Neuroprotective efficacy of lifarizine (RS-87476) in a simplified rat survival model of 2 vessel occlusion.

1. A new, modified rat two vessel occlusion model (with hypotension) was established and the neuroprotective efficacy of the novel agent lifarizine (RS-87476) was examined. 2. The two vessel occlusion model used in the study was a modification of the model described in the literature, whereby we have obviated the need to use a muscle relaxant and intubate the trachea to provide ventilatory support by providing a tight fitting face mask attached to the ventilator. Furthermore, the need to combine exsanguination and additional pharmacological means of inducing the mandatory hypotension (50 mmHg), required to decrease brain blood perfusion pressure, has been removed by simply manipulating the concentration of the already present halothane anaesthetic. 3. The appropriate level of hypotension having been reached, microvascular clips were applied to bilaterally occlude the common carotid arteries for 12 min. This resulted in a loss of the cortical EEG activity. Local cerebral blood flow was measured 6 min into the occlusion period, using the fully quantitative [14C]-iodoantipyrine autoradiographic technique, in a separate group of rats (n = 5). This illustrated the lack of any blood flow, in the areas under study, during the period when there was an isoelectric cortical EEG pattern. 4. The high grade global ischaemic lesion which occurred gave quantifiable neuronal damage in several vulnerable regions of the brain, namely, the hippocampal CA1 sub-field, cortex, thalamus, striatum, and cerebellar brain stem (Purkinje cells). 5. Following the global ischaemic insult the rats were allowed to recover for 72 h before assessment of the damage, during which time one group of rats (n = 11) received 100 micrograms kg-1 lifarizine i.a. 5 min post-occlusion, 500 micrograms kg-1 lifarizine i.p. 15 min post-occlusion, and 500 micrograms kg-1 lifarizine i.p. twice daily for 72 h. A second group of rats (n = 12) was treated with appropriate volumes of vehicle (0.4 ml kg-1 i.a. and 2 ml kg-1 i.p.) at identical time points. 6. Histopathological damage was assessed, from cresyl violet and haematoxyline/eosin stained sections, using a scoring system of 0-6 (no damage-complete neuronal death). The dosing regimen of lifarizine gave reduced damage in the hippocampal CA1 sub-field (4.1 +/- 0.3 to 2.8 +/- 0.6) and striatum (1.7 +/- 0.3 to 1.2 +/- 0.3) and significant neuroprotection in the anterior cortex (2.0 +/- 0.2 to 1.2 +/- 0.2; p < 0.05), thalamus (1.5 +/- 0.2 to 0.8 +/- 0.2; p < 0.01), posterior cortex (1.5 +/- 0.2 to 1.0 +/- 0.2; p < 0.05) and cerebellar brain stem (0.9 +/- 0.2 to 0.4 +/- 0.1; p < 0.01). The overall mean brain score was significantly reduced (from 1.5 +/- 0.1 to 0.9 +/- 0.2). 7. These data show that the newly modified 2 vessel occlusion model produced a quantifiable level of ischaemic damage and that the novel agent lifarizine is neuroprotective in the model.

Reduction by lifarizine of the neuronal damage induced by cerebral ischaemia in rodents.

