JPH11180891A - Anti-cell death agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an anti-cell death agent effective in treatment, etc., of hepatitis, ischemic brain disease, Alzheimer's disease, etc., caused by cell death by including a substance capable of inhibiting a human Caspase or a human Caspase activity inducer therein. SOLUTION: This anti-cell death agent is obtained by including a tyrosine- valine-alanine-spartic acid, aspartic acid-glutamic acid-valine-aspartic acid and glutamic acid-serine-methionine-aspartic acid or the like which is converted into an aldehyde and is a modification of a protein containing an amino acid sequence near a processing site of a Caspase as a human Caspase or/and a human Caspase activity inducer. The anti-cell death agent is useful as a therapeutic agent, etc., of diseases caused by cell death such as hepatitis, ischemic brain disease, Alzheimer's disease, and myelotoxicity and diarrhea, etc., which are adverse effects of administration of an antitumor agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention has a function of inhibiting a certain cysteine protease or the like as an effect.
The present invention relates to an anti-cell death agent containing the inhibitor, and a therapeutic agent for a disease caused by cell death, comprising the anti-cell death agent as an active ingredient.

[0002]

2. Description of the Related Art Cell death is a vital phenomenon that is important for homeostasis of a living body together with cell growth. It is said that cell death involves two types of processes, each of which involves apoptotic cell death (apoptotic cell death).
ll death) and necrotic cell death. Apoptotic cell death has a specific form, such as fragmentation of cytoplasm and cell membrane and fragmentation of chromosomal DNA, and has been observed in developmental processes, the reproductive system, the immune system, and the like. Necrotic cell death is caused by physical stimulation such as heat shock or laceration, and is observed as necrosis. Recently, it has been revealed that cell death, particularly apoptotic cell death, is closely related to diseases such as hepatitis and autoimmune diseases, and elucidation of the molecular mechanism thereof has been regarded as important in the medical field.

[0003] A cysteine protease called CED-3 was isolated from a search for factors related to apoptotic cell death using nematodes. When a factor having high homology with CED-3 was searched for in animal cells, CED-3 showed high homology with interleukin-1β converting enzyme (ICE). In addition, from a gene transfer test of differentiation antigens CD4 and ICE into cultured cells, both factors alone induce apoptotic cell death, and activation of ICE / CED-3 plays an important role in apoptotic cell death signaling. It became clear that we were doing. Fas, which has been isolated as a receptor for apoptotic cell death, and the type 1 tumor necrosis factor receptor also include the respective ligands Fas
ICE / CED-3 is activated by binding to a ligand or tumor necrosis factor α (TNF-α).

At present, 10 kinds of ICE / CED are available in humans.
-3 family cysteine proteases have been isolated and are further divided into three subfamilies: ICE, CPP32 and ICH. At present, activation by ICE subfamily cysteine protease is C
An ICE cascade that activates the cysteine protease of the PP32 subfamily (Enali, et al., Na
Nature, Vol. 380, p723-726, 1996) and the apoptotic signal transduction system (apoptotic d
eath signaling) Induction (Hasegawa J., et al., Cancer
Res. Vol.56, p1713-1718, 1996).

Recently, the name of the human ICE / CED-3 family cysteine protease has been changed to "Caspase (C
(Emad S. Alnemri et al., Cell, Vol.87 p171,199)
6) There is. In this name, human caspase-1
(Caspase-1) corresponds to ICE, and human caspase-3 (Caspase-3) corresponds to CPP32, Y
ama and apopain.

[0006]

In recent years, the detailed mechanism of apoptotic cell death signal transduction related to cell death has been elucidated. However, in addition to known pathways, it is induced by drugs such as antitumor agents. Is done. ICE / C in humans
The ED-3 homolog (Homolog) is closely involved in such drug-induced cell death, and various reports have been made (Ma
shima, et al., Biochem. Biophys. Res. Commun., Vol. 20
9, p907-915, 1995; Suzuki and Kato, Exp.Cell Res.
Vol.227, p154-159, 1996), for example, relating to etiology, medicinal properties and side effects of drugs. The inventor has ICE /
It is thought that the control of the CED-3 homologue greatly contributes to suppression of diseases and side effects.

