U.S. patent application number 17/284824 was filed with the patent office on 2021-12-16 for antipsychotic and use thereof.
The applicant listed for this patent is NATIONAL UNIVERSITY CORPORATION TOKAI NATIONAL HIGHER EDUCATION AND RESEARCH SYSTEM. Invention is credited to Yuko ARIOKA, Itaru KUSHIMA, Daisuke MORI, Taku NAGAI, Norio OZAKI, Kiyofumi YAMADA.
Application Number | 20210386755 17/284824 |
Document ID | / |
Family ID | 1000005825667 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210386755 |
Kind Code |
A1 |
OZAKI; Norio ; et
al. |
December 16, 2021 |
ANTIPSYCHOTIC AND USE THEREOF
Abstract
An object of the present invention is to provide a novel
therapeutic agent for a mental disorder with abnormal
neurodevelopment as a pathological condition. The present invention
provides an antipsychotic drug containing a Rho kinase inhibitor as
an active ingredient. The antipsychotic drug can be used, for
example, in the treatment of schizophrenia, autism spectrum
disorder, bipolar disorder, or depression.
Inventors: |
OZAKI; Norio; (Nagoya-shi,
Aichi, JP) ; YAMADA; Kiyofumi; (Nagoya-shi, Aichi,
JP) ; NAGAI; Taku; (Nagoya-shi, Aichi, JP) ;
MORI; Daisuke; (Nagoya-shi, Aichi, JP) ; ARIOKA;
Yuko; (Nagoya-shi, Aichi, JP) ; KUSHIMA; Itaru;
(Nagoya-shi, Aichi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL UNIVERSITY CORPORATION TOKAI NATIONAL HIGHER EDUCATION AND
RESEARCH SYSTEM |
Nagoya-shi |
|
JP |
|
|
Family ID: |
1000005825667 |
Appl. No.: |
17/284824 |
Filed: |
September 10, 2019 |
PCT Filed: |
September 10, 2019 |
PCT NO: |
PCT/JP2019/035488 |
371 Date: |
April 13, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/551 20130101;
A61P 25/18 20180101 |
International
Class: |
A61K 31/551 20060101
A61K031/551; A61P 25/18 20060101 A61P025/18 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 15, 2018 |
JP |
2018-194682 |
Claims
1. An antipsychotic drug comprising a Rho kinase inhibitor or a
pharmacologically acceptable salt thereof as an active
ingredient.
2. The antipsychotic drug according to claim 1, which normalizes
dysfunction of ARHGAP10.
3. The antipsychotic drug according to claim 1, wherein the Rho
kinase inhibitor is fasudil or ripasudil.
4. The antipsychotic drug according to claim 1, which is for use in
the treatment of a mental disorder with abnormal neurodevelopment
as a pathological condition.
5. The antipsychotic drug according to claim 4, wherein the mental
disorder is caused by dysfunction of ARHGAP10.
6. The antipsychotic drug according to claim 4, wherein the mental
disorder is schizophrenia, autism spectrum disorder, bipolar
disorder, or depression.
7. A method for treating a mental disorder with abnormal
neurodevelopment as a pathological condition, the method comprising
the step of administering a therapeutically effective amount of a
Rho-kinase inhibitor to a patient in need of treatment or
prevention.
8. The method according to claim 7, wherein the mental disorder is
caused by dysfunction of ARHGAP10.
9. The method according to claim 7, wherein the mental disorder is
schizophrenia, autism spectrum disorder, bipolar disorder, or
depression.
10. The antipsychotic drug according to claim 2, wherein the Rho
kinase inhibitor is fasudil or ripasudil.
11. The method according to claim 8, wherein the mental disorder is
schizophrenia, autism spectrum disorder, bipolar disorder, or
depression.
Description
TECHNICAL FIELD
[0001] The present invention relates to an antipsychotic drug (a
therapeutic agent for a mental disorder). More specifically, the
present invention relates to a therapeutic agent for a mental
disorder with abnormal neurodevelopment as a pathological
condition, and its applications, etc.