1. The objective of this study was to evaluate the broad neurocytoprotective potential of the novel sodium-calcium ion channel modulator, lifarizine (RS-87476), in two rodent 72 h survival models of forebrain ischaemia. 2. Under fluothane anaesthesia, rats were subjected to 10 min four vessel occlusion and gerbils to either (i) 5 or (ii) 10 min bilateral carotid artery occlusion. 3. Rats were dosed parenterally solely post-ischaemia (reperfusion) in a series of five studies covering a range of intra-arterial/intraperitoneal (i.a./i.p.) combination doses from 2/10, 5/20, 20/100, 50/200 and 100/500 micrograms kg-1, where the initial loading dose was injected i.a. at 5 min. An i.p. dose was given at 15 min and repeated twice daily. In a sixth study, treatment at 50/200 micrograms kg-1 was deferred for 1 h. 4. Gerbils were treated (i) 15 min pre-ischaemia with either (a) 250, (b) 500 micrograms kg-1 i.p., or (c) 5 mg kg-1 by gavage (p.o.) for 3 days then at 1 h pre-ischaemia. Animals treated as (ii) received 500 micrograms kg-1 i.p. 15 min pre-ischaemia. The above doses were repeated twice daily for 3 days post-ischaemia for the respective groups. 5. In rats, the protective effect of lifarizine was regionally and cumulatively assessed in six brain regions (anterior and posterior neocortex, hippocampal CA1 subfield, thalamus, striatum, cerebellar Purkinje cells-brain stem) at each dose level. Cumulative (total) means +/-s.e.mean neurohistopathological scores(0-4) of 1.16+/-0.09 (n=5), 1.02+/-0.10 (n=5), 0.93+/-0.06 (n=6), 0.79+/-0.09 (n=9) and 0.45+/-0.16(n = 7), respectively, were obtained for the above treatment groups compared to the control (2.01 +/- 0.17,n = 16) group (P<0.0035). The score for the 1 h deferred treatment group was also significant at 0.77 +/- 0.10, n =5 (P< 0.0035). The normal group without ischaemia showed a score of 0.52 +/- 0.09 (n = 6).6. In gerbils, (i) percentage delayed neuronal death (DND) of hippocampal pyramidal cells in the CA1subfield was prevented at 250 (a) and 500 microg kg-' i.p. (b) (27.2+/- 14.6, n=6 and 26.9+/- 10.4%, n= 10 respectively, P<0.02) compared to controls (78.3+/-8.5%, n= 12) and by 5 mg kg-1 p.o. (c) (2.9+/-0.8%,n =l1, P <0.002). Mean +/- s.e.mean total brain scores (0-4) for each of 4 different features denoting cerebral 'oedema' were lower for normal brains (1.60 +/-0.34, n =6) and reduced in animals dosed at 250(a) (3.00+/-0.79, n=6) and 500 microg kg-1 i.p. (b) (3.75 0.36, n= 10) compared to controls (6.58+/-1.00,n = 12) (P< 0.02 -0.03). There was a linear relationship (r = 0.97) between the 'oedema' scores and percentage CA1 DND. Percentage CA1 DND in response to 10 min ischaemia (ii) was reduced(53.0+/-21.0%, n=6, P<0.05) compared to controls (100.0+/-0.0%, n=7).7 The significant neuroprotection shown by lifarizine in rodents substantiates findings in other species.These observations, together with its effect on ion channels and efficacy at extremely low doses offers novelty and suggests a broad spectrum of activity in ischaemia.

The effect of lifarizine (RS-87476), a novel sodium and calcium channel modulator, on ischaemic dopamine depletion in the corpus striatum of the gerbil.

1. Unilateral ligation of the right common carotid artery in the anaesthetized gerbil for 3 h caused a 62.7% decrease in ipsilateral dopamine in the corpus striatum from 1.40 (+/- 0.13, n = 27) micrograms g-1 in the non-ischaemic hemisphere to 0.47 (+/- 0.07, n = 27) micrograms g-1 in the ischaemic hemisphere (all results are expressed as mean +/- s.e. mean). In sham-operated animals there were no differences in the dopamine levels (1.31 +/- 0.14 micrograms g-1, n = 11, left; 1.27 +/- 0.13 micrograms g-1, n = 11 in the right hemisphere). Animals with intact communicating arteries in the circulus arteriosus were excluded. 2. Lifarizine (RS-87476; 250, 500, but not 50, micrograms kg-1, i.p.) protected against this dopamine depletion showing only a 9.2% decrease at 250 micrograms kg-1, i.p. (P < 0.01) and no decrease at 500 micrograms kg-1, i.p. (P < 0.01). 3. Nicardipine (250 micrograms kg-1, p.o.) was effective when administered chronically once daily for 10 days (26.6% decrease, P < 0.05) but not when administered acutely at 50 micrograms kg-1, i.p.

