Note: Descriptions are shown in the official language in which they were submitted.
2 ~ O ~
PROPHYLACTIC AND THERAPEUTIC TREATMENT OF ALZHEIMER'S
DISEASE
The present invention relates to a prophylactic
and therapeutic treatment of Alzheimer's disease, which
comprises administering a calpain inhibitor to a patient -
suffering from, or being susceptible to, Alzheimer~s
disease. The invention also relates to the use of the
calpain inhibitor in preparing a pharmaceutical formulation
for prophylactic or therapeutic treatment of Alzheimer's
10 disease. Further, the invention relates to said
pharmaceutical formulation containing the calpain inhibitor ~
as an active ingredient.
Alzheimer's disease or Alzheimer syndrome is a
progressive presenile dementia occurring at age between 45-
15 65. Pathologically, many senile plaque and neurofibrillary
changes are observed in the brain of a patient suffering
from the disease. Since senile dementia due to natural
senescence observed in patients after 65 years old shows no
substantial pathological difference from that of
20 Alzheimer's disease, it is called senile dementia of
Alzheimer type. The number of patients suffering from
these diseases has been increasing with the increase of
senile population, and therefore, the diseases have become
serious social problem. However, the etiology of this
25 disease is not known despite of the existence of many
... . - . ,. ., . . : : . ., ....................... : ... :
.: : .. : - . :: . . . : -
- : . ,.... ... , - ::;
- ,- :. : . ~ -. , .. .. :
2 ~ 7
hypotheses, and early solution for this problem has been
desired.
It is known that the incidence of the above-
mentioned two pathological changes characteristic to
Alzheimer's disease and senile dementia of Alzheimer type
(hereafter, the term "Alzheimer's disease~ includes both of
these dementia) shows a good correlation with the degree of ~-
cognitive impairment. Therefore, many studies have been
done since the first half of 1980's to throw light on the
cause of this disease through study at molecular level of
an insoluble cumulative material which causes the above-
mentioned two pathological changes.
It is known that disappearance of synapse due to
neuronal cell death shows an extremely good correlation
with the extent of mental dysfunction (Annals of Neurology,
30, 572 (1991); Annals of Neurology, 39, 355 (1989); Annals
of Neurology, 27, 457 (1990)). Mechanism through which
this disappearance occurs is not known at molecular level.
However, the role of calcium has drawn increasing
attention, because it has been reported that the dynamic
equilibrium of intracellular calcium is destroyed in
Alzheimer's disease as well as in other disorders such as
excitatory amino acid toxication, ~-amyloid
neurotoxication, and free radical disorder (Aging, 1, 17
(1989~; Neuron, 1, 623 (1989); Journal of Neuroscience, 12,
'
.' ''` , - .
2:~0l ~17
376 (1992)). Similar phenomenon has been observed in
fibroblasts of patients of Alzheimer's disease (Brain
Research, 543, 139 (1991); New England Journal of Medicine,
312, 1063 (1985); Proc. Natl. Acad. Sci. USA, 83, 2758
S (1986); Brain Research Bulletin, 21, 825 (1988);
Neuroscience Letters, 121, 239 (1991); Biological
Psychiatry, 22, 1079 (1987); Brain Research, 565, 42
(1991); Journal of Neuroimmunology, 33, 167 (1991)).
However, it was difficult to detect the abnormality of
calcium dynamic equilibrium in human brain because the
calcium level changes after death.
It appears that the change in calcium dynamic -~
equilibrium affects Ca(calcium)-dependent neutral protease
(calpain). This enzyme is believed to participate in the
control of intracellular transmission system (Proc. Natl.
Acad. Sci. USA, 87, 3705 (1990); Cell Structure Function,
15, 1 (1990); Biochemistry International, 18, 263 (1989)).
It has been suggested that restricted degradation by
calpain is involved in the control of other important
enzymes such as Ca-dependent protein-phosphorylating
enzymes, protein-dephosporylating enzymes, and
neurotransmitter-related enzymes, and of functions of
membrane-constituting proteins and membrane-skeletal
proteins (Annals of the New York Academy of Sciences, 568,
198 (1989)). Thus, abnormal activation of calpain appears
2 ~ 0 ~
- 4 -
to give serious effects through various metabolic pathways.
