in 86Rb efflux (the 'off' response). Each kinetic response had a different dose-dependency: the 'on' response was half-maximal at tolbutamide concentrations of 0.02 mM, maximal at 0.2 mM and decreased by concentrations greater than 0.2 mM whereas the 'off' response was half-maximal at 0.07 mM, maximal at 0.7 mM, with further increases in concentration (up to 5 mM) causing no further change in magnitude. Analysis of the time- and concentration-dependency of tolbutamide action, by presenting increasing concentrations (0 to 1.4 mM) of tolbutamide as a ramp or step function, established a critical dependence of the kinetics of 86Rb efflux during and after exposure to tolbutamide upon the initial rate of increase of the tolbutamide concentration rather than its final steady state. In the presence of quinine (10 microM), D600 (50 microM), or tetraethylammonium (20 mM), the secondary increase in 86Rb following tolbutamide (0.7 mM) removal was totally inhibited. Co2+ (2.56 mM) not only blocked the secondary 'off' response but also potentiated the initial 'on' response of tolbutamide. Glibenclamide produced a rapid decrease in 86Rb efflux but at a much lower concentration (10 microM) than tolbutamide and with no 'off' response apparent over a wide range of concentration (1 to 100 microM); moreover the decrease in 86Rb efflux was sustained and only slowly reversible. It is concluded that tolbutamide has two opposing actions on islet beta-cell 86Rb efflux, and therefore PK: (i) a tendency to increase a calcium-sensitive PK by stimulating calcium entry into the cell and (ii) a decrease in PK that may be due to a direct effect on the calcium-sensitive PK itself. The more sustained pharmacological action of glibenclamide is explained by the longer-lasting decrease in PK that it produces.
 
The effect of forskolin and several H2-agonists was investigated on the activity of adenylate cyclase in homogenates of guinea-pig lung parenchyma. Histamine, 0.1 microM to 1 mM, dimaprit, 1 microM to 10 mM, 4-methyl histamine, 0.1 microM to 10 mM, impromidine, 10 nM to 10 microM and forskolin, 1 nM to 100 microM, all produced a dose-dependent stimulation of adenylate cyclase activity above the basal level. The histamine H1-receptor antagonist mepyramine, 10 microM, and beta-adrenoceptor antagonist propranolol, 10 microM, had no effect on the stimulation by histamine of adenylate cyclase. The dose-response curve for stimulation by histamine of adenylate cyclase was shifted to the right in a dose-dependent manner by increasing concentrations of several H2-antagonists. Schild plots constructed for each H2-antagonist produced straight lines with slopes not significantly different from unity. The equilibrium dissociation constants obtained for the H2-antagonists in this study were similar to those previously reported for inhibition of dimaprit-induced relaxation of the pre-contracted lung strip, inhibition of [3H]-tiotidine binding to homogenates of guinea-pig lung parenchyma and inhibition of histamine-stimulated adenylate cyclase in guinea-pig gastric mucosa.
 
1. The cardiovascular and vasorelaxant effects of (+)-glaucine and of a semisynthetic derivative (N-carbethoxysecoglaucine) were studied in rats. 2. N-carbethoxysecoglaucine did not modify either systolic arterial pressure or heart rate values in conscious (25 mg kg-1, p.o.) and anaesthetized normotensive rats (5 mg kg-1, i.v.). Furthermore, this compound showed no activity in the experiments carried out on rat isolated aorta [contractility and 45Ca2+ influx assays (5 microM)] and did not modify the rate and force of contraction in rat isolated atria (5 microM). 3. In conscious normotensive rats, oral administration of (+)-glaucine (25 mg kg-1) did not modify either systolic arterial pressure or heart rate. 4. In anaesthetized normotensive rats, (+)-glaucine (5 mg kg-1, i.v.) produced a remarkable fall in mean arterial pressure (MAP) accompanied by a significant decrease in heart rate. In the same preparation, (+)-glaucine (5 mg kg-1, i.v.) did not modify the cardiovascular effects induced by noradrenaline (NA) (5 micrograms kg-1) and 5-hydroxytryptamine (5-HT) (300 micrograms kg-1) but markedly inhibited those induced by nicotine (200 micrograms kg-1). 5. In isolated intact aorta of rat, (+)-glaucine (0.15-5 microM) competitively inhibited the contractions induced by NA (with a pA2 value of 7.14) and non-competitively those induced by 5-HT (in normal Krebs solution) and Ca2+ (in depolarizing Ca(2+)-free high-K+ 50 mM solution), with depression of the maximal response and with pD2 values of 5.56 and 5.26, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
 
1. The effects of noradrenaline, ATP, adenylyl-imidodiphosphate (AMP-PNP), adenosine, alpha,beta-methylene-ATP and the P2-purinoceptor antagonist, suramin on N'-acetyl-5-hydroxytryptamine production were studied in cultured denervated rat pineal glands. 2. Noradrenaline (3 nM-1 microM) increased N'-acetyl-5-hydroxytryptamine production as measured both in the gland and the culture medium. 3. In noradrenaline (10 nM)-stimulated pineal glands, ATP (0.03 nM-1 mM) or AMP-PNP (0.1 microM-1 mM) increased N'-acetyl-5-hydroxytryptamine production in a concentration-dependent manner. 4. Alpha,beta-Methylene-ATP at the concentration of 0.1 mM, but not 3 microM, attenuated the enhancement by ATP (0.1 mM) of noradrenaline (10 nM)-induced N'-acetyl-5-hydroxytryptamine production. 5. Suramin (0.1 mM) blocked the potentiating effect of ATP (0.1 mM), but not the potentiating effect of adenosine (0.1 mM) in glands incubated with noradrenaline (10 nM). 6. These findings suggest that the rat pineal gland possesses P2-purinoceptors which when stimulated potentiate the effect of noradrenaline but do not, by themselves, induce an increase in N'-acetyl-5-hydroxytryptamine production.
 
1. The effects of two inhibitors of nitric oxide synthase, NG-monomethyl L-arginine (L-NMMA) and NG-nitro L-arginine (L-NOARG), were examined on non-adrenergic non-cholinergic (NANC) inhibitory transmission in the rat anococcygeus, bovine retractor penis (BRP) and bovine penile artery. 2. In the rat anococcygeus, L-NMMA (10-1000 microM) produced a concentration-dependent augmentation of guanethidine (30 microM)-induced tone and inhibited NANC relaxation at all frequencies tested (0.1-20 Hz): the maximum inhibition obtained was 56 +/- 6% (n = 6). L-NOARG (0.3-30 microM) also augmented tone and inhibited NANC relaxation in a concentration-dependent manner, but unlike L-NMMA the maximum inhibition was 100%. 3. In the BRP, L-NMMA (10-100 microM) had no effect on tone or NANC-induced relaxation, but at 1000 microM tone was increased and NANC relaxation inhibited by 25 +/- 7% (n = 6). L-NOARG (0.3-30 microM) produced a concentration-dependent increase in tone and inhibition of NANC relaxation. As in the rat anococcygeus, inhibition of NANC relaxation was complete. 4. The effects of L-NMMA and L-NOARG were stereospecific since D-NMMA (10-1000 microM) and D-NOARG (1-1000 microM) had no effect on tone or NANC relaxation of the rat anococcygeus or BRP. 5. L-Arginine (10-300 microM) had no effect by itself on NANC-induced relaxation of the rat anococcygeus or BRP. It did, however, reverse the ability of L-NMMA (10-1000 microM) to augment tone and inhibit NANC relaxation in the rat anococcygeus and BRP.(ABSTRACT TRUNCATED AT 250 WORDS)
 
1. Conscious, Long Evans rats, chronically instrumented for the measurement of regional haemodynamics, were used to assess responses to 3 min infusions of the potassium channel opener, BRL 38227 (1 and 10 micrograms kg-1 min-1) or adrenaline (0.05 and 0.5 microgram kg-1 min-1) in the absence and in the presence of NG-nitro-L-arginine methyl ester (L-NAME; 3 mg kg-1 h-1), an inhibitor of nitric oxide biosynthesis. 2. In the absence of L-NAME, the low dose of BRL 38227 caused slight hypotension and tachycardia, accompanied by small increases in mesenteric and hindquarters blood flow only. However, there were increases in renal, mesenteric and hindquarters vascular conductances. L-NAME had no effect on any of these responses. 3. The high dose of BRL 38227 caused substantial hypotension and tachycardia. Renal and hindquarters flows did not change significantly, but there was a marked increase in mesenteric flow. There were only modest increases in renal and hindquarters vascular conductances but a substantial mesenteric vasodilatation. In the presence of L-NAME, there was a slight reduction of the latter but no other changes in the responses to BRL 38227. 4. In the absence of L-NAME, the low dose of adrenaline caused slight hypotension but a marked tachycardia. There were no changes in renal or mesenteric blood flow but a clear-cut increase in hindquarters flow. Renal and mesenteric vascular conductances showed only small rises, in contrast to the substantial hindquarters vasodilatation. In the presence of L-NAME, there was significant attenuation of the tachycardia and of the increases in hindquarters flow and vascular conductance in response to adrenaline.5. The high dose of adrenaline caused marked hypotension and tachycardia. Renal flow did not change, but there was a fall in mesenteric and a marked rise in hindquarters flow. Renal vascular conductance showed a slight increase but mesenteric vascular conductance did not change significantly, whereas there was a substantial hindquarters vasodilatation. In the presence of L-NAME, adrenaline caused an increase in blood pressure but no significant change in heart rate; the renal vasodilatation was abolished, there was a mesenteric vasoconstriction, and the hindquarters vasodilatation was markedly reduced. L-NAME also attenuated the tachycardia induced by adrenaline in animals with no cardiac baroreflexes.6. The present results indicate that L-NAME-sensitive mechanisms are involved in the vasodilator and tachycardic effects of adrenaline. The relative lack of effect of L-NAME on responses to BRL 38227 indicates that the changes in the responses to adrenaline were not non-specific or due to changes in haemodynamic status caused by L-NAME. The results raise the possibility that the 'hypertensinogenic' properties of endogenous adrenaline could be amplified when nitric oxide biosynthesis is impaired.
 