Drugs acting on calcium channels: potential treatment for ischaemic stroke.

Calcium subserves a ubiquitous role in the organisation of cell function. Ca2+ channels which control influx may be modified in disease states. Animal models of cerebral ischaemia do present some problems when investigating potential therapies involving Ca2+ channels. However, it is important not to be too rigid in searching for models which exactly mimic the human disease state, when even the best experimental approaches fall short of such an ideal. There are differences between different classes of calcium entry blocking drugs with regard to their activity on Ca2+ channels and transmembrane Ca2+ movement. Some calcium antagonists may also affect ion channels other than Ca2+, and this potential is exemplified by the novel ion channel modulator RS-87476, which affords experimental neurocytoprotection. Limitation of intracellular Na+ influx during ischaemia-induced depolarization may be useful.

Prevention of myocardial enzyme release by ranolazine in a primate model of ischaemia with reperfusion.

In control anaesthetized baboons subjected to 30 min occlusion of the left anterior descending coronary artery, followed by 5.5 h reperfusion, total plasma levels for creatine kinase (CK) and lactate dehydrogenase (LDH) were markedly elevated in a time-related manner. In a second group of baboons pretreated 10 min prior to ischaemia with ranolazine [(+/-)-N-(2,6-dimethyl-phenyl)-4[2-hydroxy-3-(2-methoxyphenoxy)propyl]-1 - piperazine acetamide dihydrochloride; RS-43285-193] at 500 micrograms kg-1 i.v., followed by continuous infusion of 50 micrograms kg-1 min-1, neither enzyme was significantly elevated at any time point. Similarly, serum levels of the cardiospecific isoenzyme CK2 were 8 fold greater in the controls than in the ranolazine-treated animals at 6 h. The results indicate that ranolazine pretreatment abolished cardiac enzyme release over a 5.5 h reperfusion period, indicating a potential protective effect.

Effects of enprostil on cardiovascular and respiratory parameters in the dog, and on hematologic parameters in the rat, monkey, and human.

The effects of enprostil (+/-)-7-[(1R*,2R*,3R*)-3-hydroxy-2-[(E)-(3R*)-3-hydroxy-4-phenoxy-1- butenyl]-5-oxocyclopentyl]-4,5-heptadienoate) were studied on cardiovascular and respiratory parameters in the dog and on hematologic parameters in the rat, monkey, and human. Anesthetized dogs were instrumented to allow measurement of heart rate, systemic blood pressure, respiratory rate or tracheal pressure, and ventricular contractile force after intragastric (i.g.) or intravenous (i.v.) administration of enprostil in the presence or absence of autonomic challenges. The effects of intraduodenal (i.d.) enprostil on arterial and venous PO2, PCO2, and pH; respiratory rate, flow rate, and volume; blood pressure (b.p.); and heart rate were also examined. Enprostil i.v. (0.3-10 micrograms/kg) significantly increased tracheal pressure in a dose-dependent manner, but minimally altered b.p., heart rate, and ventricular contractile force. Enprostil i.v. (1-10 micrograms/kg) significantly inhibited pressor and depressor responses to several autonomic challenge agents at the highest dose level, indicative of a nonspecific inhibitory effect on b.p. responses. Cardiovascular effects of enprostil (1-100 micrograms/kg i.g.) were absent. Enprostil (10-3,000 micrograms/kg i.d.) had no noteworthy effects on respiratory parameters in the dog. Platelet aggregation effects of enprostil were studied in vitro using platelet rich plasma (PRP) from the rat, monkey, and human; whole blood clotting time and prothrombin time after oral enprostil were studied in the rat; and ex vivo effects on platelet activation were studied in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the effects of a series of anti-anginal agents in a novel canine model of transient myocardial ischaemia.

