FR2649308A1 - ANIMAL EXPERIENCE, METHOD FOR PRODUCING THE SAME, AND CONJUGATE USING THE SAME - Google Patents

Abstract

The invention relates to an experimental animal, its method of obtaining and a conjugate which can be used for this purpose. The experimental animal according to the invention, which is a mammal with the exception of a human being, is obtained in that the cholinergic neurons which protrude from the Meynert nucleus or from the basal forebrain and which penetrate into the cerebral cortex and / or cholinergic neurons which protrude from the nucleus of the septum and enter the hippocampus have been selectively destroyed using a conjugate of a nerve growth factor and a neurotoxin. The experimental animal according to the invention is particularly intended to be used as an experimental model for the study of dementia of the Alzheimer type.

Description

ANIMAL EXPERIENCE, METHOD OF OBTAINING IT AND

CONJUGATE FOR THIS EFFECT

  The present invention relates to a new experimental animal that can be used as an animal model of dementia, its method of production and a conjugate that can be used for this purpose. The standard of living of the Japanese is among the highest in the world and the fraction of the total population that is represented by the working-age population is increasing year by year, and as a result, the number of people who suffer from senile dementia, which

  Alzheimer-type dementia is also known to increase.

  Alzheimer's dementia is pathologically characterized in that the cholinergic neurons of the Meynert nucleus or basal forebrain are destroyed.

importantly.

  In order to develop a pharmaceutical product for the therapy of Alzheimer's dementia, it is necessary to have an animal model of disease for the estimation of the pharmacological effect, and there is known an animal whose corresponding forebrain at the Meynert nucleus primates are electrically destroyed, as well as an animal whose basal brain is destroyed by the administration of a toxic substance such as kainic acid, ibotenic acid, aziridinium ion of mustard ethylcholine

(AF64A).

  However, in the animal model obtained by electrical destruction and in the animal model obtained by treatment with kainic acid or with ibotenic acid, parts of the nervous system other than cholinergic neurons, such as for example non-cholinergic neurons and the fibers that pass are also destroyed. In the animal model treated with AF64A, cholinergic neurons other than cholinergic neurons that emerge from the Meynert nucleus and enter the cerebral cortex are also harmed without distinction. For these reasons, these animal models are not necessarily suitable for evaluating the pharmacological effect. The present inventors have done extensive research to create an experimental animal for dementia, particularly an animal model for Alzheimer's dementia, and they found as a result of this research that by administering a conjugate of a nerve growth factor (NGF) and a neurotoxin, which was not known in the art, in the cerebral cortex of an animal, said conjugate is incorporated by the end of the axon cholinergic neurons in the cerebral cortex, so that cholinergic neurons that protrude from the Meynert nucleus (in the case of a primate) or basal forebrain (in the case of an animal other than a primate) enter the cerebral cortex are selectively destroyed, and also that the extent of this destruction depends on the dose of the NGF conjugate and neurotoxin, and that the extent of the destruction may be

controlled by changing the dose.

  In addition, the present inventors have found that cholinergic neurons that protrude from the septum nucleus and enter the hippocampus can be selectively destroyed and dose dependent upon administration.

  conjugate above in the hippocampus of an animal.

  Thus, based on these findings, the present invention relates to a mammal, with the exception of a human being, whose cholinergic neurons protrude from the Meynert nucleus or basal forebrain and enter the cerebral cortex and / or whose cholinergic neurons that protrude from the septum nucleus and enter the hippocampus are selectively destroyed and

its process of obtaining.

  Figure 1 is a photomicrograph of a basal forebrain band of a rat administered the NGF 2.5 Staphylococcus conjugate of diphtheria on the unilateral brain in Experimental Example 1; A, C indicate the side of the administration and B, D the side that

did not receive the conjugate.

  The mammal that may be used according to the present invention may be, for example, a primate such as a monkey, a rodent such as a rat, a mouse and a guinea pig, a canine, or a feline. It is preferred to use a rodent such as the rat

or the mouse.

  The animal according to the present invention can be obtained by administering a conjugate of NGF and -neurotoxin to the cerebral cortex and / or the hippocampus to selectively destroy the cholinergic neurons that protrude from the Meynert nucleus or the brain. basal anterior and which penetrate into the cerebral cortex and / or cholinergic neurons that protrude from the septum nucleus and which

enter the seahorse.