While rapid activation of calpain which occurs during
ischemia causes an acute neuronal cell death (Journal of
Neuroscience, 9, 1579 ~1989); Proc. Natl. Acad. Sci. USA,
88, 7233 (1991)), continuous activation at low level
appears to have slow but progressive effects on protein-
phosphorylation and protein's metabolic turnover. For
example, it participates in processing of amyloid precursor
(APP) (Proc. Natl. Acad. Sci. USA, 87, 6003 (1991); Proc.
Natl. Acad. Sci. USA, 89, 3055 (1992)), and abnormal
phosphorylation of cytoskeletal proteins (Annals of the New
York Academy of Sciences, 568, 198 (1989); Archives of
Neurology, 42, 1097 (1985)). Measurement of the change in
calpain activity was considered to provide an important
information on the etiology and treatment of Alzheimer's
disease, since activation of calpain not only reflects
dynamic equilibrium of intracellular calcium, but directly
reflects neuronal denaturation associated with increased
level of intracellular calcium.
It is known that there are two isozymes, calpain
I and calpain II. These enzymes require for their
activation ~M and mM levels of calcium concentration,
respectively. Both calpains predominantly exist within
cells in the form of an inactive precursor (~ell Structure
and Function, 15, 1 (1990); FEBS Letters, 220, 271 (1987)).
.. . .
:.. . : .
. . .. . . . .
- . : .. : ~ . ::. - .. : : .
: :: . : . . : ,
:.' :,: . . ~ . :
2 ~ ~3 .t ~ ~ 7
-- 5 --
The precursor is converted into its active form in the
presence of calcium through the self-digestion process
which occurs at the N-terminal (Journal of Biochemistry,
90, 1787 (1981); Journal of Biochemistry, 98, 407 (1985);
Biochimica et Biophysica Acta, 1078, 192 (1991)).
It is difficult to monitor in vivo action of
calpain by means of in vitro measurement of the enzyme
activity. This is because the precursor is activated in : :
the course of the measurement of activity. Thus, enzyme
activity to be measured is affected by in-stability of -the
enzyme and various intracellular suppressors or activators ~ .
(Intracellular Calcium-dependent Proteolysis (CRC Press,
Boston, MA), 1 (1990))
It has been shown using antibodies to calpain ~-
that calpain exists in senile plaque and a lesional portion
of neurofibril (Brain Research, 558, 105 (1991); Brain ~.
Research, 561, 177 (1991)), suggesting that calpain may
participate in the formation of the pathological structures
characteristic to Alzheimer's disease. However, the result
from direct measurement of calpain activity showed no
significant difference between patients suffering from
Alzheimer's disease and normal healthy subjects (Biomedical
Research, 10, 17 (1989); Journal of Neurological Science,
102, 220 (1991); Neurobiology of Aging, 11, 425 (1990)).
. - . .. :
2 ~0
-- 6
The present inventors paid special attention to
the amount of each of calpain I isoforms existing in post-
mortemh~En brains , i.e., an active form and its precursor,
which correspond respectively to the active form and the
precursor reported on platelet and erythrocyte (Biochemical
Journal, 246, 481 (1987); Biochemical Journal, 261~ 1039
(1989)). This was because, since protein degradation
at physiological calcium level requires the self-digestion
of calpain I (Biochimica et Biophysica Acta, 1094~ 249
(1991)), the ratio between the active form and the
precursor of this enzyme in tissues was considered to
provide an index of the in vivo function of calpain I.
With this index, the inventors discovered increased level
of the active form of calpain I in brain of patients
suffering from Alzheimer's disease. Thus, Alzheimer's
disease may be treated by administering a patient suffering
from the disease an effective amount of a calpain
inhibitor. The present invention i5 based on these
findings.
Thus, the present invention provides the method
of treating a subject who is suffering from, or susceptible
to, Alzheimer's disease by administering said subject a
calpain inhibitor.