CROSS REFERENCE TO RELATED APPLICATIONS
[0002] This application is a U.S. National Stage application of
PCT/JP2019/035488 filed 10 Sep. 2019, which claims priority to
Japanese Patent Application No. 2018-194682 filed on Oct. 15, 2018,
the disclosure of which is incorporated herein by reference in its
entirety.
Background Art
[0003] Schizophrenia is a mental disorder that shows positive
symptoms such as hallucination-delusion, negative symptoms
characterized by poor speech and facial expression and lack of
motivation, and cognitive dysfunction in memory and concentration,
etc. The prevalence of schizophrenia is about 1% of the general
population. In Japan alone, about 700,000 patients with
schizophrenia are undergoing treatment, but there are many
intractable cases for which current treatment is not sufficiently
effective. As a result, 153,000 of the patients in treatment are
forced to be hospitalized in psychiatric beds (2017). In addition,
the life expectancy of schizophrenic patients is 10 to years
shorter than for the general population. Taking the above into
consideration, the economic loss caused by schizophrenia in Japan
is calculated to be 2.8 trillion yen every year (2013).
[0004] Since cardiovascular disease is a common cause of early
death in schizophrenia, molecules involved in both the brain and
the heart are assumed to play a role in the pathological condition
of schizophrenia. The development of a therapeutic agent with
excellent effects and fewer side effects on the cardiovascular
system or the like is long awaited. However, the details of the
pathology of schizophrenia are still unknown, and the development
of pathology-based treatment agents has not progressed.
[0005] Although the pathology of schizophrenia is still unknown,
epidemiological studies have shown that the involvement of genetic
factors in its onset is higher than in other mental disorders. A
research group of the present inventors conducted genomic copy
number variation (CNV) analysis in Japanese schizophrenia patients
and identified rare CNVs involved in the onset of schizophrenia in
9% of all patients (Non-patent Literature (NPL) 1). As a result of
further analysis, the present inventors identified CNVs in the
ARHGAP10 gene, which belongs to the RhoGAP family associated with
neurodevelopment and cardiovascular function, in five patients
(four deletions and one duplication) and found that the CNVs are
statistically associated with the onset of schizophrenia. One of
the patients with a deletion in the ARHGAP10 gene had a single
nucleotide variant (SNV) that causes an amino acid substitution
(Ser490.fwdarw.Pro) in the RhoGAP domains of alleles simultaneously
(FIG. 1A).
[0006] ARHGAP10 protein is a GTPase-activating protein that has the
function of inactivating low-molecular-weight G proteins, such as
RhoA and Cdc42, is widely distributed in the body, including the
brain and cardiac muscle, and plays an important role in the
regulation of various physiological functions, such as actin
cytoskeleton regulation through the regulation of the activity of
effector Rho kinase or the like. It is suggested that an decrease
of the ARHGAP10 expression level due to deletion of the ARHGAP10
gene increases active forms of RhoA and Cdc42 in cells, thus
increasing the activity of Rho kinase. The present inventors
examined the influence of SNV in the RhoGAP domain identified in
one schizophrenic patient in an in vitro binding experiment. As a
result, the inventors found that the SNV mutant lost its ability to
bind to RhoA and clarified that the ARHGAP10 SNV mutant cannot
inactivate RhoA as a substrate (FIG. 1B).
CITATION LIST
Non-Patent Literature (NPL)
[0007] NPL 1: Kushima I. et al., High-resolution copy number
variation analysis of schizophrenia in Japan. Mol Psychiatry 22:
430-440, 2017
SUMMARY OF INVENTION
Technical Problem
[0008] In view of the above background, an object of the present
invention is to provide a novel therapeutic agent for mental
disorders with abnormal neurodevelopment as a pathological
condition, such as schizophrenia.
Solution to Problem
[0009] The present inventors conducted further research to develop
a novel therapeutic agent based on the pathology of schizophrenia.