  The NGF and neurotoxin conjugate can be prepared

  by reacting NGF with the neurotoxin.

  Like NGF, human placental NGF, mouse submandibular gland NGF, bull prostate NGF, bull sperm NGF, and muskrat sub-mandibular NGF can be used. NGF 7S, NGF 0 and NGF 2.5S of NGF are preferably used.

submandibular gland of mice.

  As neurotoxin, for example, protein toxins such as diphtheria toxin, cholera toxin and ricin can be used. These protein toxins generally comprise A-chain and B-chain subunits, but according to the present invention any toxin containing the A-chain can be used. The above reaction can easily be carried out in a suitable solvent. in the presence of a condensing agent by adding a solution of nerve growth factor to a neurotoxin solution and then adding a condensing agent, leaving the solution at rest and separating the conjugate. It is preferable to use an excess of NGF with respect to the neurotoxin to prevent the remaining neurotoxin remaining unreacted when the reaction is complete. For example, it is preferable to use 1.5 to 3 molecules of NGF per molecule of

neurotoxin.

  As the condensing agent, one of those used in peptide synthesis can be used. for example, it is preferable to use

  water-soluble condensation such as 1-ethyl-3- (3-L-

  dimethylaminopropyl) carbodiimide and N-cyclohexyl-N'-

  morpholinocarbodiimide. As the reaction solvent, water is preferred. The reaction can proceed at room temperature, but it is preferable to maintain during the reaction the pH of the reaction mixture between 5.5 and 8.0 at room temperature.

  using an appropriate buffer such as phosphate buffer.

  Once the reaction is complete, it is desirable to purify the conjugate formed by a conventional method used in this field, such as dialysis and filtration.

on gel.

  The NGF and neurotoxin conjugate thus obtained can be administered into the cerebral cortex or hippocampus in a conventional manner, for example by perforating the skull bone and administering it through the perforated portion. When operating in this way, it may be preferable to make one or more perforations in each cerebral hemisphere, the portion of the skull bone that contains the cruciform suture being located in the center, and to inject the conjugate. through the perforated part using a microsyringe. Similarly, in the case of an injection into the hippocampus, one or more perforations can be made for each cerebral hemisphere, and the conjugate can be injected in the same way

than above.

  You can choose the administration site. In the case

  of the rat, for example, one can choose the site -

  in the cerebral cortex among the frontal cortex, the parietal cortex, the occipital cortex by consulting the atlas of the brain of Paxinos and Watson [The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney (1986)], and the administration site located in the hippocampus

among the sites CA1, CA2 and CA3.

  The dose of conjugate may vary slightly depending on the type of animal, but may be approximately in the range of about 0.5 to 30 μg per animal. For example, when administering to the cerebral cortex of the rat or mouse, 0.5 to 6 μg per rat or mouse may be suitably administered. The extent of destruction of cholinergic neurons can be controlled by increasing or decreasing the dose so

adequate in this range.

  After administration, the experimental animal according to the present invention is obtained by normally feeding the treated animal for about 3 to 7 days. The experimental animal according to the present invention can also be obtained by administering the NGF and neurotoxin conjugate directly into the Meynert nucleus and / or

  basal forebrain and in the septum nucleus.

  The mammal according to the present invention thus obtained can be used as an experimental model for Alzheimer's dementia or as an experimental animal to examine the function of cholinergic neurons in the basal forebrain and the septum nucleus, since neurons Cholinergic cells that protrude from the Meynert nucleus or basal forebrain and / or cholinergic neurons that protrude from the septum nucleus and enter the hippocampus are selectively destroyed. Thus, the present invention also relates to a method for testing a pharmaceutical product for the therapy of Alzheimer's dementia, which comprises administering the drug product to the animal

experiment according to the invention.

  The present invention will be better understood when reading

  non-limiting examples.

