HK1001471B - Use of 1-(2-naphthylethyl)-4-(3-trifluoromethylphenyl)-1,2,3,6-tetrahydropyridine for the preparation of medicaments for the treatment of cerebral and neurona - Google Patents

Description

The present invention relates to the use of a 1- ((2-naphthylethyl) -4- ((3-trifluoromethylphenyl) -1,2,3,6-tetrahydropyridine of the following formula: - What? or its salts of addition with pharmaceutically acceptable acids, for the preparation of medicinal products for the treatment and/or prophylaxis of brain and neurological diseases.

In particular, the present invention relates to the use of a pharmaceutically acceptable compound of formula (I) or its additive salts in the preparation of drugs for the treatment of diseases associated with neuronal degeneration.

In formula (I) above, the 1-[4-(3-trifluoromethylphenyl)-1,2,3,6-tetrahydropyridinyl]ethyl group may be bound at position 1 or 2 of the naphthalene.

The formula (I) compounds, in the form of free bases or additive salts, and their preparation were described in European patent application EP-A-101381. Pharmaceutically acceptable additive salts of the formula (I) compound include those formed with mineral acids, such as hydrochloric, bromide, phosphoric or sulphuric acids, or with organic acids such as acetic, formic, propionic, benzoic, maleic, succinic, tartaric, citric, methoxylic, aspartic, methanesulfonic, ethanesulfonic, benzulfonic, p-tulsulfonic, etc.

The European patent application cited above reports anorexigenic activity of the products.

It has now been found, quite unexpectedly, that compounds of formula (I) are capable of exerting a neurotrophic action on the nervous system, similar to that of Nerve Growth Factor (NGF) and can restore the functioning of nerve cells damaged or with abnormalities in their physiological functions.

In vitro pharmacological evaluation of the active substance

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In particular, the septic region of 17-day-old rat embryos was removed under dissection microscope and the structures were maintained at 4°C during the entire sampling period in the following environment: - What?

DME/F₁₂ (v:v)
glucose	5 %
amphotéricine B	1 %
gentamycine	0,5 %

The cells are dissociated by treatment for 20 minutes at 37°C with trypsin-EDTA, then centrifuged twice and washed in PBS. The dissociation is completed at the Pasteur pipette in Hanks' medium. This step is followed by 3 centrifugations. - What?

DME/F₁₂ (v:v)
KCl 34 mM
sérum de veau foetal	5 %
sérum de cheval	5 %
glutamine	0,1 %
amphotericine B	1 %
gentamycine	0,5 %

This cell suspension is placed in a culture flask and kept in the oven for 90 minutes at 37°C at 5% CO2.

The non-nerve cells will quickly adhere to the plastic of the vial, and the suspension is therefore enriched with neurons (95 to 98%). These suspensions were centrifuged and the cocoons taken back into a non-serum culture medium (H.W. Muller and N. Seifert, J. Neurosc. Res., 8, 195-204, 1982): - What?

DME/F₁₂ (v:v)
transferrine	1 µg/ml
tri-iodothyronine	3 mM
insuline	5 µg/ml
hydrocortisone	20 µM
glutamine	0,1 %
amphotericine B	1 %
gentamycine	0,5 %

The neurons were cultured in 96-well plates (5x104 viable cells per well).

Each well was treated with poly-L-lysine (10 μg/ml) to form a matrix essential for neuronal adhesion, survival and differentiation. 130 μl samples of non-serum medium containing either the test products of formula (I) at the doses chosen or the solvent of the product (dimethyl sulfoxide) at the corresponding concentrations were distributed in the wells. After seeding the neurons, the plates were kept in the oven (37 °C - 5% CO2) for 18 hours. The cells, after fixation in a glutaraldehyde/paraformaldehyde mixture, were counted as follows: In each well seeded, the total number of cells observed and the number of cells with at least one neurite (neurite = extension) twice the diameter of the cell are counted for each microscopic field,five fields are counted per well and two wells are seeded for each dose of the tested product, so that ten values are obtained for each dose.The results are expressed as a percentage of neurite cells out of the total number of cells counted.Each batch is compared to its control by a non-parametric Kruskall-Wallis analysis.

The results obtained with the hydrochloride of 1-[2-(2-naphthyl) ethyl]-4-(3-trifluoromethylphenyl)-1,2,3,6-tetrahydropyridine (Compound A) are as follows: - What? TABLEAU I

	% DE CELLULES A NEURITES
Composé A: 2,4 nM	44,8 ± 3,45
Contrôle (DMSO 10⁻⁶)	30,2 ± 1,68
Composé A: 24 nM	39,2 ± 2,39
Contrôle (DMSO 10⁻⁵)	29,4 ± 1,64
Composé A: 240 nM	44,1 ± 2,02
Contrôle (DMSO 10⁻⁴)	34,3 ± 1,82
Dans le même test, en utilisant le NGF, on obtient les résultats suivants:

TABLEAU I

	% DE CELLULES A NEURITES
NGF: 1,6 nM	51,7 ± 1,61
Contrôle	40,6 ± 2,08

The mechanism by which Compound A exerts this neurotrophic activity has not been elucidated.