1. Coronary arteries from bovines (BCA) and pigs (PCA) were used for measuring endothelium-dependent relaxation in the presence of L-NG nitroarginine and indomethacin. As some compounds tested have been found to have an inhibitory effect on autacoid-activated endothelial Ca2+ signalling, endothelium-dependent relaxation was initiated with the Ca2+ ionophore A23187. 2. The common compounds for modulating arachidonic acid release/pathway, mepacrine and econazole only inhibited L-NG nitroarginine-resistant relaxation in BCA not in PCA. In contrast, proadifen (SKF 525A) diminished relaxation in BCA and PCA. Mepacrine and proadifen inhibited Hoe-234-initiated relaxation in BCA and PCA, while econazole only inhibited Hoe 234-induced relaxation in PCA. Due to the multiple effects of these compounds, caution is necessary in the interpretation of results obtained with these compounds. 3. The inhibitor of Ca(2+)-activated K+ channels, apamin, strongly attenuated A23187-induced L-NG nitroarginine-resistant relaxation in BCA while apamin did not affect L-NG nitroarginine-resistant relaxation in PCA. 4. Pertussis toxin blunted L-NG nitroarginine-resistant relaxation in BCA, while relaxation of PCA was not affected by pertussis toxin. 5. Thiopentone sodium inhibited endothelial cytochrome P450 epoxygenase (EPO) in PCA but not in BCA, while L-NG nitroarginine-resistant relaxation of BCA and PCA were unchanged. Protoporphyrine IX inhibited EPO in BCA and PCA and abolished L-NG nitroarginine-resistant relaxation of BCA not PCA. 6. An EPO-derived compound, 11,12-epoxy-eicosatrienoic acid (11,12-EET) yielded significant relaxation in BCA and PCA in three out of six experiments. 7. These findings suggest that L-NG nitroarginine-resistant relaxation in BCA and PCA constitutes two distinct pathways. In BCA, activation of Ca(2+)-activated K+ channels via a pertussis-toxin-sensitive G protein and EPO-derived compounds might be involved. In PCA, no selective inhibition of L-NG nitroarginine-resistant relaxation was found.
 
1. We have studied both the electrophysiological and contractile effects of the purine nucleotide, adenosine-5'-triphosphate (ATP), as well as a number of its structural analogues as agonists at P2X purinoceptors in the rat vas deferens in vitro. 2. Electrophysiological effects were investigated by a whole cell voltage clamp technique (holding potential-70 mV) with fast flow concentration-clamp applications of agonists in single isolated smooth muscle cells. ATP, 2-methylthio adenosine-5'-triphosphate (2-MeSATP) and alpha,beta methylene adenosine-5'-triphosphate (alpha,beta-meATP) all evoked inward currents over a similar concentration range (0.3-10 microM), being approximately equipotent with similar concentrations for threshold effects (0.3 microM). ADP (10 microM) also evoked a rapid current of similar peak amplitude to that seen with ATP (10 microM). 3. alpha,beta-meATP was the most potent agonist in producing concentrations of the rat vas deferens whole tissue preparation, with a threshold concentration equal to that in the electrophysiological studies (0.3 microM). However, ATP and 2-MeSATP were at least ten times less potent in studies measuring contraction than in the electrophysiological studies. Furthermore, their concentration-effect curves were shallow with smaller maximal responses than could be achieved with alpha,beta-meATP. ADP, AMP and adenosine were inactive at concentrations up to 1 mM. The rank order of agonist potencies observed for contraction was alpha,beta-meATP > ATP = 2-MeSATP. 4. Measurement of inorganic phosphate (iP), as a marker of purine nucleotide metabolism in the vasdeferens whole tissue preparation, indicated that ATP and 2-MeSATP were rapidly metabolized,whereas alpha,beta-meATP was stable for up to 2 h. Removal of divalent cations prevented breakdown of ATP and 2-MeSATP, suggesting that metabolism involved a Ca2+/Mg2+-dependent enzyme.5. It appears that in isolated preparations of rat vas deferens, the low potency of ATP and 2-MeSATP can be explained by rapid agonist breakdown by ectonucleotidases. However, this is not the case in the single cell studies where the use of rapid concentration-clamp applications revealed the true potency of the agonists. Under such conditions the three agonists were all equal in potency indicating that the rank order of agonist potencies of alpha,beta-meATP> ATP = 2-MeSATP is not in fact characteristic of smooth muscle P2x-purinoceptors as commonly believed.
 
1. A comparative study was carried out between the adenosine receptor mediating a stimulation of cyclic AMP formation in guinea-pig cerebral cortical slices with the adenosine receptor mediating relaxation of phenylephrine precontracted guinea-pig aortic rings. 2. [3H]-cyclic AMP accumulation in [3H]-adenine-prelabelled guinea-pig cerebral cortical slices was stimulated by adenosine and its analogues with the following EC50 values (microM): 5'-N-ethylcarboxamidoadenosine (3.1 +/- 0.3) > 2-chloroadenosine (10 +/- 2) > adenosine (109 +/- 15). 3. 2-Chloroadenosine and adenosine elicited maximal responses for [3H]-cyclic AMP accumulation that were 100 +/- 7 and 71 +/- 6% of the maximal response to 5'-N-ethylcarboxamidoadenosine, respectively. CGS 21680 (100 microM) and DPMA (100 microM) elicited -2 +/- 2 and 12 +/- 3% of the response to 100 microM 5'-N-ethylcarboxamidoadenosine. 4. Estimation of antagonist potencies at the A2 adenosine receptor of cerebral cortex showed a rank order of potency (K1, nM): xanthine amino congener (35 +/- 3) > 8-cyclopentyl-1,3-dipropylxanthine (130 +/- 22) > PD 115,199 (407 +/- 82) > 3,7-dimethyl-1-propargylxanthine (13 +/- 2 microM). 5. Adenosine analogues produced long-lasting relaxation of phenylephrine-precontracted aortic rings with the following rank order of potency (EC50 values, microM): 5'-N-ethylcarboxamidoadenosine (0.68 +/- 0.06) > 2-chloroadenosine (4.3 +/- 0.6) > adenosine (104 +/- 13). Maximal relaxations elicited by these agents were 71 +/- 3, 98 +/- 1, and 100 +/- 1%, respectively. CGS 21680 and DPMA at 100 microM elicited smaller relaxations of the precontracted tissues (12 +/- 2 and 43 +/- 15%, respectively). 6. Antagonism by xanthine derivatives of the 5'-N-ethylcarboxamidoadenosine-induced relaxation of aortic rings showed the following rank order of potency (Ki, nM): xanthine amino congener (17 +/- 4) > 8-cyclopentyl-1,3-dipropylxanthine (171 +/- 36) > PD 115,199 (341 +/- 64) > 3,7-dimethyl-1-propargylxanthine (5520 +/- 820). 7. We conclude that the A2 adenosine receptor mediating relaxation of phenylephrine-contracted aortic rings is an A2b adenosine receptor which exhibits certain minor differences from the A2b receptor which stimulates cyclic AMP accumulation in cerebral cortical slices.
 
1. The aim of this investigation was to study the relationship between contractile responsiveness, activation of the L-arginine pathway and tissue levels of guanosine 3':5'cyclic monophosphate (cylic GMP) in aortic rings removed from rats 4 h after intraperitoneal administration of bacterial endotoxin (E. coli. lipopolysaccharide, LPS, 20 mg kg-1). 2. LPS-treatment resulted in a reduction of the sensitivity and maximal contractile response to noradrenaline (NA). 3. Depression of the maximal contractile response was restored to control by 6-anilo-5,8-quinolinedione (LY 83583, 10 microM), which prevents activation of soluble guanylate cyclase. 4. Cyclic GMP levels in tissue from LPS-treated rats were 2 fold greater than cyclic GMP levels detected in tissue from control (saline-treated) rats. The LPS-induced increase in cyclic GMP content was observed both in the presence and absence of functional endothelium. 5. Addition of L-arginine 1 mM) to maximally contracted aortic rings produced significantly relaxation of rings from LPS-treated rats but not rings from control animals. In the LPS-treated group, addition of L-arginine was also associated with a significant increase in cyclic GMP content. L-Arginine had no effect on the cyclic GMP content of control rings. D-Arginine (1 mM) was without effect. 6. In rings from LPS-treated rats, NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), an inhibitor of nitric oxide (NO) production, increased the contractile response to NA and prevented the LPS-induced increase in cyclic GMP content. In control rings, L-NAME increased the NA sensitivity only when the endothelium remained intact and reduced the cyclic GMP content of these rings to that of control endothelium-denuded rings. 7. These results demonstrate that LPS-induced hyporeactivity to NA occurs secondarily to activation of the L-arginine pathway and subsequent activation of soluble guanylate cyclase in vascular tissue. In addition they suggest that LPS induces the production of an NO-like relaxing factor in non-endothelial cells.
 
