JPH06172211A - Prophylactic and therapeutic medicine for alzheimer's disease - Google Patents

Abstract

PURPOSE: To provide a prophylactic or therapeutic medicine for an Alzheimer's disease consisting of a material having an inhibitory action on calpain as an active component. CONSTITUTION: The medicine which inhibit the transition from the precursor of the calpain (calcium dependent neutral protease) I to activator is considered to be effective for suppressing or prophylaxis of the progress of Alzheimer's disease by preventing the cellular death or amyloid accumulation in the Alzheimer's disease. The example of the cappain inhibitor include the E-64 of formula I, the NCO-700 of formula II, the estatins A, B of formula III, the peptidyl-acyloxymethyl ketone deriv. of formula IV [R<3> is an amino acid residue or peptide residue which may have a substituent at an N terminal; R<4> is a group forming the side chain of amino acid; X is F, Cl, formula V (Ar is substitutable phenyl)], the calpastatin of the human brain, etc. The clinical dose of the calpain inhibitor for adult is 0.01 to 1,000 mg/day by oral administration and 0.001 to 100 mg/time by of an injection.

Description

Detailed Description of the Invention [Industrial applications]

The present invention relates to a prophylactic or therapeutic agent for Alzheimer's disease, and more particularly to a prophylactic or therapeutic agent for Alzheimer's disease using a calpain inhibitor, a pharmaceutical composition and a method thereof.

[Prior Art and Problems to be Solved by the Invention]

Alzheimer's disease is aged (45 to 65 years old)
It is a progressive dementia that develops in the brain. Pathologically, numerous senile plaques and neurofibrillary tangles are found in the brain. Senile dementia due to so-called natural aging, which occurs in the senile age of 65 years or older, is called Alzheimer-type senile dementia because no essential pathological difference is observed. The number of patients with this disease has increased with the increase of the elderly population, and has become a socially important disease. However, the cause of this disease is still unknown as a result of various opinions, and early clarification is desired.

It is known that the appearance amounts of two pathological changes characteristic of Alzheimer's disease and senile dementia of the Alzheimer's type (hereinafter collectively referred to as "Alzheimer's disease") correlate well with the degree of intellectual impairment. There is. Therefore, there is a study to elucidate the insoluble accumulated substances that cause these two pathological changes at the molecular level and reach the etiology of this disease.
It has been done since the first half of the 980s.

It is known that synapse loss due to death of nerve cells has a very good correlation with the degree of intellectual dysfunction in Alzheimer's disease (Annals of Neuro).
logy, 30 , 572 (1991); Annals of Neurolog
y, 39 , 355 (1989); Annals of Neurology, 2
7 , 457 (1990)). The mechanism that causes this disappearance is
Nothing is known at the molecular level. Among them, it has been reported that the dynamic equilibrium of intracellular calcium is disrupted due to Alzheimer's disease and other excitatory amino acid toxicity, β-amyloid neurotoxicity, and free radical damage (Aging, 1 , 17 (1989); Neuron, 1 , 6
23 (1989); Journal of Neuroscience, 12 , 3
76 (1992)), the role of calcium is rapidly gaining attention. Similar changes were observed in fibroblasts of Alzheimer's disease patients (Brain Research, 543 , 1).
39 (1991); New England Journal of Medi
cine, 312 , 1063 (1985); Proc. Natl. Aca
d. Sci. USA, 83 , 2758 (1986); Brain R
esearch Bulletin, 21 , 825 (1988); Neurosci
ence. Letters, 121 , 239 (1991); Biological
Psychiatry, 22 , 1079 (1987); Brain Res
earch, 565 , 42 (1991); Journal of Neuroimmu
nology, 33 , 167 (1991)). However, capturing abnormalities of calcium dynamic equilibrium in the human brain has been difficult because calcium levels change postmortem.