The invention also provides a pharmaceutical
formulation for prophylactic or therapeutic treatment of
: :::. :, : :, :.: . . :.. -. - , -
:. : ;- . .
2 ~ 1 7
Alzheimer~s disease, which contains as an active ingredient
a calpain inhibitor.
In the accompanying drawing, Fig. 1 shows the
ratio of the active form of calpain I to the calpain
precursor in brain of patients suffering from Alzheimer~s
disease (AD) or Huntington's disease (HD), or normal -~
healthy subject (control). In the Figure, a) shows the
ratio in prefrontal cortex, b) shows the ratio in putamen,
and c) shows the ratio in cerebellum.
The present invention is described in more detail
below.
The increased level of calpain I in brain of a
patient suffering from Alzheimer's disease when compared
with normal healthy subject was confirmed according to the
following procedures.
Calpain I is partially purified from post-mortem
brain. Fraction containing crude calpain I is subjected to
electrophoresis, and reacted with monoclonal antibody to
human calpain I (Biochemical Journal, 246, 481 (1987);
Biochemical Journal, 261, 1039 (1989)) to show three bands.
Molecular weights of these bands are 80KDa, 78KDa, and
76KDa, respectively, and are identical to those in
erythrocyte (Biochemical Journal, 246, 482 (1987);
Biochemical Journal, 261, 1039 (1989)). When human
erythrocyte is incubated in the presence of calcium, the
.::
: ,, ~ , - - ,: :
: :, ;, . ~ . ~ . .. : .,
2 ~ 1 7
- 8 -
80KDa band disappears and the 76KDa band appears. This
reaction is inhibited by leupeptine which is a calpain
inhibitor. Furthermore, there exists a good correlation
between the amount of the 76KDa protein and the enzyme
activity. Thus, it is believed that the 80KDa band of
calpain I represents its inactive form (precursor) and the
76KDa band represents its active form. This transformation
from 80KDa to 76KDa in the presence of calcium can also be
observed with slice specimens of post-mortem human brain.
When the ratio between the active form and the
precursor was examined in different sites of brain
(prefrontal cortex, putamen, cerebellum) of a patient
suffering from Alzheimer's disease, increased amounts of
the active form were observed in all sites mentioned above,
as described in working Example hereinafter described.
This fact suggests that the increase of the active form of
calpain I is not the secondary outcome of neuronal cell
death, but it precedes the death of cells and may be a
trigger of the death. Accordingly, an agent which inhibits
the conversion of calpain I precursor to its active form,
which is herein called a calpain inhibitor, is believed to
prevent cells from the death and to avoid accumulation of
amyloids, and therefore, to be useful for suppression or
prevention of progression of Alzheimer's disease.
:: . ,:.,'., - . :
-
- - , . . .
. - ., . , . :
: : : . - , -
. .
- . . . . -
- 9 - :
Examples of calpain inhibitors which may be used
in the present invention include epoxysuccinic acid
derivatives such as E-64 (Agricultural and Biological
Chemistry, 42, 529 (1978)), E-64-C (Journal of
5 Biochemistry, 90, 255 (1981)), E-64-d (Journal of
Biochemistry, 93, 1305 (1983)), NC0-700 (Japanese Patent
Publication (kokai) No. 126879/1983), estatine A, B (The
Journal of Antibiotics, 42, 1362 (1989)) and the like, each
having the following formulas.
~ g NH~NH2 E-64
N ~ H R =H E-64-c
H o ~ Rl=CH3CH2- E-64-d
~0 ~ N O ~ OCN NCo-7
R~ OCH3
~H NH R2=H estatine A
HO ~ H O HN~NH2 R2=oH estatine B
: : : ' ' ' - ~' ' ;. .
,
2 ~
-- 10 --
Further examples of the calpain inhibitors are
peptidyl-fluoromethylketone derivatives (Journal of
Medicinal Chemistry, 35, 216 (1992)), peptidyl-
chloromethylketone derivatives (Journal of Biochemistry,
99, 173 (1986)), peptidyl-acyloxymethylketone derivatives
(Biochemistry, 30, 4678 (1991)) having the following
general formula (I):
H 0
R3,N ~ (I) .
