IE83650B1 - Use of vincristine for the evaluation of substances at the neuronal level - Google Patents

Description

USE OF VINCRISTINE FOR THE EVALUATION OF SUBSTANCES AT THE NEURONAL LEVEL The present invention relates to a method for the pharmacological screening of neuronally active substances which comprises: - producing neuronal lesions to non-human animals intended for pharmacological experimentation by injection in the medial septum of the animals of an amount of vincristine effective to this purpose; - administering the substance to be tested to the lesioned animals according to the suitably selected schedule; - sacrificing the animals; and - evaluating the effects of the treatment by means of morphological and/or biochemical assays.

In the study conducted by these Authors, the resemblance between the neurochemical and Behavioural modifications caused by infusion of a neurotoxicant, in particular colchicine, in the hippocampus and those observed in patients affected by Alzheimer's disease has been clearly pointed out. On the basis of the results published by Y. Nakagawa et al. and bearing in mind the crucial role played by the hippocampus in memory and learning, it has been attempted to set up an experimental model for the Alzheimer's disease of improved feasibility and even closer to the physiopathologies documented in patients with Alzheimer's disease.

The experimental model which is herein provided complies with these requirements: good feasibility and irreversible lesions highly specific for the septohippocampal cholinergic system.

In particular, lesions of the septal neurons have been caused by local injection of vincristine known to be a tubuline polymerisation inhibitor.

With respect to other similar compounds (colchicine and vinblastine) and different injection sites (intraventricular and intrahippocarnpal) which have been tested, optimum results, both in terms of specific blockade of septohippocampal cholinergic transmission and irreversible lesions, have been obtained.

Operative procedures The animals (male Sprague-Dawley rats weighing about 250 g) are anesthetised with pentobarbital (10 mg/kg i.p.) and placed in a stereotaxic apparatus.

The injection in the medial septum is made at the following coordinates which are calculated according to the atlas of Paxinos and Watson: A. 8.9 L. 0 H. 6.4 wherein point 0 corresponds to lambda.

Vincristine is dissolved in artificial cephalorachidian liquid (ACSF) having the following composition NaCl 150 mM CaC12 1.8 mM MgSO4 1.2 mM K2HPO4 2 mM glucose 10 mM pH 7.4 at a concentration of 0.6 umole of vincristine per ml. lul of this solution (0.6 nrnole of vincristine) is locally injected in the medial septum over minute.

Assessment of the lesions - Evaluation of morphological changes (histoenzymatic AChE determination).

The animals are perfused with a fixating mixture (glutaral—dehyde/paraforrnaldehyde) via the aorta, with a perfusion flowof 25 ml/min for 5 minutes. Brains are removed, fixation being continued for l hour, then washed and cryoprotected with 20% sucrose in phosphate buffer. Brains are there cut on a cryostat and the cryostat sections (30 um thick) of the septum and the hippocampus are mounted on metal slides which are incubated for about 15 hours in the following medium: 200 ml of a stock solution distilled water 925 ml CuSO4 781 mg glycine 750 mg sodium acetate 2.89 g to which acetylcholine iodide 230 mg and ethopropazine 10 mg are added just before use.

The reaction is then detected by means of 2% ammonium sulfide and evidenced by 0.25% AgNO3.

The presence of acetylcholinesterase (generally associated with cholinergic synapses) is indicated as a dark precipitate.

Biochemicalobservations (ChAT) ChAT activity is determined by the method described by Fonnum (J .Neurochem. 24, 1975, 407-409). Tissue samples are homogenised at 4°C. Each sample is brought to a concentration of 1 mg of protein per ml. Aliquots of the obtained homogenates (10 ul) are incubated for 7 minutes 30 seconds at 37°C, in the presence of choline (1.5 mM), acetylCoA (70 uM),l4C- acetylCoA (30 uM) and physostigmine (0.15 mM).

The reaction is stopped by lowering the temperature by means of an ice-bath and adding phosphate buffer (5 ml). After addition of tetraphenylboron/acetone (2 ml) and of a scintillating agent (5 ml), MC-acetylcholine is counted in a scintillation spectrometer. Each sample is tested in triplicate.

Each time the result is compared with that obtained with the corresponding control by the Student test.

Behavioural studies Groups of animals kept with an inverse light-dark cycle have been employed specifically for these studies. The rats used in these tests are Winstar rats lesioned as described above.

Social memog test (A. Perio et al., Psychopharmacology, 1989, 31, 262-268). In this test a juvenile rat is placed in the home cage of an adult rat and the time spent by the adult rat in investigating the juvenile is measured in seconds (T1). The animals are then separated for 15 minutes and then the adult rat is again exposed to the same juvenile and the time of investigation during this second exposure is also measured (T2). In the case of normal animals, the recognition of the juvenile rat reduces the time of investigation (T2/T1 there is a memory impairment the T2/Tl ratio is 31.

T-maze learning test This test has been carried out according to the methodology described by P. Soubrié et al., in J . Pharmacol. (Paris), 1977,8,3,393-403 for the Y-maze test.

Holeboard test (S.E. File et a1., Pharmacol. Biochem. Behav., 1985, ~, 941-44).