Due to changes in calcium dynamic equilibrium
And calcium-dependent neutral protease (calpain)
It is thought to be affected. This enzyme is in the intracellular transmission system
It is believed to be involved in control (Proc. Natl.
Acad. Sci. USA,87, 3705 (1990); Cell
 Structure Function,15, 1 (1990); Biochem
istry Alternativeal,18, 263 (1989)). this
Limited degradation by calpain is not limited to other important enzymes such as
Calcium-dependent protein kinase, protein release
Regulation of citrate enzymes, neurotransmitter-related enzymes, and membrane organization
Involved in controlling the functions of proteins and membrane skeletal proteins
Has been suggested (Annals of the New York
Academy of Sciences,568, 198 (1989)).
Therefore, abnormal activation of calpain leads to various metabolic pathways.
It is thought to have a significant impact through During ischemia
The rapid activation of calpain that causes acute neuronal cell death
While causing (Journal of Neuroscience,9, 15
79 (1989); Proc. Natl. Acad. Sci. USA,
88, 7233 (1991)), sustained low level activity
Is associated with protein phosphorylation and protein turnover
Considered to have a slow but progressive impact
available. For example, the process of amyloid precursor (APP)
Sing and (Proc. Natl. Acad. Sci. USA,87,
6003 (1991); Proc. Natl. Acad. Sci. US
A,89, 3055 (1992)), differences in cytoskeletal proteins
Normal Phosphorylation (Annals of the New York Acade
my of Sciences,568, 198 (1989); Archives
 of Neurology,42, 1097 (1985))
ing. Calpain activation means intracellular calci
Not only does it reflect the dynamic equilibrium of um, but also intracellular calcium
Can directly reflect the phenomenon of neurodegeneration that accompanies the increase in
So, showing the change in calpain activity means that
It is thought to give important information on the etiology and treatment of Immer disease
Was given.

It is known that calpain has two isozymes, calpain I and calpain II. These are μM and m upon activation, respectively.
Requires M-level calcium concentration. Both calpains mainly exist intracellularly as inactive precursors (Cell
Structure and Function, 15 , 1 (1990); FE
BS Letters, 220 , 271 (1987)). And it becomes an activator by the autolysis process of the amino group side in the presence of calcium (Journal of Biochemistr.
y, 90 , 1787 (1981); Journal of Biochemis
try, 98 , 407 (1985); Biochemica et Biophy.
sica Acta, 1078 , 192 (1991)).

It is difficult to monitor the action of calpain in the living body by measuring the enzyme activity in vitro. This is because the precursor is activated in the process of measuring activity. That is, the observed enzyme activity is influenced by enzyme instability and various cytoplasmic suppressors or activators (Intracellular Calcium-dependent P
roteolysis (CRC Press, Boston, MA), 1 (199
0)).

A study using an antibody of calpain revealed that calpain exists in senile plaques and neurofibrillary tangles (Brain Research, 558 , 105 (199).
1); Brain Research, 561 , 177 (1991)). It has been suggested that calpain is involved in the pathological structure formation specific to Alzheimer's disease, but direct calpain activity measurement showed no significant difference between Alzheimer's disease patients and normal subjects. (Biomedical
Research, 10 , 17 (1989); Journal of Neuro
logical Science, 102 , 220 (1991); Neurobi
ology of Aging, 11 , 425 (1990)).

[Means for Solving the Problems]

The present inventors focused on the amount of each isoform of calpain I in the brain after human death. They correspond to the active forms and precursors reported in platelets and erythrocytes (Biochemical Journal, 246 , 481 (19
87); Biochemical Journal, 261 , 1039 (19)
89)). In other words, autolysis of calpain I is required for protein degradation at organized calcium levels in vivo (Biochemica et Biophysica Ac).
ta, 1094 , 249 (1991)), the ratio of the precursor to the activator of this enzyme in the tissue is considered to be an indicator of calpain I function in vivo. As a result, they have found that such activated forms of calpain I are increased in the brain of Alzheimer's disease patients, and have completed the present invention.

That is, the gist of the present invention lies in a prophylactic or therapeutic drug for Alzheimer's disease, which comprises a substance having an inhibitory action on calpain as an active ingredient. Hereinafter, the present invention will be described in detail.

The present inventors first confirmed that activated calpain I is increased in the brain of Alzheimer's disease patients as compared to normal people. The method is shown below.