1. An anaesthetized canine model of transient myocardial ischemia (TMI) has been developed in which reproducible and reversible electrocardiographic (ECG) and haemodynamic responses are exacerbated by electrical pacing. 2. The model could separate the ECG and haemodynamic effects of compounds with anti-ischaemic properties. 3. Compounds known to possess peripheral or coronary vasodilator properties did not necessarily alleviate the ECG consequences of TMI since glyceryl trinitrate was active whereas dipyridamole was not. The effects of verapamil were complicated by its adverse conduction effects while lidoflazine inhibited the ECG changes only during the ischaemic phase and the 'metabolic modulator' oxfenicine worsened the ECG response. 4. In a model considered to lack coronary reserve, improvements observed in the ischaemic ECG and global ventricular function were considered to result from a direct myocardial effect of the drugs examined rather than by a vascular influence. This was provided to the greatest degree by the Ca2+-entry blockers nifedipine and nicardipine, with flunarizine adopting an intermediate position.

Activators and inactivators of calcium channels: effects in the central nervous system.

The interactions of calcium antagonists or channel activators with the different classes of calcium channel are reviewed with particular emphasis on interactions with neuronal tissue; reasons for the failure of calcium antagonists to inhibit neurotransmitter release under normal circumstances are outlined. Calcium antagonists may be protective in several pathological situations and the possibilities of protection against ischaemic damage in the central nervous system are evaluated.

Effects of the novel class Ia and class III antiarrhythmic agent RS-87337 on myocardial conduction in the anaesthetised dog.

The effects of the novel Class Ia/III antiarrhythmic compound RS-87337 on canine myocardial conduction were compared with those of the Class I antiarrhythmic disopyramide. RS-87337 had no effects on intra-atrial (I-A) or intra-ventricular (I-V) conduction parameters up to 10 mg.kg-1 i.v. (n = 6). Only one incidence of atrioventricular (A-V) block occurred at 10 mg.kg-1 at a pacing frequency of 261 beat.min-1. Disopyramide (5-10 mg.kg-1 i.v., n = 6) produced a frequency-dependent I-A conduction block and also significantly increased resting and paced A-V conduction times. Overall, disopyramide exhibited atrioselectivity while RS-87337 appeared more selective for ventricular conduction, possibly produced by a balance of its mixed Class Ia/III properties. RS-87337 was not cardiodepressant in the normal canine myocardium and produced no adverse effects on conduction parameters at doses up to 10 mg.kg-1 i.v.

Comparative protective effects of nicardipine, flunarizine, lidoflazine and nimodipine against ischaemic injury in the hippocampus of the Mongolian gerbil.

1. Morphological changes characterizing delayed neuronal death (DND) of selectively vulnerable CA1 pyramidal cells in the hippocampus of the Mongolian gerbil brain occurred 72 h after transient (5 min) bilateral occlusion of the common carotid arteries. 2. Different groups of animals were treated 15 min before carotid artery occlusion and twice daily during the 72 h post-ischaemia period with either saline alone, nicardipine, flunarizine, lidoflazine or nimodipine at doses of 500 micrograms kg-1 intraperitoneally. 3. At 72 h the animals were killed and their brains examined histologically. Absolute cell counts were made from 5 sites distributed linearly throughout the hippocampal CA1 subfield in each hemisphere to determine the percentage DND in each group. Normal brains and those of sham-operated animals were included in the study for comparison. 4. Features of DND were distributed evenly throughout the CA1 subfield in both hemispheres in all groups of gerbils. Nicardipine, lidoflazine and flunarizine, but not nimodipine, were protective. This protection extended linearly throughout the hippocampus without altering the pattern of neuronal damage. 5. Compared to saline-treated (78.3 +/- 2.9% DND) and nimodipine-treated (76.5 +/- 3.4% DND) gerbils, the overall protection afforded by nicardipine (41.8 +/- 3.8% DND) was statistically significant. The effects of lidoflazine (53.6 +/- 7.1%) and flunarizine (55.8 +/- 3.9% DND) were of borderline significance. 6. Abnormal neurones appeared in normal and sham-operated brains to the extent of 4.5 +/- 1.0% and 4.6 +/- 0.4%, respectively. Such changes can be attributed to fixation artefacts. 7. The results demonstrate that overall protection is conferred on ischaemic hippocampal CA1 neurones by nicardipine and to a lesser extent by flunarizine and lidoflazine, but not by nimodipine.