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. British Journal of Pharmacology (1999) 127, 252–258; doi:10.1038/sj.bjp.0702541
 
Hydroxamate derivatives have been attracted considerable attention, due to their broad pharmacological properties. Recent studies reported their potential use in the treatment of cardiovascular diseases, arthritis or infectious diseases. However, the inhibitory mechanisms of hydroxamate derivatives in inflammation remain to be elucidated. In an effort to develop a novel pharmacological agent that could suppress abnormally activated macrophages, we investigated a novel aliphatic hydroxamate derivative, WMJ-S-001, and explored its anti-inflammatory mechanisms. RAW264.7 macrophages were exposed to lipopolysaccharide (LPS) in the absence or presence of WMJ-S-001. COX-2 expression and signaling molecules activated by LPS were assessed. The LPS-induced COX-2 expression was suppressed by WMJ-S-001. WMJ-S-001 inhibited p38MAPK, NF-κB subunit p65 and C/EBPβ phosphorylation in cells exposed to LPS. Treatment of cells with a p38MAPK inhibitor (p38MAPK inhibitor III) markedly inhibited LPS-induced p65 and C/EBPβ phosphorylation and COX-2 expression. LPS-increased p65 and C/EBPβ binding to the COX-2 promoter region was suppressed in the presence of WMJ-S-001. In addition, WMJ-S-001 suppression of p38MAPK, p65 and C/EBPβ phosphorylation, and subsequent COX-2 expression were restored in cells transfected with mitogen-activated protein kinase phosphatase-1 (MKP-1) dominant negative (DN) mutant. WMJ-S-001 also caused an increase in MKP-1 phosphatase activity in RAW264.7 macrophages. WMJ-S-001 may cause MKP-1 activation to dephosphorylate p38MAPK, resulting in the decrease in p65 and C/EBPβ binding to the COX-2 promoter region and COX-2 down-regulation in LPS-stimulated RAW264.7 macrophages. The present study suggests that WMJ-S-001 may be a potential drug candidate in alleviating LPS-associated inflammatory diseases. This article is protected by copyright. All rights reserved.
 
1. The effects of imidazopyrazine derivative, SCA40, on the activity of single large conductance, Ca(2+)-activated K+ (BKCa) channels in inside-out and outside-out patches from bovine tracheal smooth muscle (BTSM) cells in culture have been compared with those of two established BKCa channel openers, NS 004 and NS 1619. 2. The presence of BKCa channels on inside-out patches of BTSM membranes was confirmed by the single channel conductance (240 pS), selectivity for K+, dependence of channel activity on [Ca2+]i, and sensitivity to the selective BKCa channel blocker, iberiotoxin. 3. NS 004 and ND 1619 (3-30 microM) induced concentration-related increases in open state probability of BKCa channels when applied to either inside-out or outside-out BTSM patches, thus confirming that these compounds are activators of the BKCa channel in this preparation. 4. SCA40 (0.1-10 microM) had no effect on the activity of BKCa channels when applied to either inside-out or outside-out patches which subsequently responded to the application of NS 004 (10-20 microM). 5. It is concluded that SCA40 does not have a direct effect on BKCa channel activity in BTSM patches and that the previously reported relaxant action of SCA40 on tracheal smooth muscle is unlikely to be mediated by this mechanism.
 
The adhesion molecule mucosal addressin cell adhesion molecule (MAdCAM) plays an essential role in the recruitment of lymphocytes to specialized high endothelial venules of the gastrointestinal tract and in their excessive tissue extravasation observed in inflammatory conditions, such as Crohn's disease. We have characterized the in vitro pharmacological properties of two monoclonal antibodies blocking MAdCAM, MECA-367 and PF-00547659, and determined their pharmacokinetic/pharmacodynamic profiles in vivo. Functional adhesion assays and surface plasmon resonance were used to characterize, in vitro, the pharmacological properties of MECA-367 and PF-00547659. The in vivo effects of MECA-367 and PF-00547659 on restriction of beta(7) (+) memory T lymphocytes were determined in mice and macaques, respectively, over the pharmacological dose range to confirm pharmacokinetic/pharmacodynamic relationships. MECA-367 and PF-00547659 bound with high affinity to mouse and human MAdCAM with K(d) values of 5.1 and 16.1 pmol.L(-1) respectively and blocked the adhesion of alpha(4)beta(7) (+) leukocytes to MAdCAM with similar potency. MECA-367 and PF-00547659 induced a similar, dose-dependent two- to threefold increase in circulating populations of beta(7) (+) memory T-cells in the mouse and macaque; without affecting the beta(7) (-) populations. PF-00547659 has potential utility in the treatment of inflammatory conditions by blocking tissue homing of activated alpha(4)beta(7) (+) leukocytes. The characterization of a rodent cross-reacting antibody as a surrogate for PF-00547659 in the search for potential pharmacological biomarkers and the determination of efficacious doses was effective in addressing the restricted orthologous cross-reactivity of PF-00547659 and the challenges this poses with respect to efficacy and safety testing.
 
20-Hydroxyeicosatetraenoic acid is a potent vasoconstrictor that contributes to cerebral ischaemia. An inhibitor of 20-Hydroxyeicosatetraenoic acid synthesis, TS-011, reduces infarct volume and improves neurological deficits in animal stroke models. However, little is known about how TS-011 affects the microvessels in ischaemic brain. Here, we investigated the effect of TS-011 on microvessels after cerebral ischaemia. TS-011 (0.3 mg·kg(-1) ) or a vehicle was infused intravenously for 1 h every 6 h in a mouse model of stroke, induced by transient occlusion of the middle cerebral artery occlusion following photothrombosis. The cerebral blood flow velocity and the vascular perfusion area of the peri-infarct microvessels were measured using in vivo two-photon imaging. The cerebral blood flow velocities in the peri-infarct microvessels decreased at 1 and 7 h after reperfusion, followed by an increase at 24 h after reperfusion in the vehicle-treated mice. We found that TS-011 significantly inhibited both the decrease and the increase in the blood flow velocities in the peri-infarct microvessels seen in the vehicle-treated mice after reperfusion. In addition, TS-011 significantly inhibited the reduction in the microvascular perfusion area after reperfusion, compared with the vehicle-treated group. Moreover, TS-011 significantly reduced the infarct volume by 40% at 72 h after middle cerebral artery occlusion. These findings demonstrated that infusion of TS-011 improved defects in the autoregulation of peri-infarct microcirculation and reduced the infarct volume. Our results could be relevant to the treatment of cerebral ischaemia.
 
1. Intrathecal (i.t.) administration of prostaglandin E2 (PGE2) to conscious mice was reported to induce allodynia, a state of discomfort and pain evoked by innocuous tactile stimuli through prostaglandin E receptor subtype EP1 and hyperalgesia through prostaglandin E receptor subtypes EP2 and/or EP3. In the present study, we investigated the effects of an EP1 antagonist on these sensory disorders by use of ONO-NT-012 or AH6809. 2. ONO-NT-012 dose-dependently antagonized the PGE2-induced allodynia but had no effect on the PGE2-induced hyperalgesia by the hot plate test. On the other hand, AH6809 blocked the PGE2-induced hyperalgesia at the highest dose examined (50 micrograms kg-1) but had no effect on the PGE2-induced allodynia. The i.t. injection of AH6809 or ONO-NT-012 alone did not have any effect on the response to noxious or innocuous stimuli. 3. Increasing doses (5 pg kg(-1)-500 ng kg-1) of ONO-NT-012 produced parallel shifts to the right of the dose-response curves to PGE2. The Schild plot regression line was linear and the slope was close to unity. The pA2 value against PGE2 was calculated to be 9.96. 4. The present study demonstrates that i.t. administration of PGE2 exerts allodynia through EP1 in the mouse spinal cord and that ONO-NT-012 is a highly potent, simple competitive antagonist for the PGE2-induced allodynia.
 