R
wherein R3 is an amino acid or peptide residue which may
have a substituent on its N-terminal, R4 is an amino acid
side chain-forming group, X is a fluorine atom, a chlorine
atom, or a group -O-CO-Ar in which Ar is a phenyl group
which may be substituted, peptidyl-diazomethylketone
derivatives (Biochemical Journal, 253, 751 (1988)) having
the following generaI formula (II):
H 0 ~N
wherein R3 and R4 are as defined above in the general
formula (I), peptidyl aldehyde derivatives such as
leupeptine (The Journal of ~ntibiotics, 22, 183 (1969)),
calpeptin (Journal of Enzyme Inhibition, 3, 195 (1990)),
and MDL 28170 (Biochemical and Biophysical Research
. i . . .. . . ~ . ., -
:'"' - . : ~ ' -
~ .: . . . , . - :
:.... ~ . ' ' ~ - ' ` :'
.
2 ~ 7
Communication, 157, 1117 (1988)), each having the following
~ormulas.
H ~ H
O ~ O
leupeptine
HN~,NH2
NH
~ H ;.
N ~ N ~ H calpeptin
0 ~ H 0
N ~ ~ H
H 0 ~ MDL 28170 -
~dditional examples of the calpain inhibitors are
peptidyl-a-diketone or peptidyl-a-ketoester derivatives
(Journal of Medicinal Chemistry, 33, 11 (1990)) having the
following general formula (III):
H 0 5
R3~N~ (m) '- ':
R~
2l0~ 17
- 12 -
wherein R3 and R4 are as defined above in the general
formula (I), and R5 is an alkyl or alkoxy group. Peptide
compounds such as human brain calpastatin (U.S. Patent
Application Ser. No. 200,141) or derivatives thereof are
also included.
The compounds described above may be readily
prepared using the methods described in the above mentioned
references.
In the clinical application of the above
compound, it may be administered in the form of a
pharmaceutical formulation, in which the ratio of the
compound to carriers may vary from 1% by weight to 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, solutions or the like, or may be
administered intravenously, intramuscularly, ,
subcutaneously, intramedullarly, or intraventicularly in
the form of injections. Furthermore, the compound may be
used in the form of suppositories. Alternatively, it may be
formulated in powders for injections which are used in
preparing injections at the time of use. For preparing the
pharmaceutical formulations of the present invention, any
pharmaceutically-acceptable organic or inorganic solid or
liquid carr.ers or diluents suitable for oral, per rectum,
:' ' . ', ' ~ : ~ . ' ,
- ~ :
- ,
, :
2 i ~
- 13 -
or parenteral administration may be used. Examples of
carriers to be used for preparing solid formulations are
lactose, sucrose, starch, talc, cellulose, dextrin, kaolin,
calcium carbonate and the like. Liquid formulations for
oral administration such as emulsions, syrups, suspensions,
solutions or the like contain conventional inert diluents
such as water, vegetable oils, or the like. In addition to
the inert diluents, the formulations may further contain
adjuvants such as humectants, suspension aids, edulcorants,
aromatics, coloring agents, or preservatives. The
compounds may be formulated into solutions and filled into
capsules made of absorbable materials such as gelatin.
Examples of medium and suspending agent to be used in
preparing the formulations for parenteral administration
such as injections, suppositories, or the like, include
water, propylene glycol, polyethylene glycol, benzyl
alcohol, ethyl oleate, lecithin, and the like. Examples of
base to be used for suppositories are cacao butter,
emulsified cacao butter, laurin tallow, witepsol, and the
like. Such formulations may be prepared using any one of
conventional methods.
The clinical dose of the compound for oral `
administration is generally 0.01 - 1000 mg per day for
adult, though it is preferable to adjust appropriately for
particular case depending on the age of particular patient,
... . .. -- :-- .. : . . . ................................... .
.. : ' : .,- , ~,: . ' ' . : - -
~ :. - : . .
2 ~
conditions and symptoms of the disease to be treated. The
above daily dose of the compound may be administered as a
single dose or in two or three divisions at appropriate
intervals or intermittently.