The effects of the novel anti-anginal compound RS 43285 on myocardial conduction in the anaesthetized dog.

1. A pentobarbitone-anaesthetized canine model of myocardial conduction was developed to evaluate drug effects on intra-atrial (I-A), intra-ventricular (I-V) and atrioventricular (A-V) conduction parameters, both at rest and during electrical pacing of the right atrium or ventricle. Drug effects on the ability of the sino-atrial (SA) node to re-establish sinus rhythm on switching off electrical pacing were also considered. The effects of the novel anti-anginal compound RS 43285-193 ((+/-)-N-(2,6-dimethyl-phenyl)-4[2-hydroxy-3-(2-methoxyphenoxy)propyl] -1-piperazine acetamide dihydrochloride) were compared to those of the standard anti-anginal compounds nicardipine, nifedipine and verapamil. 2. In the dose range 15-7000 micrograms kg-1, RS 43285 had no significant effects on I-A, I-V or A-V conduction either at rest or during electrical pacing and did not affect the re-establishment of sinus rhythm. 3. Nicardipine had no effects on conduction parameters at resting heart rate. There were no effects on I-A or I-V conduction on electrical pacing but A-V conduction was increased at 200-500 micrograms kg-1 (with a 2:1 A-V conduction block in two out of six dogs); this was accompanied by a prolongation of the interval to reversion of sinus rhythm. 4. Nifedipine had no significant effects on I-A or I-V conduction but significantly prolonged A-V conduction at 1000 micrograms kg-1 and this dose also increased the interval to SA node recovery. 5. Verapamil did not effect I-A or I-V conduction. However, A-V conduction was affected with a significant prolongation occurring at resting heart rate at 100-400 Atg kg-' and a 2:1 A-V block in one dog at rest. During right atrial pacing verapamil significantly increased A-V conduction at 50- 400 fig kg-'. All dogs exhibited a 2:1 A-V conduction block at the highest frequency at 400 jig kg-'.

The potential beneficial effect of nicardipine in a rat model of transient forebrain ischemia.

In a rat 3-day survival model of 10-minute four-vessel occlusion, halothane anesthesia was used to attenuate the ictal blood pressure elevation of the cerebral ischemic response and thereby maintain an isoelectric EEG. Selectively vulnerable regions of the brain were protected by preischemia plus postischemia maintenance treatment with the calcium entry blocker nicardipine. Compared with untreated animals, repeated doses at 500 micrograms/kg IP were markedly more effective than doses of 50 micrograms/kg. Ongoing studies demonstrate a neurocytoprotective action of nicardipine when deferred treatment is given postischemia.

Nicardipine in models of myocardial infarction.

In a dog model of partial myocardial ischaemia, superimposed ST segment elevations in epicardial ECGs were inhibited by nicardipine over a cumulative i.v. dose range of 1-20 micrograms kg-1. Over the cumulative i.v. dose range of 0.5-166.5 micrograms kg-1, nicardipine had little overall effect on gross cardiac conduction, at spontaneous heart rate. Dogs that received oral 1-2 mg kg-1 nicardipine daily for 16 weeks and then survived 1 week occlusion of the left anterior descending coronary artery (LAD) developed a superior coronary collateral circulation compared with untreated animals. Nicardipine given by three different dosing schedules to baboons markedly limited myocardial infarction over a 6 h period of LAD occlusion. Compared with a group of completely untreated dogs, there was protection of the myocardium in the animals given nicardipine that survived 3 months occlusion of the LAD.

