DE4004573A1 - NEW TRIAL AND HIS PRODUCTION - Google Patents

Abstract

A test animal which is a mammal, except for human beings, in which a cholinergic neuron projecting from a Meynert nucleus or a basal forebrain into a cerebral cortex or a cholinergic neuron projecting from a septum nucleus into a hippocampus or both such cholinergic neurons have been selectively destroyed. The preparation thereof, which comprises administering a conjugate of a nerve growth factor and a neurotoxin into either one or both of the cerebral cortex and the hippocampus of a mammal except for human being, thereby destroying selectively either one or both of the cholinergic neurons projecting from the Meynert nucleus or the basal forebrain into the cerebral cortex and the cholinergic neurons projecting from the septum nucleus into the hippocampus.

Description

The invention relates to a new experimental animal, which as Animal model for dementia, and a method for his Production.

The average lifetime of the Japanese is worldwide the peak, and the relationship of the elderly to the Total population increases from year to year. It also decreases the number of patients with senile dementia, the so-called Dementia of the Alzheimer's type too. Dementia of the Alzheimer's type is pathologically characterized in that the cholinergic Neurons in the Meynert nucleus in the basal forebrain clearly are atrophied.

To develop a drug for the therapy of such Dementia of the Alzheimer's type is an infectious disease Animal model needed to estimate the pharmacological effect. There is an animal known in which the basal forebrain, the Meynert nucleus of primates corresponds, electrically destroyed is, and an animal in which the basal forebrain passes through Administration of a toxic substance, such. B. kainic acid (2-carboxy-4-isopropenyl-3-pyrrolidineacetic acid), ibotenic acid and ethylcholine diethyl sulfide aziridinium ion (AF64A) is.

Both in the animal model caused by electrical destruction as well as the animal model, kainic acid or ibotenic acid, however, is the nervous system except the cholinergic neurons, such. B. noncholinergic Neurons and continuous fibers are also destroyed. In that Animal model to which AF64A has been administered are the cholinergic Neurons derived from the cholinergic neurons that originate from the Meynert nucleus protrude into the cerebral cortex, are different, as well  damaged without being injured. For such reasons, these are compelling animal models are not necessarily used as an animal model Assessment of pharmacological effects.

Intensive studies were conducted to find a test animal for dementia, in particular an animal model for dementia of To create Alzheimer's type. It was found that by Administration of a conjugate of a nerve growth factor (NGF) and a neurotoxin, which so far according to the state of the Technique was unknown to the cerebral cortex of an animal, the conjugate through the axon end of the cholinergic neurons into the Cerebral cortex is stored, with the cholinergic neurons, those from the Meynert nucleus (in the case of primates) or the basal forebrain (in the case of animals other than primates) in the Cerebral cortex protrude, are selectively destroyed, and that the Extent of such destruction from the dose of the conjugate NGF and neurotoxin depends and that the extent of destruction can be controlled by controlling the dose.

Furthermore, it was found that the cholinergic neurons released from the Seclusion of the nucleus into the hippocampus protrude, selectively and dose-dependent by administration of the above conjugate to the Hippocampus of an animal can be destroyed.

On the basis of such discoveries, according to the invention Mammal except for humans provided at the cholinergic neurons from the Meynert nucleus or the basal forebrain protrude into the cerebral cortex, and / or the cholinergic neurons emerging from the septum of the nucleus in the Hawocampus protrude, are selectively destroyed, as well as a procedure for its production.

The invention will be more apparent from the attached figures explained. Show it:

Figure 1 is a photomicrograph of the rat forebrain stripe, to which a 2.5 s. NGF diphtheria toxin conjugate had been administered to a hemisphere of the cerebrum according to Experimental Example 1;
A, C the treated hemisphere;
B, D is the untreated hemisphere.

The mammal used in the invention may, for. A primate, like the monkey, a rodent, like the rat, the mouse and that Guinea pig, a canine animal and a feline Animal, more preferably a rodent, such as the rat and the Mouse, his.

The animal according to the invention can be produced by a conjugate of NGF and neurotoxin into the cerebral cortex and / or hippocampus of the above animal, wherein selectively, the cholinergic neurons from the Meynert nucleus or the basal forebrain protrude into the cerebral cortex, and / or the cholinergic neurons emerging from the septum of the nucleus the hippocampus protrude, destroyed.