Example 1

  (1) Preparation of NGF Conjugate and Neurotoxin: To a solution of 10 μg of 2.5S NGF (prepared by Colaborative Research Co.) dissolved in 40 μl of 0.02 M phosphate buffered saline was added a solution of 22.3 μg of diphtheria toxin (molecular weight: about 62,000, prepared by Wako Junyaku KK) dissolved in 40 μl of 0.02M phosphate buffered saline. Then, 80 pound of

  1-ethyl-3- (3-L) hydrochloride solution

  dimethylaminopropyl) carbodiimide in distilled water (100 mg / ml), and the mixture was allowed to stand at room temperature for 10 hours, while shaking the mixture periodically. Then, the mixture was dialysed against 0.02M phosphate buffered saline at 4 ° C overnight. There was thus obtained a dialysate containing the conjugate of 2.5S NGF and diphtheria toxin. Part of the dialysate was removed and SDS-polyacrylamide gel electrophoresis confirmed the presence of the conjugate band (molecular weight about 71000) and the absence of A-band (about 21000) and B chain (molecular weight

  about 37,000) of the diphtheria toxin.

  (2) Conjugate Administration: A rat (Wistar strain, male, weighing approximately g) was fixed on a stereotaxic device (manufactured by Shigemo Kagaku KK) under sodium pentobarbiturate anesthesia (administered intraperitoneally, 40 mg / kg). . Then, the skin was cut at the waist of the skull along the medial line to expose the skull bone and was perforated on both sides (1) at 2.5 mm on the front, 2.5 mm on the side, (2) 0.5 mm on the back, 2.5 mm on the side and (3) 3.5 mm on the back, 3.0 min on the side of the cruciform suture. The dialysate solution containing the conjugate of 2.5S NGF and diphtheria toxin obtained in (1) was aspirated into a microsyringe and injected with 1 μl in the cerebral cortex at a depth of 0, 5 mm. After administration, feeding the rat normally for 3 days resulted in a rat whose cholinergic neurons protruded from the basal forebrain and entered the cerebral cortex

were destroyed.

  (3) Observation of ChAT positive cells: The rat obtained above was subjected to a thoracotomy under sodium pentobarbiturate anesthesia (administered intraperitoneally, 40 mg / kg), a catheter was inserted through the left ventricle into the aorta. left, and after infusion with 100 ml of physiological saline, then with 500 ml of Zamboni solution (mixture of 4% paraformaldehyde and 0.2% picric acid) the brain was enucleated and fixed by immersion in the

  Zamboni solution for a day and a night.

  The resulting brain sample was immersed in a 30% sucrose solution for one day and one night and then frozen rapidly on dry ice, a 30 μm thick band was prepared at room temperature. using a Cryostat (Moriyasu Seisakusho KK) and we plunged the band into

  0.02M phosphate buffered saline.

  The brain band was immersed in a solution of anticholin acetyltransferase antibody (0.5 μg / ml, rat-mouse derivative, manufactured by Boehlinger-Mannheim Co.) for one day and one night, and then examined under a microscope. choline acetyltransferase (ChAT) positive cells as an index of cholinergic neurons by the antibody indirect immunofluorescence method. It was thus confirmed a selective reduction of cholinergic neurons that protrude into the cerebral cortex of both sides of the basal forebrain (reduction of choline acetyltransferase positive cells).

  and decreased immunoreactivity.

  EXAMPLE 2 As in Example 1- (2), the bone of the skull of a rat (Wistar strain, male) weighing about 150 g was exposed, the bones of the skull were perforated on both sides ( 1) at 5.5 mm to

  the back and 4.0 mm on the side of the cruciform suture.

  The dialysate containing the conjugate of 2.5S NGF and diphtheria toxin obtained in Example 1 (1) was aspirated into a microsyringe and 1 pound in the cerebral cortex was injected at a depth of 4 times. mmn. After administration, by normally feeding the rat for 3 days, a rat was obtained whose cholinergic neurons protruding from the septum nucleus and entering

  the hippocampus were selectively destroyed.

  Experimental Example 1 The treatment was carried out in the same manner as in Example 1 except that 0.5 μl of 2.5S NGF conjugate solution and diphtheria toxin were injected unilateral side of the rat cerebral cortex, and choline cells were examined

  acetyltransferase positive in the basal forebrain.

  The results are illustrated in Figure 1.