However, it cannot be ruled out that it involves a serotonergic effect because Compound A shows no affinity for the various serotonin receptors other than the 5-HT1A receptor (i.e. 5-HT1B, 5-HT1C, 5-HT1D, 5-HT2 and 5-HT3 receptors) and on the other hand compounds known as 5-HT1A agonists or partial agonists, including buspirone, lipsapirone and 8-hydroxy-2-di-n-propylamino) tetraline (8-OH-DPAT), have been found to be completely inactive in the model described above.

Compound A was also very active (at concentrations between 250 nM and 2.5 μM) in a neuronal survival protection test in which neurons are placed in a very poor environment with no growth factors.

In vivo pharmacological evaluation of the active substance

To confirm the interest of these very positive in vitro results, a new experimental model has been developed which has allowed the in vivo evaluation of the neurotrophic/neuroprotective activity of formula (I) compounds in neuronal degeneration processes.

An experimental model for this type of assessment has recently been proposed by Y. Nakagawa et al. (Brain Research, 1987, 408, 57-64).

In their study they showed the similarity between the neurochemical and behavioural changes caused by the injection of a neurotoxic agent, in particular colchicine, into the hippocampus and those found in patients with Alzheimer's disease.

The model we have developed answers the following questions: good feasibility and irreversible damage, very specific to the septohippocampal cholinergic system.

In particular, damage to septic neurons has been induced by local injection of vincristine as an inhibitor of tubulin polymerization.

Compared with other products of this type (colchicine and vinblastine) and the different injection sites (intraventricular and intrahippocampal) tested, the best results were obtained in terms of either specificity of blockade of the septohippocampal cholinergic pathway or irreversibility of lesions.

The following information is provided for the purpose of this report: Method of production of lesions

The animals (male rats, Sprague Dawley about 250 g) are anesthetized with pentobarbital (10 mg/kg i.p.) and placed on a stereotactic device. In accordance with the coordinates of the Paxinos and Watson atlas, the injection into the median spetum is made at the following coordinate point: - What?

antériorité	8,9
latéralité	0
ventralité	6,4
(le point 0 correspond au lambda)

Vincristine is dissolved in artificial cerebrospinal fluid (CSF) with the following composition: - What?

NaCl	150 mM
CaCl₂	1,8 mM
MgSO₄	1,2 mM
K₂HPO₄	2 mM
glucose	10 mM
pH 7,4

The maximum concentration of vincristine in the feed additive shall be 0,6 μmol/ml.

1 μl of this solution (0.6 nmol of vincristine) is injected topically into the median septum in 1 minute.

Method of assessment of lesions - Morphological evaluation (histogenic observation of the AchE)

The animals are infused through the aorta with a fixative mixture (glutaraldehyde/paraformaldehyde) at an infusion rate of 25 ml/min for 5 minutes.

The brain is removed, fixation continued for 1 hour; this step is followed by washing and cryoprotection in 20% sucrose in a phosphate buffer. The brain is then cut at the cryostat, 30 μm thick cuts are taken from the septum and hippocampus and mounted on a blade. The blades are incubated for about 15 hours in the following mixture: 200 ml of stock solution: to which is added ex-temporarily 230 mg acetylthiocholine iodide 10 mg of ethopropazine. The reaction is then detected by passing in 2% ammonium sulphide and amplified by AgNO3 at 0,25%.

The acetylcholinesterase sites (usually associated with cholinergic synapses) are revealed as a brown precipitate.

- Biochemical evaluation (determination of the activity of the CHAT)

The determination of ChAT activity was carried out by the Fonnum method (J. Neurochem., 24, 1975, 407-409). The tissues are homogeneous in a potter (at 4°C). Each sample is reduced to 1 mg protein/ml. 10 μl homogenate is incubated in the presence of 1.5 mM choline, 70 μM acetylCoA, 30 μM 14C marked acetylCoA, in the presence of 0.15 mM serine. The incubation lasts 7 minutes 30 sec. at 37°C. The reaction is stopped in the ice bath with 5 ml of phosphate buffer. After adding 2 ml of tetracycline/acetylbore and 5 ml of stillate, the marcoline acetylcyl 14 is counted with the corresponding dose of each batch of test is compared with the control sample three times.