As a result of detailed experiments using model mice and
dopaminergic neurons derived from patient-derived iPS cells, it was
shown that Rho kinase inhibitors exhibit excellent antipsychotic
effects and are useful as a novel therapeutic agent based on the
pathology of schizophrenia. On the other hand, as a result of
considering experimental results as well as known genomic
variations associated with various mental disorders and the scope
of application of existing schizophrenia drugs (e.g., the fact that
schizophrenia drugs are also applied to other mental disorders,
such as bipolar disorder), Rho kinase inhibitors were reasonably
expected to be useful as a novel therapeutic agent for various
mental disorders with abnormal neurodevelopment as a pathological
condition (e.g., schizophrenia, autism spectrum disorder, bipolar
disorder, and depression).
[0010] The following inventions are based on the above results and
considerations.
[1] An antipsychotic drug comprising a Rho kinase inhibitor or a
pharmacologically acceptable salt thereof as an active ingredient.
[2] The antipsychotic drug according to [1], which normalizes
dysfunction of ARHGAP10. [3] The antipsychotic drug according to
[1] or [2], wherein the Rho kinase inhibitor is fasudil or
ripasudil. [4] The antipsychotic drug according to any one of [1]
to [3], which is for use in the treatment of a mental disorder with
abnormal neurodevelopment as a pathological condition. [5] The
antipsychotic drug according to [4], wherein the mental disorder is
caused by dysfunction of ARHGAP10. [6] The antipsychotic drug
according to [4] or [5], wherein the mental disorder is
schizophrenia, autism spectrum disorder, bipolar disorder, or
depression. [7] A method for treating a mental disorder with
abnormal neurodevelopment as a pathological condition, the method
comprising the step of administering a therapeutically effective
amount of a Rho kinase inhibitor to a patient in need of treatment
or prevention. [8] The therapeutic method according to [7], wherein
the mental disorder is caused by dysfunction of ARHGAP10. [9] The
therapeutic method according to [7] or [8], wherein the mental
disorder is schizophrenia, autism spectrum disorder, bipolar
disorder, or depression.
BRIEF DESCRIPTION OF DRAWINGS
[0011] FIG. 1 shows a single nucleotide polymorphism of ARHGAP10
identified in a patient (Ser490.fwdarw.Pro) (A) and its effect (B).
The ARHGAP10 SNV mutant had reduced binding ability to RhoA.
[0012] FIG. 2 shows morphological abnormality in patient-type
ARHGAP10 mutant mouse neurons (A) and patient iPS cell-derived
neurons (B). The length of neurites and the number of branches
decreased in neurons of the primary culture of patient-type
ARHGAP10 mutant mice (KI/NHEJ). In ARHGAP10 mutant patient (case
No. 4) iPS cell-derived dopamine neurons, the length of neurites
and the number of branches were reduced as compared with those in
healthy subjects (the control).
[0013] FIG. 3 shows an experimental scheme (A) and plasma fasudil
concentrations (B) and (D) and brain fasudil concentrations (C) and
(E).
[0014] FIG. 4 shows an experimental scheme (A) and effects of
fasudil on spontaneous locomotor activity (B)-(C).
[0015] FIG. 5 shows meth-induced visual discrimination learning
impairment in C57BL/6 mice (wild type) and ARHGAP10 mutant mice
(A), and experimental scheme (B), and ameliorating effects of
fasudil (C)-(D).
[0016] FIG. 6 shows an experimental scheme (A) and inhibitory
effects of fasudil on hypermobility in MK-801-treated mice (B).
[0017] FIG. 7 shows an experimental scheme (A) and ameliorating
effects of fasudil on social behavior disorder in MK-801-treated
mice (B).
[0018] FIG. 8 shows an experimental scheme (A) and ameliorating
effects of fasudil on visual discrimination learning impairment in
MK-801-treated mice (B)-(E).
[0019] FIG. 9 shows examples of automated detection of
neurites.
[0020] FIG. 10 shows examination of effects of Rho kinase
inhibitors using human iPS cell-derived neurons.