  In the photographs of Figures 1A to 1D (A and C represent the treated side, and B and D the untreated side), it is clear that the choline acetyltransferase positive cells and the immunoreactivity on the administered side are markedly reduced in comparison to those on the unmanaged side. It can be inferred that cholinergic neurons that protrude from the basal forebrain and enter the cerebral cortex are selectively destroyed by

  conjugated with NGF and diphtheria toxin.

  Experimental Example 2 Purpose of the Assay Using a mouse to which the 2.5S NGF conjugate and diphtheria toxin were administered, the memory acquisition test was performed 3 days after administration, and the memory retention test was performed 8 days later to study the inhibition by the conjugate of 2.5S NGF and diphtheria toxin.

  the ability to acquire and retain memory.

  Experimental method (1) Memory acquisition test 3 days after administration of 2.5S NGF conjugate solution and diphtheria toxin: (1) 29 mice (6 weeks old, Slc strain: ddY) weighing 26 to 30 g for each group. The test group was administered 0.2 μl of 2.5S NGF conjugate and diphtheria toxin at the sites on the side at approximately 1.5 mm and at a depth of 0.3 to 0.5 mm from the cerebral cortex and parietal cortex on both sides of the head, using a Hamilton syringe under anesthesia with ether. In the control group, the same

  volume of physiological saline in the same way as

  above. (2) 3 days after administration, the mouse was stationarily placed in the illuminated chamber of the traversing-type device comprising a lighted chamber and a dark chamber (said device having a passage in the middle), and posteriorly facing the passage, and once the entire body of the mouse was inside the dark chamber, an electrical discharge (AC, 0.5 mA, 3s) was applied to the using an interfering device / electric shock generator (manufactured by RBS Co., brand LVE-11) to evoke darkroom avoidance memory. (3) Avoidance memory was judged to be acquired when the mouse remained in the illuminated chamber continuously for 120 seconds or more, and the degree of memory acquisition was estimated by the number of

  discharges received until the acquisition of the memory.

Results

  The results are shown in Table 1.

  Number of discharges received Treatment group 1.45 + 0.084 Control group 1.28 +0, 106 It can be seen from the table above that the mice treated with the 2.5S NGF conjugate and diphtheria toxin were received a greater number of discharges before memory acquisition than the control group. This fact demonstrates that the treated group suffers from a

  the ability to acquire memory.

  (2) Memory Retention Test: The animal having acquired the memory in the above memory acquisition test (1) was stationarily placed in the illuminated chamber of the pass-through type device. same way as above, the eighth day, the posterior part being turned towards the side of the passage. The latency required for the marker attached to the first lumbar vertebra (L1) and the two lower limbs to fully penetrate the dark chamber and the number of animals for which 300 seconds or more were required for the marker and the two lower limbs penetrate completely into the dark chamber, and from these data it was estimated the degree of retention of memory acquired 8 days before.

  0 The results are presented in Table 2.

  The results are shown in Table 2.

Table 2 (a>

  Latency (s) LI Lower limb Treatment group 151.9-16.9 256.2 + 14.4 Control group 231.3 + 14.1 285.0 + 7.85

Table 2 (b)

  Number of animals for which 300 s or more are required Li Lower limb treated group 5/29 9/29 control group 14/29 24/29 It can be seen from the tables above that mice treated with 2.5S NGF and diphtheria toxin enter the dark chamber after a remarkably shorter duration than the average duration required for the control group and the retention of dark-room avoidance memory acquired 8 days previously is lower than that of the control group, that is, the memory retention ability is degraded. Also, the number dm s? for which a duration of 300 seconds or more was required to penetrate the dark chamber was also remarkably lower in the treated group than in the control group, which can be interpreted as a disorder of the retention capacity of the patient. memory due to the conjugate of NGF 2.5S and

of toxin from diphtheria.

  Thus, it is understood from the foregoing assays that the conjugate of 2.5S NGF and diphtheria toxin clearly disrupts the ability of mice to acquire memory and the ability of mice to retain of memory. EXPERIMENTAL EXAMPLE 3 Aim of the Assay A mouse was administered immediately after memory acquisition the 2.5S NGF conjugate solution and diphtheria toxin and the memory retention test was performed on the eighth day. to examine how the ability to retain acquired memory was disrupted by the administration of the solution of

  conjugated with 2.5S NGF and diphtheria toxin.