The beneficial effect of nicardipine on the healing of myocardial infarcts in dogs.

The effect of 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methyl amino)] ethyl ester 5-methyl ester hydrochloride (nicardipine, RS-69216, YC-93), on the healing of myocardial infarcts has been examined in dogs surviving for 3 months after ligation of the left anterior descending coronary artery (LAD). Three groups of 12-14 dogs were subjected to the following treatments: a) Group X received one-month oral pretreatment (1-2 mg/kg/d) with post-ligation i.v. treatment delayed for 1 h (10 micrograms/kg followed by 2 micrograms/kg/15 min for 5 h) and oral treatment (1-2 mg/kg/d) for 3 months (n = 9 survivors). b) Group Y received oral placebo for one month, i.v. saline vehicle every 15 min for 5 h commencing 1 h postligation, and oral placebo for 3 months (n = 8 survivors). c) Group Z received oral placebo for one month as in group Y, i.v. and oral drug treatment post-ligation as for group X (n = 10 survivors). Recordings were made of general haemodynamics and at post mortem, each heart was X-rayed and sliced transversely to give 14 sections from which detailed observations were made on left ventricular (LV) geometry. Comparative morphological measurements were available from filed data for 11 normal dog hearts (designated group C) of similar type. Nicardipine treatment significantly modified the general haemodynamic responses to LAD ligation particularly in maintaining a low LV systolic pressure and inhibiting elevation of LV end diastolic pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

The gastric antisecretory activity of 3-methyl-5,6,7,8-tetrahydroquinoline-8-thiocarboxamide hydrochloride (tiquinamide).

3-Methyl-5,6,7,8-tetrahydroquinoline-8-thiocarboxamide hydrochloride (tiquinamide) possesses potent gastric antisecretory activity in a number of animal species. It causes a marked inhibition in the volume and concentration of basal and chemically-stimulated gastric acid secretion in the conscious rat and guinea-pig, and of chemically-stimulated secretion in the anaesthetised cat, dog and monkey. Marked activity is also seen in conscious Heidenhain-pouch dogs, against secretion stimulated by betazole and gastrin tetrapeptide. In the pylorus-ligated rat preparation, the antisecretory effect of 30 mg/kg tiquinamide persists for 8-9 h. The possible mode of action of this drug, in view of its wide non-selective antisecretory profile, is discussed.

The gastrointestinal and autonomic effects of 3-methyl-5,6,7,8-tetrahydroquinoline-8-thiocarboxamide hydrochloride (tiquinamide).

3-Methyl-5,6,7,8-tetrahydroquinoline-8-thiocarboxamide hydrochloride (tiquinamide), a potent gastric anti-secretory compound, affords good protection against stress- and chemically-induced gastric and duodenal erosions. It causes a dose-related delay in gastric emptying and intestinal transit, and does not have a primary effect on gastric mucosal blood flow. The only actions of tiquinamide on the peripheral autonomic nervous system are weak, but apparently specific, histamine H2-receptor blocking activity, and non-competitive inhibition of acetylcholine and histamine responses at 10(-3) mol/l. Since no other effect on isolated tissues can be detected, it seems unlikely that the antisecretory action of tiquinamide involves a direct action on the peripheral autonomic nervous system.

The renin-angiotensin system, dietary salt, and increased sensitivity to noradrenaline in mesenteric vasculature preparations from renal/salt hypertensive rats.

Exogenous angiotensin II causes noradrenaline supersensitivity in rat mesenteric vasculature preparations. The noradrenaline supersensitivity of tissues from renal/salt hypertensive rats, with low plasma renin activity, is not caused by endogenous angiotensin II since it was unaffected by Sar1 Ileu8 angiotensin II. Dietary salt-loading caused a small increase in noradrenaline sensitivity.