1. The aim of this study was to determine whether a synthetic inhibitor of the interleukin-1 beta converting enzyme (ICE) displays oral activity in models of inflammation. 2. To this end, the ICE inhibitor, SDZ 224-015, was examined in rat paw oedema, pyrexia and nociception tests. 3. SDZ 224-015 (0.3-300 micrograms kg-1) potently reduced carrageenin-induced paw oedema, with an oral ED50 of approximately 25 micrograms kg-1. This effect was independent of endogenous glucocorticoid, as shown by retention of activity upon adrenalectomy. 4. Pyrexia induced by lipopolysaccharide (0.1 mg kg-1 s.c.) or by interleukin-1 beta (100 ng i.v.) was also reduced, over a similar dose-range to oedema (oral ED50s 11 micrograms kg-1 and 4 micrograms kg-1 respectively). 5. SDZ 224-015 (0.2-5 mg kg-1, p.o.) displayed analgesic activity in the Randall-Selitto yeast-inflamed paw pressure test, significant at a dose of 1 mg kg-1, p.o. 6. Thus, SDZ 224-015 has potent oral activity in several acute models for inflammation, suggesting that ICE inhibitors may constitute a novel type of anti-inflammatory agent.
 
Peroxynitrite is a strong oxidant that results from reaction between NO and superoxide. It has been recently proposed that peroxynitrite plays a pathogenetic role in inflammatory processes. Here we have investigated the therapeutic efficacy of raxofelast, a new hydrophilic vitamin E-like antioxidant agent, in rats subjected to carrageenan-induced pleurisy. In vivo treatment with raxofelast (5, 10, 20 mg kg−1 intraperitoneally 5 min before carrageenan) prevented in a dose dependent manner carrageenan-induced pleural exudation and polymorphonuclear migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, as well as histological organ injury were significantly reduced by raxofelast. Immunohistochemical analysis for nitrotyrosine, a footprint of peroxynitrite, revealed a positive staining in lungs from carrageenan-treated rats. No positive nitrotyrosine staining was found in the lungs of the carrageenan-treated rats, which received raxofelast (20 mg kg−1) treatment. Furthermore, in vivo raxofelast (5, 10, 20 mg kg−1) treatment significantly reduced peroxynitrite formation as measured by the oxidation of the fluorescent dihydrorhodamine 123, prevented the appearance of DNA damage, the decrease in mitochondrial respiration and partially restored the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. In conclusion, our study demonstrates that raxofelast, a new hydrophilic vitamin E-like antioxidant agent, exerts multiple protective effects in carrageenan-induced acute inflammation. British Journal of Pharmacology (1999) 126, 407–414; doi:10.1038/sj.bjp.0702275
 
1 Sodium p-benzyl-4-[1-oxo-2-(4-chlorobenzyl)-3-phenyl propyl]phenyl phosphonate (N-0164) selectively inhibited the formation of thromboxane-A(2) from prostaglandin endoperoxides by human platelet microsomes in a dose-dependent manner (IC(50) 2.2 x 10(-5) M or 11.6 mug/ml).2 N-0164 was approximately 15 to 20 times as potent as indomethacin as an inhibitor of thromboxane-A(2) formation. In contrast, indomethacin was 20 times as potent as N-0164 as an inhibitor of prostaglandin endoperoxide formation from arachidonic acid (IC(50) 2.6 x 10(-5) M or 9.4 mug/ml).3 Spiral strips of dog coronary arteries relaxed in the presence of prostaglandin endoperoxides and were contracted by prostaglandin E(2) and thromboxane-A(2) and were therefore used to distinguish between prostaglandins and their intermediate precursors, the endoperoxides.4 Neither indomethacin nor N-0164 (both 50 mug/ml) significantly inhibited the formation of prostaglandin-like activity from the endoperoxides following incubation with indomethacin-pretreated rabbit kidney medulla microsomes.5 It is not known whether this action of N-0164 is related to its ability to antagonize certain actions of prostaglandins (and related compounds) or whether N-0164 can penetrate the cell membrane to inhibit thromboxane formation in the intact cell.6 Selective inhibition of thromboxane formation by drugs such as N-0164 may be useful both clinically and as a pharmacological tool to elucidate the patho-physiological roles of the thromboxanes.
 
1 The ability of sodium p-benzyl-4-[1-oxo-2-(4-chlorobenzyl)-3-phenylpropyl]phenyl phosphonate (N-0164) to antagonize contractions produced by prostaglandins E2 and F2a on isolated preparations of gerbil, rat and guinea-pig gastrointestinal muscle has been studied. 2 N-0164 was found to be a potent, partially selective prostaglandin antagonist in these isolated smooth muscle preparations. The blockade produced by N-0164 in the isolated stomach strip of the rat had some, but not all, the characteristics of a competitive antagonism. 3 N-0164 produced a dose-dependent decrease in tone in the rat stomach strip that was abolished by pretreatment of the preparation with indomethacin. 4 N-0164 prevented diarrhoea induced by prostaglandin E2 in mice when given by intraperitoneal injection but was less effective when given orally. 5 N-0164 inhibited oedema induced with croton-oil and pyridine-ether in the mouse ear. 6 N-0164 delayed the onset of erythema following ultraviolet irradiation of guinea-pig skin only when an equimolar amount of pralidoxime chloride was added to the vehicle. 7 It is concluded that N-0164 is a potent, partially selective prostaglandin antagonist on several isolated smooth msucle preparations. N-0164 exhibits activity in vivo particularly following local application when problems associated with penetration and distribution are minimized.
 
Ro60-0175 has been described as a selective agonist at the 5-HT2C receptor, yet it has only 10- fold higher affinity at the 5-HT2C compared to the 5-HT2A subtype, and equivalent affinity for the 5-HT2B receptor. The selective 5-HT2C receptor antagonist SB242,084 (0.5 mg kg−1 i.p.), blocked the hypoactivity and penile grooming induced by Ro60-0175 (1 mg kg−1 s.c.). The combination of SB242,084 (0.5 mg kg−1 i.p.) and Ro60-0175 (3 – 10 mg kg−1) produced a completely different pattern of behaviours including wet-dog shakes, hyperactivity and back muscle contractions. These latter effects were blocked by the selective 5-HT2A receptor antagonist MDL100,907 (0.5 mg kg−1 i.p.), but not the 5-HT2B receptor antagonist SB215,505 (3 mg kg−1 p.o.). The indirect 5-HT releaser/reuptake inhibitor dexfenfluramine (1 – 10 mg kg−1 i.p.) produced a mild increase in locomotor activity, penile grooming, and occasional back muscle contractions and wet-dog shakes. Pre-treatment with SB242,084 (0.5 mg kg−1), blocked the incidence of penile grooming, and markedly potentiated both the dexfenfluramine-induced hyperactivity, the incidence of back muscle contractions, and to a lesser extent wet-dog shakes. Some toxicity was also evident in animals treated with dexfenfluramine (10 mg kg−1)/SB242,084 (0.5 mg kg−1), but not in any other treatment groups. The hyperactivity and toxicity produced by the dexfenfluramine (10 mg kg−1)/SB242,084 (0.5 mg kg−1) combination was replicated in a further study, and hyperthermia was also recorded. Both hyperthermia and toxicity were blocked by MDL100,907 (0.5 mg kg−1) but not SB215,505 (3 mg kg−1). An attenuation of the hyperlocomotor response was also observed following MDL100,907. These findings suggest that 5-HT2C receptor activation can inhibit the expression of behaviours mediated through other 5-HT receptor subtypes. British Journal of Pharmacology (2001) 133, 459–466; doi:10.1038/sj.bjp.0704082
 
The aim of the study was to compare the effects of 14 day subcutaneous infusion of the 5-HT2C receptor agonists, m-chlorophenylpiperazine (mCPP, 12 mg kg−1 day−1) and Ro 60-0175 (36 mg kg−1 day−1) and the 5-HT releasing agent and re-uptake inhibitor, d-fenfluramine (6 mg kg−1 day−1), on food and water intake, body weight gain and locomotion in lean male Lister hooded rats. Chronic infusion of all three drugs significantly reduced food intake and attenuated body weight gain. In contrast, drug infusion did not lead to significant reductions in locomotor activity in animals assessed 2 and 13 days after pump implantation. In a subsequent 14 day study that was designed to identify possible tolerance during days 7–14, animals were given a subcutaneous infusion of mCPP (12 mg kg−1 day−1) or d-fenfluramine (6 mg kg−1 day−1) for either 7 or 14 days. During the first 7 days both drugs significantly reduced body weight gain compared to saline-infused controls; however, from day 7 onwards animals withdrawn from drug treatment exhibited an increase in body weight such that by day 14 they were significantly heavier than their 14-day drug-treated counterparts. Both mCPP and d-fenfluramine reduced daily food intake throughout the infusion periods. For 14-day treated animals this hypophagia was marked during the initial week of the study but only minor during the second week. In light of the sustained drug effect on body weight, the data suggest that weight loss by 5-HT2C receptor stimulation may be only partly dependent on changes in food consumption and that 5-HT2C receptor agonists may have effects on thermogenesis. These data suggest tolerance does not develop to the effects of d-fenfluramine, mCPP and Ro 60-0175 on rat body weight gain. British Journal of Pharmacology (2000) 130, 1305–1314; doi:10.1038/sj.bjp.0703443
 
1. Regional haemodynamic responses to endothelin (ET)-1, -2 and -3 and big ET-1 (all at 500 pmol kg-1) were assessed in the same conscious Long Evans rats (n = 8) in the absence or presence of the mixed ETA-, ETB-receptor antagonist, Ro 47-0203 (bosentan; 30 mg kg-1). 2. Bosentan blocked the initial depressor, tachycardic and hindquarters hyperaemic vasodilator effects of ET-1, -2 and -3, and substantially curtailed the primary renal and secondary hindquarters vasoconstrictor responses. Bosentan did not inhibit the initial mesenteric vasoconstrictor action of ET-1, but reduced the duration of the later mesenteric vasoconstriction. In contrast, bosentan delayed the rate of onset, and reduced the duration, of the mesenteric vasoconstrictor actions of ET-2 and ET-3. The most likely explanation of this finding is that ET-1, but not ET-2 or ET-3, triggered a covert mesenteric vasodilator mechanism which was antagonized by bosentan. 3. Bosentan blocked all the effects of big ET-1, and, in a separate group of rats (n = 7), blocked all the haemodynamic effects of a lower dose of ET-1 (50 pmol kg-1), with the exception of a slight mesenteric vasoconstriction. 4. The most straightforward explanation of the results is that the major haemodynamic effects of ET-1, -2 and -3, and all the effects of big ET-1, are mediated through ETA- and/or ETB-receptors that are effectively antagonized by bosentan.
 