When the compound is administered in the form of
injection, it is preferably administrated continuously or
intermittently at 0.001 - 100 mg for adult.
The following Example is illustrative only and is
not intended to limit the scope of the present invention in
any way.
Example 1: Measurement of calpain in brain of a
patient suffering from Alzheimer's disease
About 0.5g of a post-mortal brain was removed,
homogenized in 10 volumes of 20mM Tris-HCl (pH 7.4), 2mM
EGTA, lmM EDTA, lmM benzamidine, lmM dithiothreitol, lmM
phenylmethylsulfonyl fluoride (PMSF), and 0.32M sucrose
("Buffer A"), and the homogenized mixture was then
centrifuged (15,000xg, 30min, 4C). The supernatant was
applied to DEAE-cellulose column (DE-52, Whatman:
l.Oxl.5cm), and fractions were collected during the elution
with a KCl concentration gradient from 0 to 3mM. The
fraction eluted at 0.15M KCl was subjected to
electrophoresis and transferred onto PVDF membrane
(Immobilon-P, Millipore). The resulting membrane was
reacted with monoclonal calpain I antibody (Biochemical
~f
~"' . ' ' ~ ~,', ', . " ' ' ' ' ', ' '
~r ' ` ~ ' . ' ' '. ' " .. . ~ - ' -
' ~ ' ' ' ' , , ' : '
' :, . ' ' ,: . ~
. , ' ' : . ` .,
. ~' ' ' ~ . ' ' ' :
": ~ ' ' . "
2~0~'17
- 15 -
Journal, 246, 481 (1987); Biochemical Journal, 261, 1039
(1989)) and analyzed according to the method described in
Journal of Neurochemistry, 92, 526 (1992).
Three bands at 80KDa, 78KDa, and 76KDa were
detected in the human bra.in extract, and they were
identical to those in human erythrocyte (Biochemical
Journal, 246, 481 (1987); Biochemical Journal, 261, 1039
(1989)).
When calpain I in human erythrocy~e was activated
in vitro by adding calcium, N-terminal of 80KDa isoform was
cleaved to create 78KDa isoform and 76RDa isoform which was
resulted from cleavage of Gly26-Leu27 site (the cleavage
site was determined by separating 76KDa isoform resulted
from the self-digestion using SDS-PAGE, transferring it
onto PVDF membrane and then conducting amino acid
sequencing of the N-terminal using Edman degradation
method). The conversion of 80KDa isoform to 76KDa isofprm
was observed within 30 seconds. The enzyme activity was
retained even after the disappearance of 80KDa isoform, and
the activity showed a good correlation with the amount of
76KDa isoform. This was consistent with the previous report
suggesting that 76KDa isoform is an enzymatically active
form (Biochemical and Biophysical Research Communication,
123, 331 (1984); Biochemical and Biophysical Research
Communication, 138, 638 (1986)).
, . . . - . . . : . - .
: ~ ' ': ' : -' -
. . . ............ . . . . .. .
. - - - ~. . - : . .
2 ~ a ~
- 16 ~
Although human brain calpain I is considerably
unstable after extraction, the conversion of 80KDa isoform
to 76KDa isoform could be observed when corticocerebral
sections were incubated with calcium. Using polyclonal
antibodies to a synthetic peptide comprising the N-terminal
34 amino acids of the sequence reported by Imajoh et al.,
Biochemistry, 27, 8122 (1988), which was prepared according
to the method described in Journal of Neurochemistry, 47,
1039 (1986), it was confirmed that the self-digestion of
calpain I has resulted from the loss of its N-terminal
region.
In order to quantitatively determine the extent
of activation of calpain I in Alzheimer's brain, the three
isoforms were measured by means of immunoassay (Journal of
Neurochemistry, 92, 526 (1992)). Brain from patients
without neuropathy was selected as a control group, and
brain from patients who were diagnosed as having dementia
and showed neuropathologic characteristics of Alzheimer's
disease was selected as an Alzheimer group (Archives of
Neurology, 42, 1097 (1985); Neurology, 41, 479 (1991)).