A portion of calpain I from the human postmortem brain.
Purify. This fraction was subjected to electrophoresis and human calpain
Monoclonal antibody against I (Biochemical Journal
l,246, 481 (1987); Biochemical Journal,
261, 1039 (1989)).
Is recognized. These molecular weights are 80KD each
a, 78 KDa and 76 KDa, which is equivalent to that of human red blood cells.
It is the same as this (Biochemical Journal,246, 48
1 (1987); Biochemical Journal,261, 103
9 (1989)). Calpain I of human red blood cells
80 kDa band when incubated in the presence of
Disappears and a band of 76 KDa appears. This reaction
Is antagonized by leupeptin, an inhibitor of calpain
It In addition, between the amount of 76 KDa protein and the enzyme activity
Has a good correlation. Therefore, Calpine I 8
The band of 0 KDa is the inactive (precursor) band of 76 KDa.
Is considered to be the active form. This is the brain after human death
80 KD in the presence of calcium even with sliced specimens
Transition from a to 76 KDa is recognized.

When the ratio of this activator to the precursor was examined in the brain region of Alzheimer's disease patients, activation was observed in all of the frontal cortex, putamen, and cerebellum of the cerebral cortex, as shown in Examples described later. It was confirmed that the body mass was increasing. This fact is a phenomenon in which the increase in the number of activators of calpain I occurs prior to cell death, not just as a secondary result due to neuronal cell death, and it is considered that this phenomenon triggers cell death. .

Therefore, it is considered that a drug which inhibits the transition from the precursor of calpain I to the activated form thereof is effective in preventing cell death or amyloid accumulation in Alzheimer's disease and suppressing or preventing the progression of Alzheimer's disease.

Examples of calpain inhibitors that can be used in the present invention include E-64 (Agrical tural and
Biological Chemistry, 42, 529, 197
8 years), E-64-c (Journal of Biochemistry, 9
0, 255, 1981), E-64-d (Journ
al of Biochemistry, 93, 1305 pages, 198.
3 years), NCO-700 (Japanese published patent publication Sho 58-12)
6879), Estatin A, B (The Journal of An
tibiotics, vol. 42, p. 1362, 1989) etc., epoxysuccinic acid derivative:

[Chemical 1] Peptidyl fluoromethyl ketone derivative (Journal o
f Medicinal Chemistry, Volume 35, 216 pages, 19
1992), peptidyl chloromethyl ketone derivative (Jou
rnal of Biochemistry, 99, 173 pages, 198
6 years), a peptidyl acyloxymethyl ketone derivative represented by the following general formula (I) (Biochemistry, 30 volumes, 46
(P. 78, 1991):

[Chemical 2] (In the above general formula (I), R ³ represents an amino acid residue or a peptide residue which may have a substituent at the N-terminal, R ⁴ represents a group forming an amino acid side chain, and X represents Fluorine atom, chlorine atom or group represented by the following formula:

[Chemical 3] (Ar represents a phenyl group which may have a substituent)
Represents ), A peptidyl diazomethyl ketone derivative represented by the following general formula (II) (Biochemical Journal,
253, 751 pages, 1988):

[Chemical 4] (In the above general formula (II), R ³ and R ⁴ are as defined in the above general formula (I)), leupeptin (The Journal of Antibiotics, Vol. 22, 1)
83 pages, 1969), Calpeptin (Journal of
Enzyme Inhibition, Volume 3, 195 pages, 1990
Year), MDL28170 (Biochemical and Biophysi)
Cal Research Research, 157, 1117, 1988) and the like peptidyl aldehyde derivatives:

[Chemical 5] Peptidyl α-diketone, peptidyl α-keto ester derivative (Journal of Medicinal Chemistry, 3
(3, 11 pages, 1990):

[Chemical 6] (In the general formula (III), R ³ and R ⁴ are as defined above, and R ⁵ represents an alkyl group or an alkoxy group.),

Human brain calpastatin (US patent application 2
00,141) or a derivative thereof and the like. The above compound can be easily produced by the method described in the above literature.