[0007]

Means for Solving the Problems Accordingly, the inventor of the present invention
Investigations have been made on those having an inhibitory effect on caspase (Caspase) or / and human caspase (Caspase) activity inducer, and as one example, the possibility of a therapeutic agent having anti-cell death activity was clarified, and the invention was completed .

As an apoptosis inhibitor, hitherto unknown, a sequence that is a part of the N-terminal cleavage portion of caspase-3 (CPP32 / Yama which is one of the CPP32 subfamily), that is, the amino acid sequence is glutamic acid-serine -Methionine-Aspartic acid-modified protein modified with aldehyde (ESMD-CH
O).

[0009] Casspace 3 (CPP32 / Yam
a) is responsible for the conversion of the zymogen having a molecular weight of 32 Kd into an active protein having a molecular weight of 29 Kd. This 29Kd caspase-3 is capable of inducing apoptotic cell death by itself and becomes active in only 1 minute upon stimulation with Fas or the like. Therefore, it was thought that ESMD-CHO, which is an aldehyde-modified protein that is a part of the N-terminal cleavage portion of caspase-3, can inhibit an unknown caspase-3 activity inducer. In other words, it was thought that if the production of the active protein was inhibited, the transmission of the cell death signal could be surely stopped, so that a therapeutic agent for a disease caused by cell death could be used.

Therefore, the inhibitory activity of ESMD-CHO, that is, the inhibitory activity of caspase-3 activity can be measured to determine its inhibitory activity.

[0011] Human caspase-1, human caspase-3 (Caspase-3) activity inducer and the like may be similarly considered, and an anti-cell death agent having an inhibitory effect may be designed. The inhibitory effects of the active forms of caspase-1, caspase-3, and caspase-3 inducer listed here have specific inhibitory effects upstream of the cell death / signal cascade, and are more effective. And can treat various diseases caused by cell death.

[0012]

The present invention relates to a human caspase (Cas).
base) or / and human caspase (Cas)
a) a drug having an activity of inhibiting an activity inducer.

[0013] The present invention also relates to human caspase-1.
(Caspase-1) and / or a drug having an inhibitory action on human caspase-3 (Caspase-3). As described above, the ICE / CED-3 gene transfer experiment revealed that both have important significance in the apoptotic death signal. afterwards,
Numerous ICE / CED-3 related genes have been isolated,
These have been designated as ICE / CED-3 homologs (Homolog).

ICE / CED-3 homologues include IC
E, CPP32, Yama, Apopain and the like. In the name of caspase, as described above, human caspase-1 (Caspase-1) corresponds to ICE, and human caspase-3 (Caspase-
3) corresponds to CPP32, Yama and Apopain.

[0015] Drugs are a general term for chemical substances and the like that can treat and prevent diseases by utilizing useful effects for the human body. If it has the above-mentioned inhibitory action, it may be a drug.

The present invention relates to an anti-cell death agent comprising a substance that inhibits human caspase or / and a human caspase activity inducer.

Furthermore, human caspase-1 (Ca
space-1) or / and human caspase-
The present invention relates to an anti-cell death agent containing a substance inhibiting 3 (Caspase-3).

The anti-cell death agent refers to an agent capable of preventing the phenomenon of cell death by some action or reducing the appearance of the phenomenon. As this effect exhibiting an effect, it is desirable to inhibit human caspase-1 (/ Caspase-1) and / or human caspase-3 (Caspase-3). These are thought to be involved in the early events of the intracellular cascade of the cell death signal transduction mechanism, and show a more effective inhibitory effect than the effects of events such as events near the end of the cascade. Can be As an example of this effect, human caspase (Cas
base) or / and human caspase (Cas)
phase) The inhibitory action of the activity inducer may be due to deprocessing in the process of becoming a human caspase or / and an active form of the human caspase activity inducer. In addition, human caspase-1 (Casp
case-1) or / and human caspase-3
(Caspase-3) inhibits human caspase-1 (Caspase-1) or / and human
Any method may be used as long as it is deprocessing in the process of becoming an active form of Caspase-3.