The average ages of patients of Alzheimer group (N=22) and
the control group (N=17) were 75.8i2.0 and 68.9i2.6 years
old respectively when they died. The average time-
intervals between patient's death and freezing of their
''` ` ,' ~` : ,' , ..
': ' ,, . , . - .
;' ;. - ~, , ~ '.' -' ' ' ' . :
:' ': ' :' , ' ~,.: ,'- , ,.,'. ' . ` . " , . ' , '
.. . . . . . . .. . .
2lalL~7
- 17 -
brains were 12.2+1.3 hours and 12.0+1.8 hours for Alzheimer
group and control group respectively.
The amoun~ of 80KDa calpain I in cytoplasm of the
Alzheimer group was considerably lower than that of the
control group (22.7+1.5% for the control group and
37.2+2.2% for the Alzheimer group, p<0.001 under one-sided
t-test). One the other hand, the amount of 76KDa calpain
I, an active form of calpain generated by self-digestion
was considerably larger in the Alzheimer group than in the
control group (41.2+1.6% for the Alzheimer group and
26.6+2.2% for the control group, p<0.001). There was no
difference in the amounts of 78KDa calpain I between the - -
two groups.
When the ratio of 76XDa calpain I to 80KDa
calpain I in each of the brain samples was measured, it was
shown that the ratio in the Alzheimer group was three times
larger than that in the control group (2.20+0.39 for the
Alzheimer group and 0.81+0.10 for the control group,
p<0.005, see Fig. la). The total amount of the three
calpain I isoforms was identical among samples.
Then, the intracellular distribution of calpain I
- was studied. Frontal cortex of cerebrum was homogenized
and centrifuged at 15,000xg for 30min at 4C to obtain a
supernatant comprising cytoplasm and a precipitate
comprising cell membrane. The amount of each of the three
. .
.,: ~ :' ~. - ' ' ' : '
, . - .: . . . . . . : :, . .
- - .
.. . . .
- ' ~ : ~ .. . , , .. '
2 1 ~ 7
- 18 -
isoforms of calpain I was measured by immunoassay as
described above. The results showed that 27-30% of total
calpain I existed in the membrane fraction both in the
control group and the Alzheimer group, and that the
distribution patterns of the three isoforms in the membrane
fractions were nearly identical to each other.
Consequently, it was confirmed that the abnormal ratio of
calpain I isoforms in the cytoplasm was not reflection of
their intracellular distribution. In the membrane
fraction, the amount of 76KDa calpain I (40.4+1.8% for the ~ -
control group and 43.1+1.7% for the Alzheimer group, N=9
for both groups) was larger than the amount of 80KDa
calpain I (18.2+1.3~ for the control group and 17.5+1.3% -
for the Alzheimer group, N=9 for both groups). This result
was consistent with the hypothesis that calpain I may be
activated principally on the membrane (Journal of the
Biological Chemistry, 264, 18838 (1969); Biochemical and
Biophysical Research Communication, 128, 331 (1985)).
Degree of abnormal activation of calpain I observed in
Alzheimer's brain was not changed depending on the
postmortem interval. Correlation (r) between the ratio of
76KDa calpain I to 80KDa calpain I and the post-mortal
period was calculated, and r=0.355 for the control group,
r=0.155 for the Alzheimer group, and r=0.148 for a group
comprising both groups were obtained. Thus, there was no
:- . : ~- . -
:: . , :: :: ~:
- ~
,:: : : -,
- 19 -
significant correlation between the ratio and postmortem
interval. Furthermore, no significant correlation was
observed between the degree of activation of calpain I and
the patient's age at his death.