Besides targeting the well-known oncogenic c-Met, crizotinib is the first oral tyrosine kinase inhibitor inhibiting anaplastic lymphoma kinase (ALK) in clinical trials for the treatment of non-small cell lung cancer. Here, we assessed the possible reversal of multidrug resistance (MDR) by crizotinib in vitro and in vivo. 1-(4,5-Dimethylthiazol-2-yl)-3,5- diphenylformazan was used in vitro and xenografts in nude mice were used in vivo to investigate reversal of MDR by crizotinib. To understand the mechanisms for MDR reversal, the alterations of intracellular doxorubicin or rhodamine 123 accumulation, doxorubicin efflux, ABCB1 expression level, ATPase activity of ABCB1 and crizotinib-induced c-Met, Akt and ERK1/2 phosphorylation were examined. Crizotinib significantly enhanced the cytotoxicity of chemotherapeutic agents which are also ABCB1 substrates, in MDR cells with no effect found on sensitive cells in vitro and in vivo. Additionally, crizotinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and inhibited the drug efflux in ABCB1-overexpressing MDR cells. Further studies showed that crizotinib enhanced the ATPase activity of ABCB1 in a concentration-dependent manner. However, expression of ABCB1 was not affected, and reversal of MDR by crizotinib was not related to the phosphorylation of c-Met, Akt or ERK1/2. Importantly, crizotinib significantly enhanced the effect of paclitaxel against KBv200 cell xenografts in nude mice. Crizotinib reversed ABCB1-mediated MDR by inhibiting ABCB1 transport function without affecting ABCB1 expression or blocking the Akt or ERK1/2 pathways. These findings are useful for planning combination chemotherapy of crizotinib with conventional chemotherapeutic drugs.
 
Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by the absence of the cytoskeletal protein dystrophin. This leads to muscle cell death accompanied by chronic inflammation. Cyclosporin A (CsA) is a powerful immunosuppressive drug, which has been proposed for DMD treatment. CsA also directly regulates the mitochondrial permeability transition pore (mPTP), which participates in cell death pathways through the inhibition of cyclophilin D. Here, we evaluated whether Debio 025, a cyclophilin inhibitor with no immunosuppressive activity, improves the dystrophic condition in a mouse model of DMD, through regulation of mPTP. The potency of Debio 025 to protect mouse dystrophic cells against mitochondria-mediated death was assessed by caspase-3 activity and calcium retention capacity assays. Mdx(5Cv) mice (3-week-old) were treated daily by gavage for 2 weeks with Debio 025 (10, 30 or 100 mg kg(-1)), CsA (10 mg kg(-1)) or placebo. The effects on muscle necrosis and function were measured. In vitro investigations showed protective effect of low concentrations of Debio 025 against cell death. Histology demonstrated that Debio 025 partially protected the diaphragm and soleus muscles against necrosis (10 and 100 mg kg(-1), respectively). Hindlimb muscles from mice receiving Debio 025 at 10 mg kg(-1) relaxed faster, showed alteration in the stimulation frequency-dependent recruitment of muscle fibres and displayed a higher resistance to mechanical stress. Debio 025 partially improved the structure and the function of the dystrophic mouse muscle, suggesting that therapies targeting the mPTP may be helpful to DMD patients.
 
We have investigated the therapeutic effects of the selective cyclophilin inhibitor D-MeAla(3)-EtVal(4)-cyclosporin (Debio 025) in myopathic Col6a1(-/-) mice, a model of muscular dystrophies due to defects of collagen VI. We studied calcineurin activity based on NFAT translocation; T cell activation based on expression of CD69 and CD25; propensity to open the permeability transition pore in mitochondria and skeletal muscle fibres based on the ability to retain Ca(2+) and on membrane potential, respectively; muscle ultrastructure by electronmicroscopy; and apoptotic rates by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays in Col6a1(-/-) mice before after treatment with Debio 025. Debio 025 did not inhibit calcineurin activity, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevented the mitochondrial dysfunction and normalized the apoptotic rates and ultrastructural lesions of myopathic Col6a1(-/-) mice. CONCLUSIONS AND IMPLICAtions: Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1(-/-) myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025. They represent an essential step towards an effective therapy for Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy, because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression.
 
Ro 03-7894 (0.6 mM) produced a non-parallel shift to the right of dose-response curves to (-)-isoprenaline in K+ depolarized uterine preparations from the guinea-pig. The displacement of the curves was readily reversed by washing. A rightward shift of similar magnitude was also produced by Ro 03-7894 in transmurally stimulated ileal preparations. The relaxant effects of fenoterol in carbachol-contracted guinea-pig tracheal preparations (in the presence of 2 microM atenolol) were not altered by 0.6 mM Ro 03-7894. In the three tissues there was no evidence of a reduction in the maximal inhibitory response to the agonists. In uterine and tracheal preparations, Ro 03-7894 (0.6 mM) depressed contractile responses to exogenous calcium. The depression of responses was enhanced after washout of Ro 03-7894 for 80 min. Contractile responses of ileal preparations to transmural stimulation were also depressed by Ro 03-7894. Concentration-effect curves for the positive inotropic effects of (-)-isoprenaline in guinea-pig left atrial preparations were markedly shifted to the right and the maximum response depressed by 0.6 mM Ro 03-7894. Although the rightward shift of the curves was fully reversed during the 120 min washout period, the maximal responses remained depressed. In similar experiments, Ro 03-7894 produced a washout-resistant depression of inotropic responses to histamine and calcium. The results of radioligand binding studies in left atria using (-)-[125I]-iodocyanopindolol indicated that, when compared to the untreated atria, there was no reduction in the maximal density of binding sites 120 min after washout of 0.6 mM Ro 03-7894. 5 On the basis of the present results it is concluded that Ro 03-7894 induces a non-specific depressant effect on smooth and cardiac muscle preparations during exposure to the drug. This depressant effect persists following washout of the drug. There is no evidence for an irreversible effect of Ro 03-7894 at beta-adrenoceptor sites.
 
The positive inotropic responses of guinea-pig left atria and papillary muscles and positive chronotropic responses of right atria to sympathomimetic amines were examined at 38 degrees and 30 degrees C. At the lower temperature, supersensitivity to orciprenaline and isoprenaline was exhibited as shifts of the dose-response curves to the left and significant reductions in EC50 values. This supersensitivity could not be attributed to reduced metabolism since the experiments were performed in the presence of metanephrine (10(-5)M) and U-0521 (3',4'-dihydroxy-2-methylpropiophenone) (10(-4)M) as inhibitors of extraneuronal uptake and catechol-O-methyltransferase (COMT) respectively, and the agonists are not susceptible to neuronal uptake. After incubation of the tissues with Ro 03-7894 (1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol), followed by its prolonged washout (greater than 2h), the maximum responses to isoprenaline and orciprenaline were depressed, confirming the apparently irreversible beta-adrenoceptor antagonism. Dissociation constants (KA) for isoprenaline and orciprenaline were determined from the equiactive concentrations obtained before (A) and after (A') incubation with Ro 03-7894, plotted as 1/A against 1/A' (KA = (slope-1)/intercept). KA values were the same for orciprenaline in the three cardiac preparations and for isoprenaline in the atria. This applied at 38 degrees and 30 degrees C and indicates that the beta-adrenoceptors mediating the inotropic and chronotropic responses of the guinea-pig heart do not differ. The KA values of both agonists were, however, consistently and significantly lower at 30 degrees than at 38 degrees C, indicating an increase in affinity. 8 It is concluded that hypothermia-induced supersensitivity of cardiac tissue to sympathomimetic amines is associated with an increase in their affinity for the B-adrenoceptors.
 