In the deprocessing, a modified protein containing an amino acid sequence at the caspase processing site can inhibit the enzyme that processes and activates the caspase, thereby inhibiting the caspase activity.

That is, any modified protein containing the amino acid sequence of the caspase processing site can be used as an anti-cell death agent.

More specifically, the present invention relates to an anti-cell death agent containing an aldehyde-modified protein of one or more proteins selected from the group consisting of the following amino acid sequences.

Tyrosine-valine-alanine-aspartic acid, aspartic acid-glutamic acid-valine-aspartic acid, and glutamic acid-serine-methionine-
Aspartic acid.

[0023] Preferably, in this group, the aldehyde-modified protein is a protein whose amino acid sequence is glutamic acid-serine-methionine-aspartic acid.

These amino acid sequences are considered to include the amino acid sequence of the processing site of the human-derived caspase, or to include the sequence near the site.

The effect of the present invention can be achieved by a modified protein having this action. As the protein, a protein that binds to the substrate binding site of the processing enzyme but exhibits the inhibitory activity by remaining at the binding site without being affected by the enzyme is desirable. Modified proteins may be mentioned as having such inhibitory activity, and the modification method is not limited as long as the modified protein can be stably present without significantly changing the spatial form of the protein. Examples include aldehyde conversion, amidation, addition of sugar to amino acid side chain, phosphorylation, and the like. Of these, aldehyde conversion is preferable. The protein to be modified does not need to have a long (large) length (large), and it is sufficient that the effect can be achieved. Specifically, the number of amino acids may be 2 to 10, preferably 3 From 5
And particularly preferably 4.

Further, those having the above-mentioned effects do not need to have a protein structure. Compounds obtained by chemical synthesis or the like can also be designed and synthesized with reference to the spatial coordination in consideration of the amino acid residues of the aldehyde-modified protein having an effect.

In the present invention, ICE / CED-
It inhibits the post-translational control step in the process of protein production of the three homologs, preventing the appearance of its activity. More specifically, aldehyded tyrosine-valine-alanine-
Aspartic acid, aldehyde-modified aspartic acid-
The amino acid sequences of glutamic acid-valine-aspartic acid and aldehyde-modified glutamic acid-serine-methionine-aspartic acid are part of the sequence near the process in protein processing. By coexisting the modified proteins having these sequences in the processing, the recognition and binding of the processing enzyme is disturbed, and the enzyme action is reduced or prevented, thereby achieving the inhibitory effect.

[0028] The present invention provides the above-mentioned anti-cell death agent,
The present invention also relates to a therapeutic agent for a disease caused by cell death.

The present invention relates to a therapeutic agent for a disease selected from hepatitis, ischemic brain disease, Alzheimer's disease, bone marrow toxicity and diarrhea, which are side effects of administration of an antitumor agent, among diseases caused by cell death. The present invention relates to a therapeutic agent for hepatitis, among diseases caused by cell death.

The present invention also relates to a therapeutic agent for hepatitis caused by cell death, which comprises an aldehyde-modified protein having an amino acid sequence of glutamic acid-serine-methionine-aspartic acid.

[0031]

BEST MODE FOR CARRYING OUT THE INVENTION The effect of the inhibitor of the present invention can be examined as follows. 1. Investigation of the effect of inhibiting Fas antibody-induced cell death 1) D98AH2-Fas cell, which is an in vitro (in vitro) model cell of acute Fas-mediated apoptosis [inhibitor domain was added to human cervical cancer cell, Hela cell (Hela cell)] Cell prepared by transfecting a defective human Fas mutant FD-5]] into ESMD-CHO
Human Fas antibody (Fas A
b) MTT assay (Suzuki and K)
ato, Exp. Cell Res. Vol. 227, p154-159, 1996) and Hoechst 33342 staining method.

2) D98AH2-Fas cells treated with ESMD-CHO or untreated were treated with FasAb, and the CPP32 / Yama activity in the cytoplasm was measured using DEVD-MCA.
(Hasegawa J., et al., Cancer Res. Vol.56, p1713-1
718, 1996) as a substrate and measurement with a fluorescence spectrophotometer.