  Experimental method 26 mice (weighing 26 to 6 weeks, strain Slc: ddY) were used for each group, which were considered as having acquired the dark chamber avoidance memory in the memory acquisition test performed. in the same way as in Example 2. The solution of 2.5S NGF conjugate and diphtheria toxin to the treated group and physiological saline to the control group of

  the same way as in Experimental Example 2.

  Then, on the eighth day, the retention test was performed in the same manner as in Experimental Example 2 to examine the retentic. of memory acquired eight days ago.

  The results are shown in Table 3.

Table 3 (a)

  Latency (s) L1 Lower limb treated group 105.0 + 20.7 192.1 + 19.8 control group 179.2 + 22.7 252.3 + 15.3

Table 3 (b)

  Number of animals for which 300 s or more are required LI Lower limb treated group 3/26 10/26 control group 11/26 17/26 It can be seen from the previous tables that mice treated with NGF conjugate 2 , 5S and diphtheria toxin have a remarkably shorter mean latency before entering the dark chamber than the control group, and that the retention of darkroom avoidance memory acquired eight days earlier is lower than that of the control group, that is to say that

  the memory retention ability is disturbed.

  - Also, the number of mice for which 300 seconds or more were required to enter the dark chamber was also remarkably lower in the treated group than in the control group, which can be attributed to the fact that the NGF conjugate 2 , 5S and diphtheria toxin interferes with the ability to retain memory. Thus, the conjugate of Nc-2,5S and diphtheria toxin clearly disrupts the ability of mice to acquire memory and their ability to retain

of memory.

  Experimental Ex. 4 Choline Acetyltransferase Activity Assay: Experimental Method Six or seven rats (Wistar strain, males) weighing about 150 g were used in each group. The solution of 2.5S NGF conjugate and diphtheria toxin prepared in the same manner as in Example 1 was administered to the treated group at a rate of 0.2 μl each side at about 1.5 μg. mmn and at a depth of 0.3 to 0.5 mm in the anterior brain cortex and upper brain cortex on both sides of the head, using a Hamilton syringe under sodium pentobarbiturate anesthesia

  (40 mg / kg, intraperitoneal administration).

  Serum was administered in the same way

physiological to the control group.

  After a normal feeding period of 5 days following administration, the animal's head was cut to collect the brain, and immediately after the weight measurement, the frontal lobe cortex was collected and the basal forebrain. The tissue was homogenized in 20 times its volume with a solution of 100 mM tris-EDTA (pH 7.4), and 10 μl was used to test the activity of choline acetyltransferase, and part of the remainder for perform a quantitative analysis of the proteins. The activity of choline acetyltransferase was measured using the Fonum method (Journal of Neurochemistry, vol.24, pages 407-409 (1975), and the protein concentration using a reagent of protein assay produced by Nippon Biorad Laboratory KK, and the activity was calculated

for 1 mg of protein.

Résunta

  The results are shown below in Table 4.

Tableau_4

  Activity of choline acetyltransferase (nmol ACh / h / mg) Cerebral Cortex Anterior basal brain Treated group 8.78 0.26 17.56 0.88 Control group 9.78 + 0.30 19.28 + 0.87 see clearly from the table above that the activity of choline acetyltransferase in the cerebral cortex and basal forebrain of animals in the treated group is decreased by about 10% and 9%, respectively, by

  compared to animals in the control group.

REVIATIONS

  An experimental animal which is a mammal, except for a human being, characterized in that the cholinergic neurons which protrude from the Meynert nucleus or basal forebrain and which enter the cerebral cortex and / or cholinergic neurons that protrude from the septum nucleus and penetrate into

  the hippocampus were selectively destroyed.

  2. Experimental animal that is a mammal, with the exception of a human being, for testing the pharmacological effect of chemical compounds for the treatment of Alzheimer's dementia, characterized in that the protruding cholinergic neurons of the Meynert nucleus, in the case of a primate, or of the basal forebrain, in other cases, which penetrate into the animal's cerebral cortex, and / or the cholinergic neurons which project from the septum nucleus and who enter

  the hippocampus of the animal were selectively destroyed.