DESCRIPTION OF EMBODIMENTS
[0021] A first aspect of the present invention relates to an
antipsychotic drug. The term "antipsychotic drug" refers to a
pharmaceutical agent that has a therapeutic or prophylactic effect
on mental disease or mental disorder. Therapeutic effects include,
for example, alleviating (ameliorating) conditions characteristic
of mental disease/mental disorder or accompanying symptoms, and
preventing or delaying the worsening of symptoms. The latter can be
regarded as a prophylactic effect because it prevents aggravation
of illness. Thus, therapeutic effects and prophylactic effects are
partially overlapping concepts; it is difficult to clearly
distinguish one from the other, and there is little benefit in
doing so. A typical prophylactic effect is to prevent or delay the
recurrence (onset) of symptoms that are characteristic of a mental
disease or mental disorder. As long as a drug (a substance) has
some therapeutic or prophylactic effect, or both effects, on a
mental disease or mental disorder, the drug (the substance) is
categorized as an antipsychotic drug.
[0022] The antipsychotic drug of the present invention contains a
Rho kinase inhibitor as an active ingredient. Rho kinase inhibitors
selectively inhibit Rho kinase (ROCK), which is one of the protein
phosphatases. Rho kinase, which is serine/tyrosine kinase, is a
target of the low-molecular-weight GTP-binding protein Rho and is
involved in various cellular functions. Examples of Rho kinase
inhibitors include fasudil, hydroxyfasudil, ripasudil, netarsudil,
thiazovivin, (1-benzylpyrrolidin-3-yl)-(1H-indazol-5-yl)amine,
(1-benzylpiperidin-4-yl)-(1H-indazol-5-yl)amine,
N-[2-(4-fluorophenyl)-6,7-dimethoxy-4-kinazolinyl]-N-(1H-indazol-5-yl)ami-
ne,
N-4-(1H-indazol-5-yl)-6,7-dimethoxy-N-2-pyridin-4-yl-quinazoline-2,4-d-
iamine, 4-methyl-5-(2-methyl-[1,4]diazepan-1-sulfonyl)isoquinolin,
Y-27632
((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide),
RKI 1447
(N-[(3-hydroxyphenyl)methyl]-N'-[4-(4-pyridinyl)-2-thiazolyl]
urea), GSK429286A
(N)-(6-fluoro-1H-indazol-5-yl)-1,4,5,6-tetrahydro-2-methyl-6-oxo-4-[4-(tr-
ifluoromethyl)phenyl]-3-pyridinecarboxamide), Y-30141
(4-(1-aminoethyl)-N-(1H-pyrrolo
(2,3-b)pyridin-4-yl)cyclohexanecarboxamide), and Y-39983
(4-[(1R)-1-aminoethyl]-N-1H-pyrrolo[2,3-b]pyridin-4-ylbenzamide
dihydrochloride). In the present invention, fasudil and ripasudil
are particularly preferable Rho kinase inhibitors. A pharmaceutical
preparation containing fasudil hydrochloride hydrate
(hexahydro-1-(5-isoquinolinesulfonyl)-1H-1,4-diazepine
monohydrochloride hemihydrate) as an active ingredient (Elill,
registered trademark) is applied to improve cerebral vasospasm and
associated cerebral ischemic symptoms after subarachnoid hemorrhage
surgery vasoconstriction, whereas a pharmaceutical preparation
containing lipasudil hydrochloride hydrate
(4-fluoro-5-{[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl}isoquinoline
monohydrochloride dihydrate) as an active ingredient (Granatek
(registered trademark)) is applied to the treatment of glaucoma and
ocular hypertension.