3) The cytoplasmic proteins extracted from the cells treated as described above are separated by SDS-polyacrylamide gel electrophoresis, and immunoblotting using an anti-human CPP32 antibody is performed. By this experiment, CPP32 / Yama
ESMD-C for molecular shift by Fas Ab
Consider the effect of HO.

2. Examination of the effect of ICE / CED-3 related factor inhibitor on fulminant hepatitis model mouse ESMD-CHO
After examining the ability to inhibit PP32 / Yama activity, a protein in which the amino acid sequence which is a known inhibitor of the ICE / CED-3 homolog was modified with an aldehyde of a protein represented by tyrosine-valine-alanine-aspartic acid [ YV
AD-CHO (ICE subfamily inhibitor)] and a protein whose amino acid sequence has been modified with an aspartic acid-glutamic acid-valine-aspartic acid aldehyde [DE
VD-CHO (CPP32 subfamily inhibitor)], the in vitro extrapolation properties of the three kinds of anti-ICE / CED-3 homologue synthetic peptides may be examined by the following experiment.

1) Intravenous administration of anti-mouse Fas antibody (Jo2 clone) to mice via the tail vein has been established as a model of fulminant hepatitis (Lacronique, V., et al. Nature).
Med.vol.2, p80-86, 1996; Rodrigues, I., et al. J.
Exp. Med. Vol.183, p1031-1036, 1996). This is hepatocellular death resulting from the induction of apoptotic cell death transmission by Fas Ab stimulation upon expression of hepatocyte surface (Ogasawara, J., et al. Nature vol.364, p8
06-809, 1993). Using this model, the life and death of mice,
The presence or absence of the anti-ICE / CED-3 homolog synthetic peptide's ability to suppress apoptosis in vitro may be examined using blood biochemical tests, chromosomal DNA tests, and histological tests as indices.

3. From the confirmation of the effect to the application to treatment The effect is confirmed from the results of the examination, and the application in the treatment method is possible from the following concept. Although most of the results are from in vitro, the following observations have been made in viral hepatitis.

Normally, hepatocytes express Fas. This causes the antigen expressing the virus infection to be expressed on the cell membrane surface by the virus infection. Cytotoxic T cells (Cytotoxi
c Tlymphocyte (CTL) recognizes this antigen and contacts virus-infected hepatocytes. By this contact, Fas binds to Fas ligand on CTL, and hepatocytes are removed by apoptotic cell death. As a result, the death of a large number of infected hepatocytes causes symptoms in the form of hepatitis.

In addition, administration of LPS (lipopolysaccharide), which induces liver damage similarly to administration of antibodies,
Knockout mice showed resistance, which also indicates direct involvement of the ICE / CED-3 homolog (Li, et al. Cell Vol. 80, p401-411, 1995). Thus, in Fas-mediated apoptosis, ICE / CE
We believe activation of the D-3 homolog is essential.

The above-mentioned viral fulminant hepatitis model is also suppressed by transgene transduction of an oncogene bcl-2 having an apoptosis-suppressing effect. Bcl-2 is I
It is thought to suppress the pre-stage of CE / CED-3 homolog activation (Simizu, S., et al. Oncogene vol. 13, p.
21-29, 1996), it is considered that the results clearly show that suppression of apoptotic cell death, that is, suppression of the activity of the ICE / CED-3 homolog, is effective against hepatitis.

The mechanism of apoptosis is thought to be conserved during evolution. In particular, in mammals, it is considered that they are the same regardless of species, and the above experimental results of mice and the like may be considered that apoptosis occurs in humans by the same mechanism.

At present, the above-mentioned various hepatitis, ischemic brain disease, and the like, which are caused mainly by apoptotic cell death,
Alzheimer's disease and the like can be considered, and bone marrow toxicity and diarrhea due to administration of an antitumor agent can be considered as side effects due to drug administration. These are phenomena that occur because cells that should have been alive have suddenly died. The inventor believes that protecting cells from such accidental death can be a treatment for the above diseases and side effects.