Assessment of the lesions Intraseptal vincristine administration affords a rapid and significant decrease (from 60 to 70% within one week after the injection) of the cholinergic markers of the hippocampus (choline acetyltransferase (ChAT) and acetylcholine esterase (AChE)), as well as a degeneration of the medial septum neurons which reaches its maximum two weeks after the injection, and which is associated with a reduction of the cholinergic markers. Said degeneration seems to be irreversible as it is still present three months after vincristine injection and involves functional disorders.

Among those functional alterations which result from vincristine administration, the major finding is a consistent and irreversible memory impairment (social memory test). Parallel assays have brought up i.a. a reduction of the explorative capabilities (T-maze learning test and hole-board test).

Treatment schedules The effects of administering l-[2-(2-naphthyl)ethyl](3-trifluoromethylphenyl)-l,2,3, -tetrahydropyridine hydrochloride (Compound A) to the animals lesioned as above have been compared with those obtained by administering NGF .

Compound A is administered orally, 2 to 3 hours after Vin- cristine injection, as a 1% carboxymethylcellulose suspen- sion, 10 ml of suspension per kg of body weight. The controls receive the vehicle only. The treatment is chronic, once a day for 11 days.

Compound A is administered at three different doses: 25 mg/kg, 5 mg/kg, and 10 mg/kg to groups of 8 animals each, and the animals are sacrificed 24 hours after the end of the treatment.

On the contrary NGF is administered by intraventricular in- fusion, dissolved in artificial cerebrospinal fluid (ACSF) containing 0.01% rat albumin and gentamycin (1 ml/ 15 ml) according to the method described by w. Fisher et al., in Nature, 1987, 329 (6l34),65-8.

NGF concentration in the solution is calculated in View of the selected diffusion flow rate (0.44 t 0.02 ul/h) so as to provide the animals (7 rats) with an overall amount of 0.105 pg, 1.05 pg, or 10.5 ug of NGF over two weeks of infusion. In the controls, NGF is replaced by a protein of similar molecular weight (about 130.000) which has no neurotrophic activity: cyto-chrome C. Two weeks after the lesions have been placed and the treatment has begun, the animals are sacrificed. One group of sacrificed animals is perfused for histoenzymatic determination of AChE, the other animals are on the other hand employed for the assay of ChAT activity in both hippo- campus and septum.

Morphologicalobservations In the lesioned, untreated, animals no dark precipitate is seen while the hippocampal buddings observed in the animals treated with 5 mg/kg of Compound A are quite similar to those seen in normal animals. These results are analogous to those obtained in the NGF- treated animals.

Biochemical evaluation The lesions provoke a marked decrease of ChAT activity in the hippocampus whose extent is reduced in a dose-dependent manner by Compound A, up to a complete recovery with the dose of 10 mg/kg. Analogous results are obtained in the NGF perfused animals.

Most particularly the obtained results are summarised in the following table ChAT activity (pmols/mg/mn) non-lesioned normal animals 277 j 13 lesioned controls 115 j 18 NGF 0.105 pg/rat/2 weeks 168 j 27 NGF 1.05 pg/rat/2 weeks 336 j 28 NGF 10.5 ug/rat/2 weeks 293 4; 10 non-lesioned normal animals 242 j 19 lesioned controls 97 : 18 Compounds A 2.5 mg/kg/day 164 _-t 42 Compounds A 5 mg/kg/day 204 _+_ 24 Compounds A 10 mg/kg/day 242 1‘ 27 In the septum, the lesions afford reduction of ChAT activity which is restored, in a dose- dependent manner, by the administration of Compound A. In the NGF-treated animals the results are not significant probably because in addition to the septal necrosis produced by the vincristine injection, an additional necrosis associated with the implantation of a cannula in a ventricle near the septum develops.

Behavioural observations Social memory test The vincristine lesioned animals show alterations in social memory which seem to be irreversible (said alterations are still present 50 days after vincristine injection).

Compound A has been tested at the dose of 10 fig/kg per os in comparison with NGF at the dose of 10.5 ug and the test has been performed on day 7 from placement of the lesions.

In both cases a very important protecting effect has been elicited with a T2/T1 ratio of between 0.6 and 0.7.

T—maze test The results obtained in the lesioned, untreated, animals show an alteration in the exploratory activity which is however restored to the normal level by administration of l0 fig/kg of Compound A.

This test has been carried out in blind, 7 days after the end of the treatment.

Holeboard test In the controls, most of the animals (6 rats) completely lost their exploratory capacity, while only two rats showed an exploratory hyperactivity. Treatment with Compound A at the dose of 10 fig/kg leads to a normalisation of the explorative behaviour compared with the average results obtained with unlesioned animals.

Said test too has been carried out in blind, 11 days after the end of the treatment.

Claims (2)

CLAIMS:

1. A method for the pharmacological screening of neuronally active substances which comprises: - producing neuronal lesions to non—human animals intended for pharmacological experimentation by injection in the medial septum of the animals of an amount of vincristine effective to this purposes; — administering the substance to be tested to the lesioned animals according to the suitably selected schedule; - sacrificing the animals; and - evaluating the effects of the treatment by means of morphological and/or biochemical assays.

2. A method according to Claim 1, substantially as herein described. MACLACHLAN & DONALDSON, Applicants’ Agents, 47 Merrion Square, DUBLIN. 2.