  3. Experimental animal according to claim 1 or claim 2, characterized in that it is a

  rodent, canine or feline.

  4. Experimental animal according to claim 3,

  characterized in that the rodent is a rat.

  5. Rat according to claim 4, characterized in that the cholinergic neurons protruding into the cortex

  brain were selectively destroyed.

  6. Rat according to claim 4, characterized in that the cholinergic neurons which protrude into

  the hippocampus were selectively destroyed.

  7. Process for obtaining an experimental animal according to claim 1 or claim 2, characterized in that it comprises the administration of a conjugate of a nerve growth factor and a neurotoxin in the cerebral cortex and / or the hippocampus of the experimental animal to selectively destroy the cholinergic neurons that protrude from the Meynert nucleus or the basal forebrain and that penetrate into the cerebral cortex and / or cholinergic neurons that cause protruding from the septum nucleus

  and who enter the hippocampus.

  8. The method of claim 7, characterized in that the nerve growth factor is 2.5S NGF, the

o10 NGF ouleNGF7S.

  9. Process according to claim 7 or claim 8, characterized in that the neurotoxin is the

  Diphtheria toxin, cholera toxin or ricin.

  10. Process according to claim 9, characterized in that the conjugate is a conjugate of the 2.5S NGF and the

toxin of diphtheria.

  He. Method according to one of claims 7 to 10,

  characterized in that the experimental animal is a rodent, a canine or a feline.

  12. Process according to claim 11, characterized in

what the rodent is a rat.

  13. A method according to r-cation 12, characterized in that it comprises the administration of a conjugate of nerve growth factor and diphtheria toxin to the cerebral cortex of rats to thereby selectively destroy neurons cholinergic that protrude from the brain

  basal anterior and penetrate into the cerebral cortex.

  14. The method of claim 12, characterized in that it comprises the administration of a conjugate of nerve growth factor and diphtheria toxin in the hippocampus of rats to thereby selectively destroy the cholinergic neurons that make protruding from the septum nucleus

  and which enter the hippocampus.

  15. A method for testing a pharmaceutical product intended for the treatment of Alzheimer's dementia, characterized in that it co-ordinates the administration of the pharmaceutical product to an experimental animal according to one of the

Claims 1 to 6.

  16. Conjugate characterized in that it comprises a

  nerve growth factor (NGF) and a neurotoxin.

  17. Conjugate according to claim 16, characterized in that the NGF is chosen from the NGF of the human placenta, the NGF of the submandibular gland of the mouse, the NGF of the snake venom, the NGF of the prostate guinea pig, the NGF of the rabbit prostate, the NGF of the bull prostate, the

  NGF of the bull's sperm and NGF of the sub-gland

mandibular of the muskrat.

  18. Conjugate according to claim 17, characterized in that the NGF of the submandibular gland of the mouse

  is NGF 7S, NGF f or NGF 2,5S.

  Conjugate according to claim 16, characterized in that

  that the neurotoxin is a protein toxin.

  20. Conjugate according to claim 19, characterized in that the protein toxin is selected from the group consisting of diphtheria toxin, cholera toxin and ricin. Conjugate according to claim 20, characterized in that

  what the neurotoxin is the toxin of diphtheria.

  Conjugate according to one of Claims 16 to 21,

  characterized in that the ratio of nerve growth factor to neurotoxin is from 1.5 to 3 molecules of NGF

for 1 molecule of neurotoxin.

  23. Process for preparing the conjugate according to one of the

  Claims 16 to 22, characterized in that it comprises the

  the steps of adding a nerve growth factor solution to a neurotoxin solution and then adding a condensing agent, allowing the mixture to

  rest and separate the conjugate.

  24. Process according to claim 23, characterized in that the condensing agent is water-soluble and can be

  1-ethyl-3- (3-L-dimethylaminopropyl) carbodiimide or N-

  cyclohexyl-N'-morpholinocarbodiimide.

  25. Method according to one of claims 23 and 24,

  characterized in that the conjugate is separated by dialysis or

gel filtration.