The dose of the compound of the present invention as the above-mentioned therapeutic agent is from 0.02 to 1 g / g from the viewpoint of the effects described later.
kg. As for the administration form, intravenous injection is desirable.

The compound of the present invention can be produced by a solid phase method for producing a protein (Merrifield, RB, J. Am. Chem. Soc., Vol. 8).
5, 2149, 1963), and then a protein having a desired amino acid sequence may be produced and then subjected to modification such as aldehyde conversion by a conventional method. From the derivative of the compound of the present application, in which the side chain of the amino acid and the aldehyde are protected, the side chain protecting group is removed with a strong acid and then the semicarbazone of the aldehyde protecting group is removed with a formaldehyde / acetic acid / methanol mixture. I do. The obtained deprotected product was reverse-phase HPL
After purification with C or the like, it can be used as the compound of the present application.

Hereinafter, the present invention will be described with reference to examples.
The present invention is not limited to only these examples.

[0045]

Example 1 Example 1 Cell Death Inhibition Experiment of Aldehyde-Modified Protein 1) Induction of Cell Death with Fas Antibody and Intravenous Injection of Inhibitor Male Slc / ddy mouse, 5 weeks old (Shizuoka experimental animal) 1 group 1
Six animals were used. Anti-mouse Fas antibody (Jo2 clone) (manufactured by Pharmingen) is a phosphate buffered saline (P
BS). 10 μg of Fas antibody was injected intravenously, and hamster IgG diluted in PBS was injected simultaneously intravenously as a control.

After the injection, the progress was observed, and the lung, heart, liver and thyroid were separated every hour and used for chromosomal DNA analysis, histological analysis and measurement of CPP32 subfamily activity.

An inhibitor of the CPP32 subfamily, DE
VD-CHO (manufactured by Peptide Lab.) Was dissolved in dimethyl sulfoxide (DMSO). Furthermore, it is dissolved in PBS (phosphate buffered saline) (DMSO at a final concentration of 1%).
%) And 0.1-0.5 mg of DEVD-CHO was injected intravenously. Intravenous injection of FAS antibody was performed 2 hours later.

Each experiment was repeated three times.

2) Analysis of Chromosomal DNA In order to determine whether apoptosis was induced in each tissue by injection of Fas antibody, fragmentation of chromosomal DNA was determined by a known method (Suzuki A., et al., EMBO J. Vol. .15, p21
1-215, 1996).

The tissue was prepared using Lysis Buffer (100 mM NaCl, 25 mM E
DTA, 100 mM Tris-HCl pH 8.0, 0.5% SDS and 0.3 mg / ml
50 with proteinase K). C, digested for 15 hours. The sample was extracted three times with a phenol-chloroform-isoamyl alcohol (25: 24: 1) solution, and extracted once with chloroform.
Extracted times. Add the same amount of isopropanol to the aqueous layer and add D
NA is -20. C for 1 hour. DNA to 10 mM T
ris-HCl pH 8.0-Dissolved in 10 mM EDTA and 37 with 50 μg RNase. C1 hours treatment.

The protein was separated and the DNA was precipitated as described above. The purified DNA is A260 / A28
The 0 ratio was measured with a spectrophotometer and the concentration was calculated.

About 1 μg of the DNA was applied to a 2% agarose gel in a Tris-borate buffer for electrophoresis at 100 V
Per hour and separated by electrophoresis. After electrophoresis, run gel
After staining with g / ml ethidium bromide for 5 minutes, rinsing with distilled water, the electrophoresis image was observed by ultraviolet irradiation.

3) Histochemical analysis Mouse tissues were fixed in formalin, embedded in paraffin, and serially cut at 8 μm. Sections were from delafeed hematoxylin and eosin or Hoechst 3334.
Stained with 2. Hoechst 33342 staining used was a modification of the conventional method.

The sections were treated with xylene and ethanol.
Washed with BS. After washing, 1 μM Hoechst 33342
For 18 hours.