This study describes the in vitro characterization of two potent and selective 5-HT6 receptor antagonists at the rat and human recombinant 5-HT6 receptor. In binding assays with [3H]-LSD, 4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) and 4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulphonamide (Ro 63-0563) had mean pKi values ±s.e.mean at the rat 5-HT6 receptor of 7.35±0.04 and 7.83±0.01, respectively and pKi values at the human 5-HT6 receptor of 7.26±0.06 and 7.91±0.02, respectively. Both compounds were found to be over 100 fold selective for the 5-HT6 receptor compared to 23 (Ro 04-6790) and 69 (Ro 63-0563) other receptor binding sites. In functional studies, neither compound had any significant effect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5-HT6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5-HT6 receptor. However, both Ro 04-6790 and Ro 63-0563 behaved as competitive antagonists with mean ±s.e.mean pA2 values of 6.75±0.07 and 7.10±0.09, respectively. In rats habituated to observation cages, Ro 04-6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5-HT6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing. Ro 04-6790 and Ro 63-0563 represent valuable pharmacological tools for the identification of 5-HT6 receptors in natural tissues and the study of their physiological function.
 
The sodium–calcium exchanger (NCX) was considered to play an important role in arrhythmogenesis under certain conditions such as heart failure or calcium overload. In the present study, the effect of SEA-0400, a selective inhibitor of the NCX, was investigated on early and delayed afterdepolarizations in canine ventricular papillary muscles and Purkinje fibres by applying conventional microelectrode techniques at 37°C. The amplitude of both early and delayed afterdepolarizations was markedly decreased by 1 μM SEA-0400 from 26.6±2.5 to 14.8±1.8 mV (n=9, P<0.05) and from 12.5±1.7 to 5.9±1.4 mV (n=3, P<0.05), respectively. In enzymatically isolated canine ventricular myocytes, SEA-0400 did not change significantly the L-type calcium current and the intracellular calcium transient, studied using the whole-cell configuration of the patch-clamp technique and Fura-2 ratiometric fluorometry. It is concluded that, through the reduction of calcium overload, specific inhibition of the NCX current by SEA-0400 may abolish triggered arrhythmias. British Journal of Pharmacology (2004) 143, 827–831. doi:10.1038/sj.bjp.0706026
 
Fibril formation of amyloid β peptide (Aβ) is considered to be responsible for the pathology of Alzheimer's disease (AD). The Aβ fibril is formed by a protein misfolding process in which intermolecular β-sheet interactions become stabilized abnormally. Thus, to develop potential anti-AD drugs, we screened an in-house library to find compounds which have a profile as a β-sheet breaker. We searched for a β-sheet breaker profile in an in-house library of approximately 113,000 compounds. From among the screening hits, we focused on N,N′-bis(3-hydroxyphenyl)pyridazine-3,6-diamine (named RS-0406), which had been newly synthesized in our laboratory. This compound (10–100 μg ml−1) was found to be capable of significantly inhibiting 25 μM Aβ1–42 fibrillogenesis and, furthermore, disassembling preformed Aβ1–42 fibrils in vitro. We then investigated the effect of RS-0406 on 111 nM Aβ1–42-induced cytotoxicity in primary hippocampal neurons, and found that 0.3–3 μg ml−1 RS-0406 ameliorates the cytotoxicity. Moreover, 3 μg ml−1 RS-0406 reversed 1 μM Aβ1–42-induced impairment of long-term potentiation in hippocampal slices. In this study, we have succeeded in identifying RS-0406 which has potential to inhibit Aβ1–42 fibrillogenesis, and to protect neurons against Aβ1–42-induced biological toxicity in vitro. These results suggest that RS-0406 or one of the derivatives could become a therapeutic agent for AD patients. British Journal of Pharmacology (2002) 137, 676–682. doi:10.1038/sj.bjp.0704911
 
BACKGROUND AND PURPOSE Airway sensory nerves play a key role in respiratory cough, dyspnoea, airway hyper-responsiveness (AHR), all fundamental features of airway diseases [asthma and chronic obstructive pulmonary disease (COPD)]. Vagally mediated airway reflexes such as cough, bronchoconstriction and chest tightness originate from stimulation of airway sensory nerve endings. The transient receptor potential vanilloid 1 receptor (TRPV1) is present on peripheral terminals of airway sensory nerves and modulation of its activity represents a potential target for the pharmacological therapy of AHR in airway disease. EXPERIMENTAL APPROACH As guinea pig models can provide some of the essential features of asthma, including AHR, we have established the model with some classical pharmacological agents and examined the effect of the TRPV1 antagonists, SB-705498 and PF-04065463 on AHR to histamine evoked by ovalbumin (OA) in unanaesthetized sensitized guinea pigs restrained in a double chamber plethysmograph. Specific airway conductance (sGaw) derived from the airflow was calculated as a percentage of change from baseline. KEY RESULTS Cetirizine and salbutamol significantly inhibited OA-evoked bronchoconstriction [sGaw area under the curve (AUC): 70 and 78%, respectively]. Atropine, SB-705498 and PF-04065463 significantly inhibited OA-evoked AHR to histamine in unanaesthetized, OA-sensitized guinea pigs (sGaw AUC: 94%, 57% and 73%, respectively). Furthermore, this effect was not related to antagonism of histamine's activity. CONCLUSION AND IMPLICATIONS These data suggest that TRPV1 receptors located on airway sensory nerves are important in the development of AHR and that modulation of TRPV1-receptor activity represents a potential target for the pharmacological therapy of AHR in airway disease.
 
1. The aims of the present study were to characterize the pharmacological profile of a new endothelin (ET) receptor antagonist, TAK-044 and to consider whether it limits the extension of myocardial infarct size in rats. 2. Binding of [125I]-ET-1 to ET receptors on rabbit ventricular and cerebellar membrane fractions was inhibited by TAK-044 with IC50 values of 3.8 nM and 130 nM, respectively. 3. It inhibited ET-1, ET-2 and ET-3-induced vasoconstriction of porcine isolated coronary arteries in a competitive (ET-1, ET-2) and a non-competitive (ET-3) manner. 4. In the rat in vivo, the ET-1-induced blood pressure changes including transient hypotension followed by sustained hypertension, were inhibited by TAK-044 (0.1-10 mg kg-1, i.v.) in a dose-dependent manner. 5. Acute myocardial infarction induced by 1 h coronary occlusion followed by 24 h reperfusion in rats caused an infarct size of 60 +/- 2% (n = 12) of the area-at-risk by weight. 6. Intravenous injection of TAK-044 10 min before coronary occlusion reduced the infarct size in a dose-dependent manner: 32% and 54% reductions at 1 and 3 mg kg-1, respectively. 7. TAK-044 administered 10 min before or 1 h after reperfusion (1 mg kg-1, i.v.) showed similar inhibitory effects: 34% and 23% reductions, respectively. 8. We conclude that TAK-044 is an ETA/ETB receptor antagonist which shows strong inhibitory effects on the extension of myocardial infarct size after coronary artery occlusion-reperfusion in rats.
 
1. We examined the effects of systemic infusion, in healthy human volunteers, of the endothelin antagonist TAK-044 on the plasma concentrations of mature endothelin, big endothelin-1 and the C-terminal fragment of big endothelin-1, by selective solid-phase extraction and specific radioimmunoassays. 2. Unlabelled TAK-044 competed with specific [125I]-endothelin-1 binding to human left ventricle tissue in a biphasic manner giving KD values of 0.11 nM and 26.8 nM at the ETA and ETB receptor subtypes, respectively, indicating a 244 fold selectivity for the ETA receptor subtype. 3. A 15 min intravenous infusion of placebo or 30 mg TAK-044 (giving a serum concentration of 2 nM, calculated to block > 95% of ETA but < 5% ETB receptors) had no effect on the immunoreactive plasma concentrations of the three peptides. 4. At the higher dose of 750 mg TAK-044 (giving a serum concentration of 80 nM, calculated to block > 99% of ETA and > 75% ETB receptors), the immunoreactive plasma endothelin concentrations were increased 3.3 fold over basal levels (P < 0.01). The concentrations of big endothelin-1 or C-terminal fragment of big endothelin-1 were unchanged. 5. At both doses of TAK-044, there were significant decreases in diastolic blood pressure, and peripheral vascular resistance, with corresponding increases in cardiac index and stroke index. There were no changes in systolic or mean arterial blood pressures or heart rate. 6. Since only the concentrations of the mature peptide were increased, we conclude that the most likely sources of endothelin contributing to the observed rise were displacement of receptor-bound peptide and reduction in plasma clearance rather than peptide synthesis.
 