- Behavioural assessment

This study involves groups of animals reserved for this purpose, with rats being reversed, and the rats used in this case are Wistar strains, harmed by the same method described above.

- Social memory

The test is based on the observation that a juvenile rat is placed in the cage of an adult rat and the duration of the social relationship is measured in seconds (T1). The animals are separated for 15 minutes and the same juvenile rat is introduced to it again. The time of contact T2 is measured in the same way. In the case of normal animals, the memory of the first encounter is translated into a T2/T1<1 ratio. In the case of memory impairment the ratio is T2/T11.

- Test of the T-labyrinth

Conducted according to the method described by P. Soubrié et al., J. Pharmacol. (Paris), 1977, 8, 3, 393-403 for the Labyrinth test in Y.

- Testing of the punchboard

The results of the study were published in the journal Pharmacochemical and Biochemical Sciences.

Assessment of injury

Intraseptic administration of vincristine results in a rapid (one week post-injection) and significant (by 60 to 70%) decrease in choline acetyltransferase (ChAT) and acetylcholinesterase (AchE) in the hippocampus, as well as degeneration of median septum neurons, which occurs at most two weeks post-injection and is associated with decreased cholinergic markers.

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Treatments

In this model, the effects of Compound A administration to injured animals were evaluated in comparison to the effects obtained with NGF.

The treatment is chronic, the product is administered once daily for 11 days. Compound A was administered at three different doses: 2.5 mg/kg, 5 mg/kg and 10 mg/kg to groups of 8 animals each, and 24 hours after stopping treatment the animals were slaughtered.

NGF, on the other hand, is administered by intraventricular infusion into the ACSF containing 0.01% rat albumin and gentamycin (1 ml/15 ml) in solution, according to the method described by W. Fisher et al., Nature, 1987, 329 (6134), 65-68. The NGF concentration of the solution is calculated so that, depending on the diffusion rate (0.44 ± 0.02 μl/h), the animals (7 rats) receive 0.105 μg, 1.05 μg, or 10.5 μg of NGF in a total infusion dose over two weeks. For the control batch, NGF is replaced by an equivalent molecular weight protein (about 130,000) with no neurotrophic activity: cytochrome C. Two weeks after initiation of treatment, the animals (7 rats) are sacrificed for evaluation in a histocampa and the other animals are sacrificed for a dose of CHAMP.

The results Morphological evaluation

In animals with injury and untreated, almost no brown precipitate is observed, while in animals treated with 5 mg/kg of Compound A, the observed hippocampal buds give a picture of structure close to that seen in a normal animal, giving similar results to those obtained with NGF.

The following information is provided:

The lesion causes a very significant drop in the activity of ChAT in the hippocampus which increases with the dose of Compound A administered up to a total recovery at 10 mg/kg, comparable to the recovery obtained in the NGF infused group. TABLEAU II

	Activité ChAT (pmole/mg/mn)
Animaux normaux non lésés	277 ± 13
Contrôles lésés	115 ± 18
NGF 0,105 µg/rat/2 semaines	168 ± 27
NGF 1,05 µg/rat/2 semaines	336 ± 28
NGF 10,5 µg/rat/2 semaines	293 ± 10
Animaux normaux non lésés	242 ± 19
Contrôles lésés	97 ± 18
Composé A 2,5 mg/kg/jour	164 ± 42
Composé A 5 mg/kg/jour	204 ± 24
Composé A 10 mg/kg/jour	242 ± 27

In the septum, the lesion causes a drop in ChAT activity, the recovery of which is dependent on the dose of the administered Compound A. In the case of NGF infused animals, the results are not significant; this may be due to the fact that the septic necrosis associated with the vincristine injection is overshadowed by additional necrosis following the canulation into a ventricle near the septum.

Behavioural assessment - Social memory

Once injured with vincristine, animals exhibited impairments in social memory that appear to be irreversible (still present 50 days after the vincristine injection). Compound A was tested at 10 mg/kg per bone compared to NGF at 10.5 μg and this test was performed on day 7 of injury. In both cases a very strong protective effect was observed with a T2/T1 ratio of 0.6 to 0.7.

- Labyrinth in T

The values obtained in untreated injured animals are significant for impaired exploratory abilities which are, however, recovered to a normal level by treatment with Compound A at 10 mg/kg. This test was performed in the blind 7 days after stopping treatment.

- The hole board .

In the control batch, the majority (6 rats) of the animals lost all exploratory ability, only two rats showed exploratory hyperactivity. Treatment with Compound A at 10 mg/kg brought about normalization of exploratory behaviour by reference to average values of normal animals. This test was performed, also in the blind, 11 days after stopping treatment.