[0023] The active ingredients of the antipsychotic drugs of the
present invention can take various forms, such as salts, hydrates,
and solvates, as long as they are pharmacologically acceptable. For
example, various salts, such as salts with inorganic bases, salts
with organic bases, salts with inorganic acids, salts with organic
acids, salts with basic amino acids, and salts with acidic amino
acids, can be used. Examples of salts with inorganic bases include
alkali metal salts (e.g., sodium salts, potassium salts, etc.),
alkaline earth metal salts (e.g., calcium salts, magnesium salts,
etc.), aluminum salts, and ammonium salts. Examples of salts with
organic bases include salts with trimethylamine, triethylamine,
pyridine, picoline, ethanolamine, diethanolamine, triethanolamine,
dicyclohexylamine, and N,N'-dibenzylethylenediamine. Examples of
salts with inorganic acids include salts with hydrochloric acid,
hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and
the like. Examples of salts with organic acids include salts with
formic acid, acetic acid, trifluoroacetic acid, fumaric acid,
oxalic acid, tartaric acid, maleic acid, citric acid, succinic
acid, malic acid, methanesulfonic acid, benzenesulfonic acid,
p-toluenesulfonic acid, and the like. Examples of salts with basic
amino acids include salts with arginine, lysine, ornithine, and the
like. Examples of salts with acidic amino acids include salts with
aspartic acid, glutamic acid, and the like. These salts can be
prepared by conventional means. The above examples should not be
used for limited interpretation of "pharmacologically acceptable
salts." That is, "pharmacologically acceptable salt" should be
interpreted in a broad sense and is a term that includes various
salts.
[0024] The active ingredient of the antipsychotic drug of the
present invention may be in the form of a prodrug. A "prodrug" is a
compound in a form that is inactive or has low activity, and that
is converted to an active substance and exhibits pharmaceutical
efficacy when administered to a living body. For example, prodrugs
are used for the purpose of improving bioavailability and reducing
side effects. Examples of prodrugs include compounds obtained by
subjecting an amino, sulfide, or like group of an original
pharmaceutical active substance to sulfonylation, acylation,
alkylation, phosphorylation, boration, carbonation, esterification,
amidation, urethanization, etc.
[0025] The antipsychotic drug of the present invention can also be
used in combination with one or more other antipsychotic drugs that
have a mechanism of action different from that of the Rho kinase
inhibitor (referred to below as "other active ingredients"). The
number of other active ingredients may be two or more. Examples of
other active ingredients include aripiprazole, asenapine,
olanzapine, quetiapine, clozapine, paliperidone, perospirone,
blonanserin, risperidone, chlorpromazine, levomepromazine,
perphenazine, fluphenazine, propericiazine, haloperidol,
bromperidol, spiperone, moperone, pimozide, sulpiride, sultopride,
tiapride, nemonapride, carpipramine, clocapramine, mosapramine, and
zolpidem.
[0026] The form for combined use is not limited. Examples include a
combination drug in which active ingredients are mixed together and
a kit comprising a first component containing a Rho kinase
inhibitor and a second component containing one or more other
active ingredients (when two or more other active ingredients are
used, various modes can be used, which include for example, a mode
in which all of the other active ingredients can be combined into a
second component, and a mode in which the two or more other active
ingredients constitute individual components). When a kit is used,
each component is administered at least once during the treatment
period. The administration schedule for each component can be
individually set. It is also possible to administer both components
simultaneously. "Simultaneously" as referred to herein does not
require strict simultaneity. Accordingly, the concept of
simultaneity includes not only cases in which the both components
are administered under conditions in which there is no time
difference in their administration, such as when both components
are mixed and then administered to the target, but also cases in
which both components are administered under conditions in which
there is no substantial time difference in their administration,
such as when one component is first administered and then the other
is administered immediately after the first administration.
[0027] Not to be confined to theory, but in view of the
experimental results described below (in the Examples section), the
antipsychotic drug of the present invention can provide its
pharmaceutical efficacy (through normalization of dysfunction of
ARHGAP10). Therefore, typically, mental disorders caused by
dysfunction of ARHGAP10 (in other words, mental disorders caused by
mutation in the ARHGAP10 gene) are targets of the treatment or
prevention by the antipsychotic drug of the present invention
(referred to below as "target disorder"). The target disorder is
preferably a mental disorder with abnormal neurodevelopment as a
pathological condition. Examples of mental disorders with abnormal
neurodevelopment as a pathological condition include schizophrenia,
autism spectrum disorder, bipolar disorder, and depression.
