Brian J. Alps

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 μg 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.

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 μg kg−1 lifarizine i.a. 5 min post‐occlusion, 500 μg kg−1 lifarizine i.p. 15 min post‐occlusion, and 500 μg 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.

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) μg g−1 in the non‐ischaemic hemisphere to 0.47 (± 0.07, n = 27) μg 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 μg g−1, n = 11, left; 1.27 ± 0.13 μg 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, μg kg−1, i.p.) protected against this dopamine depletion showing only a 9.2% decrease at 250 μg kg−1, i.p. (P < 0.01) and no decrease at 500 μg kg−1, i.p. (P < 0.01). 3 Nicardipine (250 μg 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 μg kg−1, i.p.

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‐dimethylphenyl)‐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 μg 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 μg 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 μg 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 μg kg−1 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 μg kg−1. All dogs exhibited a 2:1 A‐V conduction block at the highest frequency at 400 μg kg−1.

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 μ 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 μg kg−1 was deferred for 1 h. 4 Gerbils were treated (i) 15 min pre‐ischaemia with either (a) 250, (b) 500 μg 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 μg kg 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) mean ± 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 CA1 subfield was prevented at 250 (a) and 500 μg kg−1 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 =11, 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 μg 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 = 1). 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.