In clinical application of such compounds, the ratio of therapeutically active ingredient to carrier ingredient may be varied between 1% and 90% by weight. For example, the compound may be orally administered in the form of granules, fine granules, powders, tablets, hard capsules, soft capsules, syrups, emulsions, suspensions or solutions, or as an injection. It may be administered intravenously, intramuscularly, subcutaneously, intrathecally, or intracerebroventricularly. It can also be used as a suppository. Also,
It may be made into powder for injection and prepared before use. Pharmaceutical, organic or inorganic solid or liquid carriers or diluents suitable for oral, enteral and parenteral administration can be used for preparing the medicament of the present invention. As the excipient used when producing the solid preparation, for example, lactose, saccharose, starch, talc, cellulose, dextrin, kaolin, calcium carbonate and the like are used. Liquid preparations for oral administration, that is, emulsions, syrups, suspensions, solutions and the like, contain a commonly used inert diluent such as water or vegetable oil. In addition to an inert diluent, this formulation can contain auxiliary agents such as wetting agents, suspension auxiliary agents, sweetening agents, aromatic agents, coloring agents or preservatives. Liquid formulations may be included in capsules of absorbable material such as gelatin. Examples of the formulation for parenteral administration, that is, a solvent or suspending agent used for producing injections, suppositories, etc. include water, propylene glycol, polyethylene glycol, benzyl alcohol, ethyl oleate, lecithin and the like. Examples of bases used for suppositories include cocoa butter,
Examples thereof include emulsified cacao butter, laurin butter and Witepsol. The preparation method of the preparation may be a conventional method.

When used by oral administration, the clinical dose is generally 0.01 to 1000 mg per day as a compound for adults, but it may be appropriately increased or decreased depending on the age, disease state and symptoms. More preferable. The above-mentioned daily dose of the drug of the present invention may be administered once a day or divided into two or three times a day at appropriate intervals, or may be administered intermittently.

When used as an injection, it is desirable that a single dose of 0.001 to 100 mg as a compound be continuously or intermittently administered to an adult.

【Example】

Hereinafter, the present invention will be described in detail with reference to examples, but the invention is not limited thereto unless it exceeds the gist. Example 1 (Measurement of calpain in the brain of Alzheimer's disease patient) About 0.5 g of human post-mortem brain was taken, and a 10-fold amount of 20 mM Tr
is-HCl (pH 7.4), 2 mM EGTA, 1 mM ED
TA, 1 mM benzamidine, 1 mM dithiothreitol,
The mixture was homogenized with 1 mM phenylmethylsulfonylfluoride (PMSF) and 0.32 M sucrose (hereinafter, abbreviated as "buffer A") and centrifuged (15,0).
00 × g, 30 minutes, 4 ° C.). The supernatant is treated with DEAE-cellulos
E-column (DE-52. Whatman: 1.0 x 1.5 cm) is applied, and each fraction is collected while increasing the KCl concentration from 0 to 3 mM. The fractions eluted with 0.15M KCl were electrophoresed, and the PVDF membrane (Immobilon-P, Mi
transfer to llipore). Monoclonal calpain I antibody (Biochemical Journal, 246 , 481 (19
87); Biochemical Journal, 261 , 1039 (19)
89)) is reacted, and Journal of Neurochemistry, 9
The analysis was performed according to the method described in 2,526 (1992).

As a result, in human brain extract, 80 KDa,
Three bands of 78 KDa and 76 KDa were detected,
It was identical to that of human red blood cells (Biochemical
Journal, 246 , 481 (1987); Biochemical
Journal, 261 , 1039 (1989)). When human erythrocyte calpain I was activated by adding calcium in vitro, it was cleaved at the N-terminal side of 80 KDa isoform, resulting in a 78 KDa isoform and a 76 KDa isoform cleaved at the Gly ²⁶ -Leu ²⁷ part (cleavage). The site is the SDS-derived 76 KDa isoform produced by autolysis.
Separated by PAGE and transferred to PVDF membrane, which is then Edm
It was identified by performing amino acid analysis from the N terminus by the an degradation method). 80kDa isoform to 76kDa is
The transition to oform was confirmed within 30 seconds. The enzyme activity was maintained even after the 80 KDa isoform disappeared, and a good correlation was observed with the 76 KDa isoform amount. This is consistent with the report that the 76 KDa isoform is enzymatically active (Biochemical and Biophysical Res.
earch Communication, 123 , 331 (1984): Bio
chemical and Biophysical Research Communica
tion, 138 , 638 (1986)).