[0028] The antipsychotic drug of the present invention can be
formulated according to usual methods. When the antipsychotic drug
is formulated, other pharmaceutically acceptable components (e.g.,
carriers, excipients, disintegrants, buffers, emulsifiers,
suspending agents, soothing agents, stabilizers, preservatives,
antiseptics, surfactants, lubricants, diluents, coating agents,
sugar coating agents, taste and odor masking agents, emulsifiers,
solubilizers, dispersants, pH-adjusting agents, isotonic agents,
solubilizing agents, flavors, coloring agents, dissolution aids,
and saline) can be included. Examples of excipients that can be
used include lactose, starch, sorbitol, D-mannitol, white sugar,
and the like. Examples of disintegrants that can be used include
starch, carboxymethyl cellulose, calcium carbonate, and the like.
Examples of buffers that can be used include phosphates, citrates,
acetates, and the like. Examples of emulsifiers that can be used
include gum arabic, sodium alginate, tragacanth, and the like.
Examples of suspending agents that can be used include glyceryl
monostearate, aluminum monostearate, methyl cellulose,
carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl
sulfate, and the like. Examples of soothing agents that can be used
include benzyl alcohol, chlorobutanol, sorbitol, and the like.
Examples of stabilizers that can be used include propylene glycol,
ascorbic acid, and the like. Examples of preservatives that can be
used include phenol, benzalkonium chloride, benzyl alcohol,
chlorobutanol, methylparaben, and the like. Examples of antiseptic
agents that can be used include benzalkonium chloride,
p-hydroxybenzoate, chlorobutanol, and the like.
[0029] There are no limitations on the dosage form used in
formulation. Examples of usable dosage forms include tablets,
powders, fine granules, granules, capsules, syrups, liquids,
suspensions, emulsions, jellies, injections, external preparations,
inhalants, nasal drops, eye drops, and suppositories. The
antipsychotic drug of the present invention contains the active
ingredient in an amount necessary to achieve the expected
therapeutic (or preventive) effect (i.e., a therapeutically
effective amount). The amount of active ingredient in the
antipsychotic drug of the present invention generally varies
according to the dosage form, but the amount of active ingredient
is set within the range of, for example, about 0.01% to 95% by
weight so that the desired dose can be achieved.
[0030] The antipsychotic drug of the present invention is applied
to the subject by oral or parenteral administration (e.g., through
an intravenous, intra-arterial, subcutaneous, intramuscular, or
intraperitoneal injection; or transdermal, intranasal,
transmucosal, or like administration) depending on its dosage form.
These routes of administration are not mutually exclusive, and two
or more of any routes selected can be used in combination (for
example, an intravenous injection can be performed simultaneously
or at a specific time after the oral administration). Instead of
systemic administration, local administration can also be used. The
administration can also be performed using a drug delivery system
(DDS) so that the active ingredient is delivered specifically to
the target tissue.
[0031] A further aspect of the present invention is a method for
treating a mental disease or mental disorder using the
antipsychotic drug of the present invention (the concept of
therapeutic method includes prophylactic treatment). The
therapeutic method of the present invention includes the step of
administering the antipsychotic drug of the present invention to a
patient in need of treatment or prevention. The "patient in need of
treatment or prevention" includes, for example, a person who is
diagnosed as having, or is suspected of having, a mental disease or
mental disorder (target disorder) that is the target of treatment
or prevention; a person who presents characteristic pathological
conditions or symptoms or shows signs of pathological conditions or
symptoms; a person who shows signs of relapse; and the like. The
dose of antipsychotics generally varies depending on the patient's
symptoms, age, gender, weight, etc. However, a person skilled in
the art would be able to set an appropriate dose. For example, an
administration schedule of once to several times a day, once every
two days, or once every three days can be used. When the
administration schedule is set, the patient's symptoms and the
duration of effect of the active ingredient can be taken into
consideration.