Based on the results obtained with this model, formula (I) compounds and their pharmaceutically acceptable additive salts are suitable for use in the treatment and/or prophylaxis of all diseases involving neuronal degeneration, in particular, formula (I) compounds and their pharmaceutically acceptable additive sets are suitable for use in the following indications: memory disorders, vascular dementia, post-encephalic disorders, post-apoptotic disorders, post-traumatic syndromes due to brain injury, disorders arising from brain anoxia, Alzheimer's disease, dementia, subcorporeal dementia such as Huntington's disease and dementia, neurodegenerative disorders caused by Parkinson's disease, neurodegenerative disorders or neurological disorders such as Alzheimer's disease, neurodegenerative disorders, neurodegenerative disorders or neurological disorders, neurodegenerative disorders caused by neurological or sympathetic neurological disorders, and neurological disorders such as Alzheimer's disease, neurodegenerative disorders and neurodegenerative disorders.

The amount of active substance to be administered in the treatment of brain and neurological disorders according to the method of the present invention, as always, depends on the nature and severity of the conditions to be treated and the weight of the patients. However, the preferred unit doses will generally be from 2 to 300 mg, preferably from 5 to 150 mg, e.g. 5 to 50 mg, i.e. 5, 10, 20, 30, 40 and 50 mg, of product. These unit doses will normally be given one or more times, e.g. 2, 3, 4, or 5 times, preferably once a day, for example, in humans, and will vary from 900 to 300 mg, for example, per day, or from 2 to 500 mg, for example, per day, or more than 500 mg, for example, three times a day, for example, four times a day, or three times a day.

For therapeutic and/or prophylactic use according to the present invention, formula (I) compounds and their pharmaceutically acceptable additive salts are preferably prepared in pharmaceutical formulations. The pharmaceutical formulations of the invention contain an effective amount for the treatment or prophylaxis of brain and neurological disorders of at least one product selected from the pharmaceutically acceptable formula (I) compounds and their pharmaceutically acceptable additive salts, in combination with an inert pharmaceutical vehicle.

For oral or sublingual administration, tablets, whether single or double-stranded, granules with delayed release, drops or liposomes are used; for intravenous, subcutaneous or intramuscular administration, sterile or sterilizable solutions are used, while conventional patches can be used for transdermal administration.

The pharmaceutical compositions of the present invention may be prepared by commonly used methods, such as those described in EP-101381 or Remington's Pharmaceutical Sciences, 18th Ed., Mack Publ. Co. The active substance may be incorporated into excipients commonly used in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, aqueous or non-aqueous vehicles, animal or vegetable fats, paraffin derivatives, glycols, various moisturizing, dispersant, or emulsifying agents, preservatives, etc.

The pharmaceutical formulations of the invention may advantageously contain a compound of formula (I) or one of its pharmaceutically acceptable additive salts in combination with one or more other known and commonly used drugs in the same therapeutic indications.

Claims (9)

1. Use of at least one compound of formula (I)    or of a pharmaceutically acceptable acid addition salt thereof for the preparation of medicaments intended for the treatment and/or the prophylaxis of cerebral and neuronal diseases.
2. Use according to claim 1 for the preparation of medicaments intended for the treatment and/or the prophylaxis of diseases which involve neuronal degeneration.
3. Use according to claim 2 for the preparation of medicaments intended for the treatment and/or the prophylaxis of memory disorders, vascular dementia, post-encephalitic disorders, post-apoplectic disorders, post-traumatic syndromes caused by injury to the heed, disorders associated with cerebral anoxia, Alzheimer's disease, senile dementia, sub-cortical dementia, such as Huntington's chorea and Parkinson's disease, AIDS dementia, neuropathies caused by morbidity or lesions of the sympathetic or sensory nerves, cerebral oedema or spinocerebellar degenerations.
4. Use according to claim 3 for the preparation of medicaments intended for the treatment of vascular dementia, senile dementia or Alzheimer's disease.
5. Use according to any one of claims 1 to 4, in which the compound of formula (I) is chosen from 1-[2-(2-naphthyl)ethyl]-4-(3-trifluoromethylphenyl)-1,2,3,6-tetrahydropyridine and the pharmaceutically acceptable acid addition salts thereof.
6. Use according to any one of claims 1 to 5, where the medicament is a pharmaceutical composition in unit dosage form.
7. Use according to claim 6, in which the unit dosage form comprises from 2 to 300 mg of active principle.
8. Use according to claim 7, in which the unit dosage form comprises from 5 to 150 mg of active principle.
9. Use according to claim 8, in which the unit dosage form comprises from 5 to 50 mg of active principle.