4) Extraction of protein and measurement of CPP32 subfamily activity Protein extraction and enzyme assay were performed by a conventional method (Hasegawa J., e).
tal., Cancer Res. vol.56, p1713-1718, 1996). The tissue is separated, washed with PBS, and chopped in PBS containing 1 mM EDTA. To this was added a final concentration of 10 μM digitonin (manufactured by Sigma) for 30 minutes 37 minutes.
Incubate at C. In addition, 15000 rpm,
The precipitate was collected by centrifugation for 5 minutes and used as an assay sample.

The sample was prepared using a DC protein assay kit (Bi
o Rad).

CPP32 subfamily activity was determined by adding 10 μl of 50 μM DEVD-MCA (Peptide Lac
b.) and incubated, and the release of amino-4-methylcoumarin was monitored with a spectrophotometer.

As a result of these experiments, it was found that the administration of the Fas antibody rapidly caused the activity of the CPP32 subfamily in the liver and lungs.

Further, as shown in FIG.
CHO inhibited cell death and significantly increased individual viability.

[0060]

EFFECT OF THE INVENTION The anti-cell death agent of the present invention provides
It can inhibit cis-derived cell death and can be a therapeutic agent for hepatitis and the like derived from cell death.

[Brief description of the drawings]

FIG. 1 is a graph showing the survival rate at the time of passage showing that cell death can be inhibited by DEVD-CHO.

[Sequence list] SEQ ID NO: 1 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Hypothetical sequence: Yes Origin: Chemical synthesis Sequence features Other information: C-terminal Asp Modified with aldehyde. Sequence Tyr Val Ala Asp SEQ ID NO: 2 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Hypothetical sequence: Yes Origin: Chemical synthesis Sequence features Other information: C-terminal Asp Has been modified with an aldehyde. Sequence Asp Glu Val Asp SEQ ID NO: 3 Sequence Length: 4 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Hypothetical Sequence: Yes Origin: Chemical Synthesis Sequence Features Other Information: C-terminal Asp Has been modified with an aldehyde. Sequence Glu Ser Met Asp

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/55 ACS A61K 37/64 ABR AED ABS 45/00 ACJ C07K 5/107 ZNA ACS 5/113 AED

Claims (11)

[Claims] 1. Human caspase
And / or an anti-cell death agent containing a substance that inhibits a human caspase activity inducer. 2. The human caspase-1 (Caspas)
e-1), human Caspase-3
3) An anti-cell death agent comprising a substance that inhibits at least one of the human caspase-3 activity inducers. 3. A human caspase.
And / or anti-cells containing an inhibitory substance, wherein the inhibition of the human caspase activity inducer is due to deprocessing in the process of becoming a human caspase or / and an active form of the human caspase activity inducer. Death killer. 4. A human caspase-3 (Caspas).
e-3) Inhibition of the activity inducer is caused by human caspase-
An anti-cell killing agent containing an inhibitory substance, which is due to deprocessing in the process of becoming an active form of 3 (Caspase-3) activity inducer. 5. An anti-cell death agent comprising a modified protein containing an amino acid sequence near the caspase processing site. 6. A member selected from the group consisting of the following amino acid sequences:
An anti-cell death agent containing one or more aldehyde-modified proteins. Tyrosine-valine-alanine-aspartic acid, aspartic acid-glutamic acid-valine-aspartic acid, and glutamic acid-serine-methionine-aspartic acid. 7. An anti-cell death agent comprising an aldehyde-modified protein whose amino acid sequence is glutamic acid-serine-methionine-aspartic acid. 8. A therapeutic agent for diseases caused by cell death, comprising the anti-cell death agent according to claim 1 as an active ingredient. 9. The disease according to claim 8, wherein the disease caused by cell death is selected from the group consisting of hepatitis, ischemic brain disease, Alzheimer's disease, bone marrow toxicity and diarrhea, which are side effects due to administration of an antitumor agent. A drug for treating diseases caused by cell death. 10. A therapeutic agent for hepatitis caused by cell death, comprising the anti-cell death agent according to any one of claims 1 to 7 as an active ingredient. 11. A therapeutic agent for hepatitis caused by cell death, comprising an aldehyde-modified protein having an amino acid sequence of glutamic acid-serine-methionine-aspartic acid.