The conjugate of NGF and neurotoxin may be converted by reaction of NGF be prepared with the neurotoxin.

As NGF, human placenta NGF, mouse submucosal gland NGF, snake venom NGF, guinea pig NGF, rabbit and bovine, bovine seminal NGF and muskrat mandibular gland NGF may be used. Preferably, 7S.NGF, β- NGF and 2.5 S.NGF can be used from NGF of the submaxillary gland of mice.

As neurotoxins z. Protein toxins such as diphtheria toxin, Cholera toxin and ricin are preferably used. These Protein toxins generally consist of an A and a B chain as a subunit, but according to the invention, any A chain-containing Toxin can be used.  

The above reaction can easily be in a suitable solvent be carried out in the presence of a condensing agent. at the reaction is preferably carried out using an excess NGF based on the neurotoxin used so that no unreacted neurotoxin after completion of the reaction remains. Preferably, 1.5 to 3 molecules of NGF per Used molecule neurotoxin.

As condensing agents can usually for peptide synthesis used condensing agents are used. For example may preferably water-soluble condensing agents, such as. B. 1-Ethyl-3- (3-L-dimethylaminopropyl) carbodiimide and N-cyclohexyl N'-morpholinocarbodiimid be used. As a solvent for the reaction is water preferred. The reaction can preferably at room temperature, but during the Reaction is preferably the pH of the reaction mixture at about 5.5 to 8.0 by means of a suitable buffer z. B. one Held phosphate buffer.

After completion of the reaction, it is expedient to purify the reaction resulting conjugate according to a person skilled in the known Method, such. As dialysis and gel filtration.

The resulting conjugate of NGF and neurotoxin can be attached to the Cerebral cortex or the hippocampus after a known Methods are administered, for. B. by the skull bone drilled and administered through the drilled section performs. Specifically, when administered through the drilled Section of the skull bone to the cerebral cortex you can preferably proceed in such a way that one to several places per Brain hemisphere with the cross - shaped adhesion line of the Skull bone as a center and the conjugate through the drilled portion injected by means of a microsyringe. Also when injected into the hippocampus can have one to several sites per brain hemisphere, and the conjugate can injected as described above.  

The site of administration can be suitably selected. In the case of the rat z. For example, the site of administration in the Cerebral cortex in the area of the frontal, parietal and Occipital cortex according to the brain atlas of Paxinos and Watson [The Rat Brain at Sterotaxic Coordinates, Academic Press, Sydney (1986)] and the site of administration in the Hippocampus in the range of CA₁, CA₂ and CA₃.

The dose of the conjugate may vary slightly depending on the species vary, but is conveniently in the range of about 0.5 to 30 μg per animal. So z. B. when administered to the Cerebral cortex of rat or mouse about 0.5 to 6 μg per rat or mouse is suitably administered. By taking the dose in Increasing or decreasing this range accordingly may Controlled extent of destruction of cholinergic neurons become.

After administration, by normal feeding of the animal for about 3 to 7 days the experimental animal according to the invention can be obtained.

The experimental animal according to the invention can also be produced by introducing the conjugate of NGF and neurotoxin directly into the Meynert nucleus or the basal forebrain and / or the Divided wall of the nucleus.

Since in the mammal thus obtained according to the invention cholinergic neurons derived from the Meynert nucleus or the basal forebrain protrude, and / or the cholinergic neurons that protrude from the septum of the nucleus into the hippocampus, are selectively destroyed, it can be used as an experimental model for dementia of the Alzheimer type or as a test animal for the investigation of the Function of cholinergic neurons in the basal forebrain and be used in the septum of the nucleus.

The present invention will become more apparent from the examples explained.