1. The inhibitory effects of the endothelin (ET) receptor antagonist, TAK-044, on ET-induced vasoconstriction in various canine arteries and porcine coronary arteries were studied and were compared to those of selective ETA and ETB receptor antagonists. 2. ET-1 (0.1 nM-0.3 microM) caused vasoconstriction in canine coronary, femoral, renal, mesenteric and basilar arteries, and the strongest responses were obtained in coronary and basilar arteries. TAK-044 (10 nM, 100 nM) inhibited this ET-1-induced vasoconstriction except in the case of mesenteric arteries. The strongest inhibitory effects were obtained in coronary arteries; an EC50 value for ET-1 was 5.2 +/- 0.77 nM (n = 12) in the control and 24 +/- 3.8 nM (n = 4) in the presence of TAK-044 at 10 nM. BQ-123 (1 microM) inhibited the vasoconstriction in coronary and femoral arteries but did not in renal, mesenteric or basilar arteries. 3. TAK-044 (10-100 nM) inhibited the ET-1-induced vasoconstriction in porcine coronary arteries to a degree similar to that in canine coronary arteries. In contrast, BQ-123 (10 microM) did not inhibit the contraction completely, and a BQ-123-insensitive component was identified. Although BQ-788 (1 microM) did not modify the concentration-response curve at all, it abolished the BQ-123-insensitive component when applied together with BQ-123 (10 microM). 4. Sarafotoxin S6c (10 pM-30 nM) caused vasoconstriction in porcine coronary arteries with the maximum amplitude of the contraction being 39% of that with ET-1. Both TAK-044 (10 nM, 100 nM) and BQ-788 (1 microM) inhibited this vasoconstriction, while BQ-123 (3 microM, 10 microM) did not. 5. Vasoconstriction induced by ET-3 (0.1 nM-0.3 microM) in porcine coronary arteries showed a concentration-response curve with two distinct phases in contrast to that seen with sarafotoxin S6c. TAK-044 (0.3 nM-10 nM) inhibited both phases in a concentration-dependent manner. BQ-123 (1 microM, 3 microM) inhibited only the second phase, while BQ-788 (1 microM) inhibited the first phase. 6. We concluded that the inhibitory effects of TAK-044 on ET-1-induced vasoconstriction were the strongest in coronary arteries among the canine arteries examined. In addition, we showed that both ETA and ETB receptors mediate vasoconstriction in porcine coronary arteries and TAK-044 inhibits the vasoconstriction mediated by both of these receptors.
 
Understanding the role of the EP2 receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP2 receptor antagonist, PF-04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF-04418948 for the EP2 receptor over other EP receptors using a range of isolated tissue systems. PF-04418948 was profiled on a range of isolated tissues to assess its EP receptor potency and selectivity: ONO-DI-004-induced contraction of guinea pig trachea (EP1); ONO-AE1-259 and PGE2- induced relaxation of mouse and guinea pig trachea (EP2); PGE2-induced depolarization of guinea pig isolated vagus (EP3); PGE2-induced relaxation of human and rat trachea (EP4). PF-04418948 was also profiled in functional murine TP, IP, DP and FP receptor assays. In bioassay systems, where assessment of potency/selectivity is made against the ‘native’ receptor, PF-04418948 only acted as an antagonist of EP2 receptor-mediated events. PF-04418948 competitively inhibited relaxations of murine and guinea pig trachea induced by ONO-AE1-259 and PGE2 respectively. However, the affinity of PF-04418948 was not equal in the two preparations. Using a wide range of bioassay systems, we have demonstrated that PF-04418948 is a selective EP2-receptor antagonist. Interestingly, an atypically low affinity was found on the guinea pig trachea, questioning its utility as an EP2 receptor assay system. Nevertheless, this compound should be an invaluable tool for investigating the biological activity of PGE2 and the role of EP2 receptors in health and disease.
 
Studies of the role of the prostaglandin EP(2) receptor) have been limited by the availability of potent and selective antagonist tools. Here we describe the in vitro/in vivo pharmacological characterization of a novel EP(2) receptor antagonist, PF-04418948 (1-(4-fluorobenzoyl)-3-{[(6-methoxy-2-naphthyl)oxy]methyl} azetidine-3-carboxylic acid). Functional antagonist potency was assessed in cell-based systems expressing human EP(2) receptors and native tissue preparations from human, dog and mouse. The selectivity of PF-04418948 was assessed against related receptors and a panel of GPCRs, ion channels and enzymes. The ability of PF-04418948 to pharmacologically block EP(2) receptor function in vivo was tested in rats. PF-04418948 inhibited prostaglandin E(2)(PGE(2))-induced increase in cAMP in cells expressing EP(2) receptors with a functional K(B) value of 1.8 nM. In human myometrium, PF-04418948 produced a parallel, rightward shift of the butaprost-induced inhibition of the contractions induced by electrical field stimulation with an apparent K(B) of 5.4 nM. In dog bronchiole and mouse trachea, PF-04418948 produced parallel rightward shifts of the PGE(2)-induced relaxation curve with a K(B) of 2.5 nM and an apparent K(B) of 1.3 nM respectively. Reversal of the PGE(2)-induced relaxation in the mouse trachea by PF-04418948 produced an IC(50) value of 2.7 nM. Given orally, PF-04418948 attenuated the butaprost-induced cutaneous blood flow response in rats. PF-04418948 was selective for EP(2) receptors over homologous and unrelated receptors, enzymes and channels. PF-04418948 is an orally active, potent and selective surmountable EP(2) receptor antagonist that should aid further elaboration of EP(2) receptor function.
 
1. No specific regimen has been developed to treat post-traumatic amnesia in man. In the present study, we examined the effects of (+)-eburnamenine-14-carboxylic acid (2-nitroxyethyl) ester (VA-045), a novel derivative of apovincaminic acid, on learning and memory deficits associated with a mild traumatic brain injury (TBI) in mice. 2. Two kinds of amnesia, TBI-induced retrograde amnesia (TRA) and anterograde amnesia (TAA), were produced by means of post- and pre-acquisition head injury, respectively, by a simple weight-drop device. A novel procedure of water-finding task was used to assess learning and memory functions. 3. Both TRA and TAA mice were dramatically impaired in the task performance, with prolonged latencies for finding and drinking in either retention test or retest, indicating that retention was impaired in TRA mice while learning and retention were impaired in TAA mice. 4. VA-045 administered 30 min post-trauma in TRA mice dramatically shortened the prolonged latencies for finding and drinking in both retention test and retest, indicating that VA-045 significantly improved the retention deficit observed in TRA mice. 5. VA-045 administered 30 min post-trauma in TAA mice dramatically attenuated the prolonged latencies for finding and drinking in both retention test and retest, indicating that VA-045 significantly improved the learning and retention deficits observed in TAA mice. 6. Administration of VA-045 30 min pre-trauma in normal mice markedly attenuated the delay of latencies for finding and drinking after trauma in both retention test and retest, which shows that VA-045 significantly prevented learning and retention deficits after TBI. 7. Motor activities were not significantly affected by either the TBI or the chemical treatment at the time of task examination in either experimental model. 8. It is concluded that VA-045 may have potential effects on learning and memory deficits observed in either TBI-induced retrograde or anterograde amnesia.
 
1. The pharmacological actions of T-0509, a 3-hydroxy derivative of denopamine, were studied in various guinea-pig tissues; these effects were compared with those of isoprenaline, denopamine and xamoterol. 2. The intrinsic activities of the positive inotropic actions of T-0509, denopamine and xamoterol compared with isoprenaline (= 100%) in the papillary muscle were 99%, 83% and 28%, respectively, while their relative potencies (EC50 agonist EC50 isoprenaline) were 0.23, 33 and 1.4, respectively. The intrinsic activities of T-0509, denopamine and xamoterol as positive chronotropic agents in the right atria were 98%, 69% and 48%, respectively, and their equipotent concentrations (isoprenaline = 1) were 0.24, 50 and 4, respectively. 3. The positive chronotropic actions of T-0509 and denopamine were antagonized by bisoprolol (3 x 10(-8) M), but not by ICI 118,551 (3 x 10(-8) M). 4. The intrinsic activity of T-0509 in histamine-contracted tracheae was similar to that of isoprenaline, but its equipotent concentration was 38; the effects of both agents were antagonized by ICI 118,551 (3 x 10(-8) M), but not by bisoprolol (3 x 10(-8) M). Denopamine and xamoterol did not show any agonist activity on guinea-pig trachea. 5. Denopamine and xamoterol antagonized the positive chronotropic (pA2, denopamine: 6.98, xamoterol: 7.75) and tracheal relaxant (pA2, denopamine: 5.39, xamoterol: 6.25) effects of isoprenaline. 6. Isoprenaline, T-0509 and denopamine, but not xamoterol, contracted the guinea-pig aorta in a decreasing order in the presence of propranolol (10(-6) M).7. Based on the above studies, T-0509 appears to be a highly selective betaI-adrenoceptor agonist with full agonist properties, while denopamine and xamoterol appear to be selective, but partial betaI-adrenoceptor agonists.
 