Human brain calpain I is quite unstable after extraction, but when cerebral cortex slices were incubated with calcium, the 80 KDa isoform yielded 76 KDa.
I was able to approve the transition to the isoform. This is Im
Report of ajoh et al (Biochemistry, 27 , 8122 (1
988)) according to the sequence of Journal of Neurochem
According to the method described in istry, 47 , 1039 (1986), a synthetic peptide consisting of 34 amino acids on the N-terminal side was prepared, and by using a polyclonal antibody against this peptide, autolysis of calpain I disappeared on the N-terminal side. It was confirmed that it was caused by.

In order to quantify the degree of activation of calpain I in the Alzheimer's disease brain, the Immunoassay method (Jo
urnal of Neurochemistry, 92, 526 (1992))
The three isoforms were quantified by. As a control group, patients with no clinical neurological disease were selected, and as an Alzheimer's disease group, patients with a diagnosis of prenatal dementia and neuropathologically characterized Alzheimer's disease were selected (Archives of N
eurology, 42 , 1097 (1985); Neurology, 41 ,
479 (1991)). The Alzheimer's group (N = 22) and the control group (N = 17) each have a death age of 75.8 ±
At the age of 2.0, 68.9 minutes ± 2.6 years, the time until freezing the post-mortem brain sample (post-mortem time) was 12.2 ± 1.3 hours and 1
It was 2.0 ± 1.8 hours.

The amount of 80 KDa calpain I in the cytoplasm was considerably lower in the Alzheimer group than in the control group (22.7 ± 1.5% in the control group, 37.2 ± 2.2 in the Alzheimer group). %, P <0.001 by unpaired t-test). On the other hand, calpain I of 76 KDa activated by autolysis was significantly higher in the Alzheimer group than in the control group (41.2 ± 1.6 in the Alzheimer group).
%, The control group was 26.6 ± 2.2%. p <0.001). In addition, no change was observed in calpain I of 78 KDa.

Next, 80 KD for each brain sample
The ratio of calpain I of 76 KDa to calpain I was found to be about 3 times higher in the Alzheimer group than in the control group (2.20 in the Alzheimer group.
± 0.39, control group 0.81 ± 0.10. p <0.00
5. (See Figure 1a). The total amount of the three calpain I isoforms was unchanged in all samples.

Then, the intracellular distribution of calpain I was examined. The frontal cortex of the cerebral cortex was homogenized and centrifuged at 15,000 × g for 30 minutes at 4 ° C. to obtain a supernatant as a cytoplasmic fraction and a precipitate as a cell membrane fraction. Calpine I three
The amount of isoform was measured by the Immunoassay method similar to the above. As a result, 27 to 30% of the total calpain I was present in the membrane fraction in both the control group and the Alzheimer's group, and the distribution of each isoform was almost the same. Therefore, the calpain I isoform in the cytoplasm was almost the same. The abnormal ratio is
It was confirmed that it was not due to the shift of their intracellular distribution. In the membrane fraction, calpain I of 76 KDa
Abundant (40.4 ± 1.8% in the control group, 43.1 ± 1.7% in the Alzheimer group, both N = 9), 80K
Calpain I in Da was low (18.2 ± 1.
3%, 17.5 ± 1.3% in Alzheimer's group, both N = 9). This result was consistent with the hypothesis that calpain I is predominantly activated on the membrane (Journal of the
Biological Chemistry, 264 , 18838 (196
9); Biochemical and Biophysical Research C
ommunication, 128 , 331 (1985)). The abnormal activation of calpain I in the brain of Alzheimer's disease patients was constant regardless of the time of death. When the correlation between the ratio of 76 KDa and 80 KDa calpain I and post-mortem time was examined, r = 0.355 in the control group, r = 0.155 in the Alzheimer's group, and r = 0.14 in the combined group.
It was 8 and there was no significant correlation. Further, the degree of activation of calpain I did not show any significant correlation with the age at death.

Since it has been confirmed that a considerable amount of nerve is degenerated in the neocortical region of Alzheimer's disease patients,
Areas with little or no neurodegeneration (Nuerol
ogy, 30 , 820 (1980); The Neurobiology of
The degree of activation of calpain I in Aging, 51 (1983)) was investigated. The ratio of calpain I at 76 KDa to calpain I at 80 KDa was found to be putamen (46%, p <0.005) and cerebellum (58%) in Alzheimer's disease patients.
%, P <0.005) compared to the control group (see FIGS. 1b and c). Meanwhile, Huntington's disease
Normal distribution of calpain I isoforms in the frontal cortex and cerebellum of stage III patients
(See Figures 1a and c). However, the putamen, which has been found to have neuronal cell death in Huntington's disease (Journal of Neuropatholo
gy and Experimental Neurology, 44 , 559 (1
985); Journal of Neurology, Neurosurgery an
d Psychiatry, 48 , 422 (1985)), 50%
(p <0.05) There was an increased proportion of calpain I activators (see Figure 1b).