Example 1 (1) Preparation of the conjugate of NGF and neurotoxin:

To a solution of 10 μg 2.5 S.NGF (manufactured by Colaborative Research Co.) dissolved in 40 μl of 0.02M phosphate buffered saline, became a solution of 22.3 μg Diphtheria toxin (molecular weight about 62,000, manufactured by Wako Kunyaku K.K.) dissolved in 40 μl of 0.02M phosphate buffered physiological saline, added. Subsequently were 80 μl of a solution of 1-ethyl-3- (3-L) Dimethylaminopropyl) carbodiimide hydrochloride in distilled Water (100 mg / ml) was added and the mixture was allowed to stand for 10 hours allowed to stand at room temperature with periodic shaking. Subsequently, the reaction mixture against 0.02 m phosphate buffered saline at 4 ° C Dialyzed night. Thus, a dialysate containing the 2.5 S.NGF diphtheria toxin conjugate contained. At a part of the Dialysate was analyzed by SDS-polyacrylamide gel electrophoresis confirms that the band of the conjugate (molecular weight um 71,000) and no A chain band (molecular weight around 21,000) and the B chain (molecular weight around 37,000) of the Diphtheria toxins is present.

(2) Administration of the conjugate:

The rat (Wistar strain, male, weighing about 150 g) was raised a stereotactic device (made by Shigemo Kagaku K.K.) under pentobarbital sodium anesthesia (intraperitoneal administered, 40 mg / kg). Subsequently, the skin was on Vertex of the head cut open along the midline to expose the skull bone. The skull bone was opened both sides at (1) front 2,5 mm, side 2,5 mm, (2) back 0,5 mm, Side 2.5 mm and (3) rear 3.5 mm, side 3.0 mm from the drilled cross-shaped intergrowth line.

The dialyzed solution containing the 2.5 S.NGF diphtheria toxin conjugate was in a  Microcrew mounted and each 1 ul was 0.5 mm deep in injected the cerebral cortex. After administration was at normal feeding for 3 days a rat at which the cholinergic neurons from the basal forebrain in the Cerebral cortex protrude, are selectively destroyed.

(3) observation of choline acetyltransferase (ChAT) positive cells:

On the rat as described above was under Pentobarbital sodium anesthesia (administered intraperitoneally, 40 mg / kg) made a chest wall cut. A catheter was inserted through the left ventricle into the left aorta and after rinsing with 100 ml of physiological saline and then 500 ml Zamboni solution (a mixture of 4% para-formaldehyde and 0.2% picric acid), the brain was peeled off and by immersion in the Zamboni solution over day and night fixed.

The resulting brain preparation was in a 30% Sucrose solution immersed over day and night and then quickly frozen on dry ice. A strip with one Thickness of 30 μm was measured by a cryostat (Moriyasu Seisakusho K.K.), and the strip was cut in 0.02 m immersed in phosphate buffered saline.

The brain strip was transformed into an anti-choline acetyltransferase antibody solution (0.5 μg / ml, derived from rat mouse, manufactured by Boehringer-Mannheim Co.) over day and night dipped, and then the choline acetyltransferase positive Zelen as an index for the cholinergic Neurons under the microscope after the indirect Immunofluorescent antibody method investigated.

As a result, was a selective reduction of cholinergic Neurons that enter the cerebral cortex on both sides of the basal Forebrain (reduction of choline acetyltransferase  positive cells) and a decrease in immunoreactivity approved.

example 2

Similarly, or in the same manner as in Example 1- (2) was the Rat bones of the rat (Wistar strain, male, weight approximately 150 g) exposed. The skull bone was added on both sides (1) rear 5.5 mm, side 4.0 mm from the cruciform Grooving seam drilled. The dialysate containing the 2.5 S.NGF diphtheria toxin conjugate according to Example 1- (1) was in mounted a microsyringe and 1 μl each was 4 mm deep in injected the cerebral cortex. After administration was under normal feeding for 3 days a rat at which the cholinergic neurons emerging from the septum of the nucleus in the Hippocampus protrude, are selectively destroyed.

Experimental Example 1

The treatment was carried out as in Example 1 except that 0.5 μl each of the 2.5 S.NGF diphtheria toxin conjugate solution was injected only into the one hemisphere of the rat cerebral cortex and the choline acetyltransferase positive cells in the basal forebrain were examined. The results are shown in FIG .

From the photographs in Figs. 1A to 1D (A and C, the treated hemisphere, B and D represent the untreated hemisphere), it is clear that the choline acetyltransferase positive cells and the immunoreactivity on the treated hemisphere are significant compared to the untreated hemisphere are reduced. From this fact, it can be concluded that the cholinergic neurons protruding from the basal forebrain into the cerebral cortex are selectively destroyed by administration of the NGF diphtheria toxin conjugate.