Since it had been recognized that a considerable
amount of neuron was denatured in neocortex of patients
suffering from Alzheimer's disease, degree of activation of
calpain I was measured at the site in which neuronal
denaturation was little or not observed (Neurology, 30, 820
(1980); The Neurobiology of Aging, 51(1983)). The ratio of
76KDa calpain I to 80KDa calpain I of the patients was
significantly larger both in putamen (46%, p<0.005) and
cerebellum (58%, p<0.005) than those of the control group. -~
On the other hand, in frontal region of cerebral cortex and
cerebellum of patients suffering from Huntington's disease `
(stage III), the distribution of calpain I isoforms was
normal (see Fig. la and c). However, in putamen of
patients suffering from Huntington's disease in which
neuronal cell death has been observed (Journal of
Neuropathology and Experimental Neurology, 44, 559 (1985);
Journal of Neurology, Neurosurgery and Psychiatry, 48,
422(1985)), the ratio of the active form of calpain I had
been increased by 50% (p<0.05) (see Fig lb).
Based on the largely increased level of calpain I
observed in Alzheimer's neocortex and similar observation
--:: . : . -. .- .: -- - . , , -
:.,. . -: - ~ , . . . . .
: - : ` :
' . ~'. :...................................... :
2~01~7
- 20 -
at the site in which neuronal denaturation was little, it
is very likely that this calpain I activation doesn~t
merely reflect the last stage of neuronal degeneration, but
precedes the death of cells and possibly reflects wide
range of abnormal metabolism associated with the death.
Test 1: calpain inhibitory activity
According to the method previously described
(Journal of Biological Chemistry, 259, 12489 (1984)),
inhibitory activity of several compounds were determined
with m-calpain which was extracted and purified from rat
brain by means of the method described in Journal of
Biological Chemistry, 259, 3210 (1984). The results are
summarized in the following table in which the data for
NCO-700 (Arzneimittel Forschung/Drug Research 36, 190
(1986)) and for MDL 28170 (Biochemical and Biophysical
Research Communications, 157, 1117 (1988)) are those
disclosed in the references.
.
2 ~
- 21 -
calpain inhibitory activitv
E-64 IC50=0 96~M
NCO-700 ICso=46~M
leupeptine IC50=0.36~M
carpeptine IC50=0.046~M
MDL 28170 Ki=0.007~M .
Test 2: Acute toxicity
Acute toxicity data for the compounds previously
described are reported in various published literatures as
follow.
(1) E-64, E-64-c, E-64-d (Japanese Patent Publication
(kokoku) No. 48/1992)
LD50>2000mgtkg (po, mouse)
(2) NC0-700 (Japanese Patent Publication (kokai) No.
126879/1983)
LD50=374mg/kg (iv, mouse)
(3) estatine A, B (Japanese patent Publication (kokai) No.
76/1978)
LD50>400mg/kq (ip, mouse)
Formulation 1: Tablet
The following ingredients were admixed using a
conventional method and compressed on a conventional
equipment to form a tablet.
E-64 30mg
.: ~
:-, ; ,.
.
.. . . ~
:;; - .
~2 ~
- 22 -
crystalline cellulose60mg
corn starch lOOmg
lactose 200mg
magnesium stearate 4mg
Formulation 2: Granule
The followir.g ingredients were admixed using a
conventional method, compressed on a conventional equipment
to form tablets, grinded, and granulated. Granules of 20-
50 mesh were selected.
E-64 lOg
lactose 20g
magnesium stearate lg
Formulation 3: Soft capsule
The following ingredients were admixed using a
.5 conventional method and filled into a soft capsule.
E-64 30mg
olive oil 300mg
lecithin 2Omg
Formulation 4: Injection
Twenty mg of vitellary lecithin and 3mg of E-64
were dissolved in chloroform thoroughly and then the
chloroform was removed through lyophilization. Two ml of
distilled water for injections was then added to prepare a
2ml ample.
Formulation 5: Suppository
., . . , j - . ~ - ` -
- - . ..
2 ~
- 23 -
To 2 g of heat-melted witepsol, 30mg of E-64 was
added, well mixed, filled into a rectal suppository mold
and cooled to obtain a suppository.
Formulat on 6
Tablets, granules, soft capsules, injections, and
suppositories were obtained according to the above -
procedures using leupeptine or calpeptin instead of E-64.
! ~ , .
- ' : : . , : ' '
- '' . : ' ' " ' . , :,
., ' ' ' : ' ,' .. " , ~' , " . '