Spiroxatrine was identified as a moderately potent (Ki=118 nM) but non-selective agonist at the human nociceptin/orphanin FQ receptor, ORL1. This compound was subject to chemical modification and one of the resulting compounds, (8-naphthalen-1-ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl)-acetic acid methyl ester (NNC 63-0532) was shown to have high affinity for ORL1 (Ki=7.3 nM). NNC 63-0532 showed only moderate affinity for the following receptors (Ki values in parentheses): μ-opioid (140 nM), κ-opioid (405 nM), dopamine D2S (209 nM), dopamine D3 (133 nM) and dopamine D4.4 (107 nM) out of 75 different receptors, ion-channels and transporters. In functional assays, NNC 63-0532 was shown to be an agonist at ORL1 (EC50=305 nM), a much weaker agonist at the μ-opioid receptor (EC50>10 μM) and an antagonist or weak partial agonist at dopamine D2S (IC50=2830 nM). Thus, NNC 63-0532 is a novel non-peptide agonist with ∼12 fold selectivity for ORL1 and may be useful for exploring the physiological roles of this receptor owing to its brain-penetrating properties. British Journal of Pharmacology (2000) 131, 903–908; doi:10.1038/sj.bjp.0703661
 
Recombinant tissue-type plasminogen activator (rtPA) is the only globally approved treatment for acute ischaemic stroke. Other potential treatments might be administered with rtPA, making it important to discover whether compounds interfere with rtPA-induced lysis. We evaluated methods for examining the effect of the neuroprotectant NXY-059 on the lytic property of rtPA. Plasma clot formation and lysis in the presence of rtPA and NXY-059 was measured as the change in plasma turbidity. The effect of NXY-059 on rtPA-induced lysis was similarly assessed on preformed clots. Lysis of the thrombus formed in a Chandler loop measured release of fluorescent-tagged fibrinogen that had been incorporated during thrombus formation. Thrombi were exposed to both rtPA and NXY-059 throughout lysis in the presence of 80% autologous plasma and the release of label during lysis was measured. Data interpretation is limited in the clot lysis experiments because either the rtPA was present during clot formation or the drug was added to a clot formed in static conditions. In contrast, thrombi were formed in dynamic flow conditions in the Chandler loop and the time course of lysis in plasma was examined. rtPA increased thrombolysis and the antifibrinolytic trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCA) inhibited lysis. Lysis induced by rtPA was unaltered by NXY-059. The Chandler loop method provides a reliable technique for examining the effect of compounds on rtPA-induced lysis in vitro and demonstrated that NXY-059 does not alter rtPA-induced lysis at clinically relevant concentrations of either drug.
 
Disodium 2,4-disulphophenyl-N-tert-butylnitrone (NXY-059) was neuroprotective in experimental stroke models but ineffective in a large clinical trial. This first-ever individual animal meta-analysis was used to assess the preclinical studies. Studies were obtained from AstraZeneca and PubMed searches. Data for each animal were obtained from the lead author of each study and/or AstraZeneca. Published summary data were used if individual data were not available. Infarct volume and motor impairment were standardized to reflect different species and scales. Standardized mean difference (SMD), coefficients from multilevel models and 95% confidence intervals (95% CI) are presented. Fifteen studies (26 conditions, 12 laboratories) involving rats (544), mice (9) and marmosets (32) were identified (NXY-059: 332, control: 253) with individual data for 442 animals. Four studies were unpublished. Studies variably used randomization (40%), blinding of surgeon (53%) and outcome assessor (67%). NXY-059 reduced total (SMD -1.17, 95% CI -1.50 to -0.84), cortical (SMD -2.17, 95% CI -2.99 to -1.34) and subcortical (-1.43, 95% CI -2.20 to -0.86) lesion volume; efficacy was seen in transient, permanent and thrombotic ischaemia, up to 180 min post occlusion. NXY-059 reduced motor impairment (SMD -1.66, 95% CI -2.18 to -1.14) and neglect. Evidence for performance, attrition and publication bias was present. NXY-059 was neuroprotective in experimental stroke although bias may have resulted in efficacy being overestimated. Efficacy in young, healthy, male animals is a poor predictor of clinical outcome. We suggest the use of preclinical meta-analysis before initiation of future clinical trials.
 
The efficacy of the free radical trapping agent NXY-059 in reducing the infarct volume following both transient and permanent focal ischaemia has been examined in rats. In the transient ischaemia model, rats were subjected to a 2 h occlusion of the middle cerebral artery (MCA). Intravenous infusion of NXY-059 (1, 10 and 30 mg kg−1 h) for 21.75 h starting 2.25 h after the occlusion, produced a dose-dependent decrease in both neurological impairment and the histologically measured infarct volume (a mean 59% decrease at 10 mg kg−1 h). In the permanent ischaemia model, animals were injected (s.c.) with a loading dose of NXY-059 of 32.5, 53.8 or 75.4 mg kg−1 and osmotic minipumps were implanted which had been primed to deliver respectively 30, 50 or 70 mg kg−1 h. When treatment was initiated 5 min after MCA occlusion there was a dose dependent protection of both cortical and sub-cortical tissue (cortex: 63% at the mid-range dose). Protection was related linearly to plasma concentration (plasma unbound NXY-059 concentration at 1 h: 37±16 μmol l−1 at the mid-range dose). When the mid range dose was administered between 5 min – 4 h after MCA occlusion, a marked and statistically significant protection was seen at all time points (44% protection in cortex at 4 h). These data demonstrate the substantial neuroprotective efficacy of NXY-059 at plasma concentrations that can be achieved clinically and indicate that NXY-059 also has a therapeutic window of opportunity that is clinically relevant. British Journal of Pharmacology (2002) 135, 103–112; doi:10.1038/sj.bjp.0704449
 
1. It has been shown that adenosine is able to reduce the severity of arrhythmias induced by myocardial ischaemia. In isolated preparations, the antiarrhythmic effect of adenosine on ventricular myocardium is known to antagonize the catecholamine-induced stimulation of intracellular cyclic AMP production, an effect mediated via adenosine A1 receptors. 2. The aim of this study was to evaluate the antiarrhythmic effect of BN-063 (1-cyclopropylisoguanosine), a newly synthesized selective adenosine A1 agonist, on ventricular arrhythmias in rats. 3. Arrhythmias were induced by left coronary artery ligation or by administration of isoprenaline (7 mg kg-1) subcutaneously. Pretreatment with BN-063 (0.25, 0.5 and 1.0 mg kg-1) 10 min prior to occlusion significantly delayed the onset of ventricular arrhythmias, reduced the total number of ventricular premature contraction (VPC) and ventricular tachycardia (VT), decreased the incidence of VT and ventricular fibrillation (VF) and mortality during the first 30 min following left coronary artery ligation. In contrast, pretreatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 antagonist, was arrhythmogenic during the ischaemic period. The rate-pressure product, an index for indirect measurement of myocardial oxygen consumption, was also significantly reduced by BN-063 during ligation time. 4. The incidence of VT, VF and mortality was also significantly reduced when BN-063 was administered after left coronary artery ligation. 5. BN-063 converted the VF induced by isoprenaline to normal sinus rhythm and improved the survival rate. 6. It is concluded that, through activation of adenosine A1 receptors, BN-063 can suppress ventricular arrhythmias induced by myocardial ischaemia and catecholamines. The antiarrhythmic actions of BN-063 may be mediated by reducing heart rate and antagonizing the stimulatory effects of catecholamine in myocardial ischaemia.
 
The sites of interaction between the vasoconstrictor angiotensin II (A II) and the calcium antagonists PY 108-068 (PY) (a dihydropyridine derivative) or verapamil (V) in different peripheral vascular beds were investigated using the microsphere method in chloralose-urethane anaesthetized open-chested cats. A II was infused intravenously into 27 cats at a rate of 0.15 microgram kg-1 min-1. Systemic haemodynamic variables and regional blood flow were measured immediately before and 10 min after the start of the infusion. While the infusion of A II continued, PY (3 micrograms kg-1 min-1), V (30 micrograms kg-1 min-1) or the vehicle was infused for 10 min into 9 cats each and the effects of this combined infusion were again measured at the end of the 10 min period. A II increased mean arterial blood pressure but decreased peripheral conductance and, to a smaller but still significant degree, cardiac output and peak acceleration of blood in the aorta (an ejection phase parameter of myocardial contractility). The calcium antagonists reversed these effects. Cardiac output and total peripheral conductance were increased even beyond the pre-A II level by PY. A II constricted the vascular beds of the kidney, small intestine, liver and skin. Arterio-venous shunt flow decreased. Vasoconstriction was also found in the stomach, spleen and in different parts of the heart with the exception of the subendocardial layer of the left ventricle, where blood flow increased and conductance remained unchanged. A II did not decrease conductance in different parts of the brain or in skeletal muscle. The vasoconstrictor effects of A II persisted or tended to be increased in most of the vascular beds of placebo treated animals. PY 108-068 and verapamil abolished the vasoconstrictor effects of A II in most of the vascular beds with the exception of the liver, the spleen, the skin and the arterio-venous shunts and caused vasodilatation in the heart. PY also induced vasodilatation in the brain and skeletal muscle, where A II had not induced vasoconstriction. The pattern of attenuation of A II effects was different from the pattern of vasodilatation induced by these and other calcium antagonists in the same cat preparation not treated with a vasoconstrictor. The sites of action of this dihydropyridine derivative (PY) on the peripheral circulation thus, appear to depend not only on the vascular bed but also on the presence of a vasoconstrictor influence at the time of investigation.
 
Top-cited authors
Stephen Paul Alexander
  • University of Nottingham
John Christie Mcgrath
  • University of Glasgow
Vincenzo Di Marzo
  • Laval University
John Anthony Peters
  • University of Dundee
Roger G Pertwee
  • University of Aberdeen