Experimental Example 2 <Task of the experiment <

In a mouse infected with the 2.5 S.NGF diphtheria toxin conjugate was treated, a memory formation test on performed the third day after administration and a Memory retention test was also on the eighth day performed to investigate how the ability to Memory formation and memory preservation Administration of the 2.5 S.NGF diphtheria toxin conjugate could be.

<Experimental procedure < (1) Memory formation test on the third day after administration of the Solution with 2.5 S.NGF diphtheria conjugate

(1) 29 mice (6 weeks old Slc: ddY strain) weighing 26 to 30 g were used as a group. The treated Groups were each 0.2 μl of the 2.5 S.NGF diphtheria toxin conjugate about 1.5 mm laterally and 0.3 to 0.5 mm deep of the forebrain cortex and partial cortisum on both Head sides by means of a Hamilton syringe under ether anesthesia injected. The control group became the same volume physiological saline in the same manner as above mentioned administered.

(2) On the third day after administration, the mouse became stationary into a light chamber of the passage-type device (stepthrough type) consists of two light and dark chambers (the device is provided with a path in the middle), wherein the Tail pointed to the side of the way. At the point where the Whole body of the mouse had entered the dark room was an electric shock (alternating current, 0.5 mA, 3 seconds) by means of of an electric shock generator / speech distorter (manufactured by RBS Co., trade name: LVE-11) given to the memory, the to avoid dark chamber emerge.  

(3) The memory of avoidance was considered acquired when the Mouse in the bright chamber continuously for 120 seconds or stayed longer and the extent of memory formation was at the Shock count that was obtained until memory formation when Index estimated.

<Results <

The results obtained are shown in Table 1.

Number of shocks received treated group 1.45 ± 0.084 control group 1.28 ± 0.106

The above table shows that with the 2.5 S.NGF diphtheria toxin conjugate treated mouse more shocks than that Control group received before it came to a memory formation. This fact shows that the treated group is suffering from a disorder the capacity for memory formation suffers.

(2) memory preservation test

The animal having acquired the memory in the above memory formation test (1) was stationarily placed in the light chamber of the passage-type device similar to the above-described on the eighth day with the tail against the way side. The latency needed until the marker attached to the first lumbar vertebra (L ₁) and the two hind limbs had fully entered the dark chamber and the number of individuals who needed 300 seconds or more before the laryngeal period Marker and the two hind limbs were fully entered the dark chamber was examined and the extent of the preservation of the memory acquired eight days previously was estimated from it.

<Results <

The results given in Table 2 were obtained.

Table 2 (a)

Table 2 (b)

The above tables show that the mouse containing the 2.5 S.NGF diphtheria toxin conjugate was treated, on average one significantly shorter time until entry into the dark chamber needed as the control group and that the preservation of the eight Days previously acquired memory, the dark room too avoid, compared to the control group is lower, d. H., the ability to maintain memory is disturbed.

Likewise, the number of mice is 300 seconds or longer until entry into the dark chamber, clearly lower in the treated group than in the control group, as a disorder in the ability to maintain memory through 2.5 S.NGF diphtheria toxin conjugate can be understood.

Thus, it can be deduced from the above experiments that 2.5 S.NGF diphtheria toxin conjugate clearly demonstrates both the ability to Memory formation as well as memory retention in mice disturbs.  

Experimental Example 3 <Task of the experiment <

Immediately after memory formation, the mouse became the 2.5 S.NGF Diphtherietoxinkonjugatlösung administered and the Memory preservation test was performed on the eighth day to examine how the ability to preserve the acquired Memory by administration of the 2.5 S.NGF diphtheria toxin conjugate solution was disturbed.

<Experimental procedure <

26 mice (weight 26 to 30 g, age 6 weeks, Slc: ddY strain), the proven to increase the memory, the dark chamber too avoid in the memory formation test, similar to the one in the Experimental Example 2 was carried out used as a group. The solution of 2.5 S.NGF diphtheria toxin conjugate became the treated and physiological Saline group as in Experimental Example 2 administered.

Subsequently, on the eighth day of the memory retention test similar to that carried out in Experimental Example 2 to preserve of the memory acquired eight days previously.

<Results <

The results given in Table 3 were obtained.