From the similar results found in the large number of calpain I activators in the Alzheimer's disease neocortex and in the sites where there is little neurodegeneration, the activation of calpain I reflects the result at the end stage of mere neuronal degeneration. Rather, it is believed to reflect a more extensive metabolic change that precedes cell death and is probably directly involved in cell death.

Test Example 1 Calpain Inhibitory Activity The method described in the literature (Journal of Biological Chemist
ry, 259, 3210, 1984) using m-calpain purified from rat brain (Journal of Biological Chemistry, 25).
9), 12489, 1984), and its inhibitory activity was measured. However, NCO-700 (Arzneimitt
el Forschung / Drug Research, 36, 190 pages, 1986) and MDL28170 (Biochemical).
and Biophysical ResearchCommunications, 15
7, Vol. 117, 1988), the values described in the literature are described. Calpain inhibitory activity _{E-64 IC 50 = 0.96μM NCO} -700 IC 50 = 46μM leupeptin IC ₅₀ = 0.36μM calpeptin IC ₅₀ = 0.046μM MDL28170 Ki = 0.007μM

Test Example 2 Acute toxicity test The results of the acute toxicity test described in the literature are shown below. (1) E-64, E-64-c, E-64-d (Japanese Patent Publication No. 4-4
LD ₅₀ > 2000 mg / kg · po (2) NCO-700 in mice (JP-A-58-126879) LD ₅₀ = 374 mg / kg · iv (3) Estatin A, B (in mice) Japanese Unexamined Patent Publication No. 62-76) LD ₅₀ > 400 mg / kg ip in mice

Formulation Example (1) Tablet The following ingredients were mixed according to a conventional method and tableted by a conventional apparatus. E-64 30 mg Crystalline cellulose 60 mg Corn starch 100 mg Lactose 200 mg Magnesium stearate 4 mg

(2) Granules The following ingredients were mixed according to a conventional method, tableted by a conventional apparatus, crushed, sized and 20-50 mesh particles were selected to obtain granules. E-64 10g Lactose 20g Magnesium stearate 1g

(3) Soft capsules The following ingredients were mixed according to a conventional method and filled into soft capsules. E-64 30g Olive oil 300g Reticin 20g

(4) Injection After dissolving 20 mg of egg yolk lecithin and 3 mg of E-64 in chloroform completely, the chloroform was completely distilled off by freeze-drying. Next, 2 ml of distilled water for injection was added to prepare an ampoule containing 2 ml.

(5) Suppository E-64 30 mg in a heated melt of Witepsol 2 g
And mix well, inject this into a rectal suppository mold,
Upon cooling, a suppository was obtained.

(6) E-in the formulation of (1) to (5) above
The same operation was performed using leupeptin and calpeptin instead of 64 to obtain tablets, granules, soft capsules, injections and suppositories.

[Brief description of drawings]

FIG. 1 is a drawing showing the ratios of calpain I and activator to calpain I precursor in the brain of Alzheimer's disease (AD), Huntington's disease (HD) and normal people (control). In the figure, a) represents the ratio in the frontal region of the cerebral cortex, b) represents the ratio in the putamen, and c) represents the ratio in the cerebellum.

 ─────────────────────────────────────────────────── ————————————————————————————————————————— Inventor Ralph A. Nixon 02174 Pleasant Street, Arlington, MA 02174 USA

Claims (3)

[Claims] 1. A preventive or therapeutic drug for Alzheimer's disease, which comprises a substance having an inhibitory effect on calpain as an active ingredient. 2. A pharmaceutical composition for preventing or treating Alzheimer's disease, which comprises a substance having an inhibitory effect on calpain and a pharmaceutically acceptable carrier. 3. A method for preventing or treating Alzheimer's disease using a substance having an inhibitory effect on calpain.