Table 3 (a)

Table 2 (b)

The above tables show that with 2.5 S.NGF diphtheria toxin conjugate treated mice on average one significantly shorter latency before entering the dark chamber show as the control group and that the conservation of the eight Days previously acquired memory, the dark room too avoid, compared to the control group is lower, d. H., the ability to maintain memory is disturbed.

Likewise, the number of mice is 300 seconds or longer needed to enter the dark room, clearly smaller in the treated group than in the control group, which as a disturbance in the ability to maintain memory by 2.5 S.NGF diphtheria toxin conjugate can be understood.

Therefore, the 2.5 S.NGF diphtheria toxin conjugate clearly interferes with both the ability to form memory as well as to Memory retention in mice.

Experimental Example 4 Determination of choline acetyltransferase activity <Experimental procedure <

6 to 7 rats (Wistar strain, male) weighing About 150 g was used as a group. The as in Example 1 prepared solution of 2.5 S.NGF diphtheria toxin conjugate 0.2 μl of the treated group was approximately 1.55 mm in each case laterally and 0.3 to 0.5 mm deep into the forebrain and in the cerebral cortex of the vertex on both sides of the head by means of  a Hamilton syringe under pentobarbital sodium anesthesia (40 mg / kg, administered interaperitoneally).

Similarly, the control group was similar physiological saline solution.

After administration, the animal was fed normally for 5 days. Then it was beheaded, the brain taken out and immediately after weighting were the frontal lobes the bark and the basal forebrain won. The tissue was in a 20-fold volume 100 mm Tris-EDTA solution (pH 7.4) homogenized and 10 μl thereof were used to analyze choline acetyltransferase activity and part of the rest to used quantitative protein analysis. The choline acetyltransferase activity became after the method of Fonum (Journal of Neurochemistry, Vol. 24, pages 407-409 (1975)) determined and the protein concentration using a Protein determination reagent manufactured by Nippon Biorad Laboratory K.K., and the activity per 1 mg of protein was calculated from it.

<Results <

The results given in Table 4 were obtained.

Table 4

The above table clearly shows that the choline acetyltransferase Activity in the cerebral cortex and in the basal forebrain of the treated group is reduced by about 10% and 9%, respectively compared to the control group.

Claims (14)

1. Mammal except human, in which the cholinergic neurons derived from the Meynert nucleus or the basal forebrain protrude into the cerebral cortex, and / or the cholinergic neurons resulting from the Septum of the nucleus into the hippocampus protrude, are selectively destroyed. 2. Mammal according to claim 1, characterized that it's a rodent, a canine or feline animal. 3. mammal according to claim 2, characterized that the rodent is a rat. 4. rat, in which the projecting into the cerebral cortex cholinergic neuron is selectively destroyed. 5. rat, in which the protruding into the hippocampus cholinergic neuron is selectively destroyed. 6. Method of Making an Animal Model characterized in that one Conjugate of a nerve growth factor and a Neurotoxin at least to the cerebral cortex and / or the Hippocampus of a mammal, except man, administering selectively the cholinergic neurons, those from the Meynert nucleus or the basal forebrain sticking out into the cerebral cortex, and / or the cholinergic Neurons emerging from the septum of the nucleus in the Hippocampus protrude, destroyed.   7. The method according to claim 6, characterized that the mammal is a rodent, a dog or feline animal. 8. The method according to claim 7, characterized that the rodent is a rat. 9. The method according to claim 6, characterized in that the nerve growth factor 2.5 S.NGF, β -NGF or 7S.NGF. 10. The method according to claim 6, characterized that the neurotoxin Diphtheria toxin, cholera toxin or ricin. 11. The method according to claim 8, characterized that the conjugate is a conjugate from 2.5 S.NGF and diphtheria toxin. 12. Method for producing an animal model, characterized in that one Conjugate of a nerve growth factor and Diphtheria toxin of the cerebral cortex of a rat administering selectively the cholinergic neurons, which protrude from the basal forebrain into the cerebral cortex, destroyed. 13. Method for producing an animal model, characterized in that one Conjugate of a nerve growth factor and diphtheria toxin administered to the hippocampus of a rat, wherein selectively the cholinergic neurons that are derived from the  Septum of the nucleus into the hippocampus protrude, destroyed. 14. Conjugate of nerve growth factor and Diphtheria toxin.