EXAMPLES
1. Background and Purpose
[0032] In order to clarify the pathophysiological significance of
ARHGAP10 gene mutation based on genetic-statistical and biochemical
findings as well as previous reports and research results (see the
Background Art section), the present inventors newly created
genetically modified mice by genome editing technology using the
genotype of a patient who had both ARHGAP10 deletion and SNV as a
model. Behavioral analysis of the ARHGAP10-deficient/SNV model mice
showed that the mice had a schizophrenia-like phenotype. In vitro
morphological analysis of cultured neurons showed a reduction in
neurite branching of the cultured neurons and a tendency toward
immature neurodevelopment (FIG. 2, panel A). The results of the
phenotypic analysis using the animal models also strongly suggested
the possibility that the ARHGAP10 gene is associated with the onset
of schizophrenia.
[0033] In addition, using iPS cells established from a patient with
ARHGAP10 mutation (deletion/SNV), the present inventors analyzed
the influence of the mutation on human neurons. As in the mice, the
length of neurites and the number of neurite branches were reduced
in human neurons with ARHGAP10 mutation (FIG. 2, panel B).
[0034] These findings suggest that mutations in the ARHGAP10 gene
identified in patients may be involved in pathogenesis of
schizophrenia by causing constant Rho kinase activation. Therefore,
the present inventors decided to examine whether an Rho kinase
inhibitor is applicable as an antipsychotic drug by using model
mice and patient-derived iPS cells.
[0035] Stimulants that stimulate dopamine receptors, such as
methamphetamine (meth), are known to cause hallucinations and
delusional symptoms similar to those of schizophrenia (Lieberman et
al., 1987). Further, since antipsychotic drugs generally have
effects of blocking dopamine D2 receptors, excess dopamine
transmission is considered to be involved in the onset of
schizophrenia (Howes et al., 2015). However, since negative
symptoms are resistant to conventional antipsychotics and are
rarely observed in methamphetamine psychosis induced by
methamphetamine or amphetamine, a mechanism different from
increased dopamine transmission is suggested to be involved in the
onset of negative symptoms. On the other hand, phencyclidine (PCP),
which is an N-methyl-D-aspartate (NMDA) receptor antagonist,
induces schizophrenia-like positive and negative symptoms and
cognitive dysfunction in healthy subjects, and also exacerbates
psychiatric symptoms in schizophrenic patients (Javitt, D. C. et
al, 1991; Volkow, N. D. et al. 1992). Ketamine, which is an
anesthetic agent, also induces schizophrenia-like symptoms in
healthy subjects (Krystal et al., 1994). Accordingly, glutamatergic
neurotransmission is considered to play an important role in the
pathogenesis, pathophysiology, and clinical conditions of
schizophrenia (Tamminga et al., 1995). MK-801 is a non-competitive
antagonist of the NMDA receptor, which is one of the glutamate
receptors. MK-801 has been used as a pharmacological model of
schizophrenia because experimental animals treated with MK-801
exhibit behavioral abnormality that resembles positive and negative
symptoms of schizophrenia and cognitive dysfunction, such as
hyperactivity, social behavior disorder, and cognitive impairment
(Neill et al., 2010; Cadinu et al., 2017). In order to develop a
method for treating schizophrenia based on ARHGAP10 mutation, which
is involved in the onset of mental disorder, the present inventors
investigated the effect of an Rho kinase inhibitor, fasudil, on
behavioral abnormalities observed in genetic mutant model mice with
a patient-type ARHGAP10 mutation (deletion/SNV) and in
pharmacological model mice produced by administration of meth and
MK-801 based on the dopamine and glutamate hypothesis.
2. Method
2.1. Experimental Animal
[0036] Genetically mutant model mice with a patient-type ARHGAP10
mutation were originally created by the TALEN method (data
unpublished). For the measurement of blood fasudil and brain
fasudil concentrations and creation of pharmacological model mice
treated with meth or MK-801, seven-week-old male C57BL/6J slc mice
(Japan Slc, Shizuoka, Japan) were used. The animals were kept at
room temperature of 23.+-.1.degree. C., humidity of 50.+-.5%,
light/dark cycle with a light period of 9:00-21:00, and were
allowed free access to water and food.
2.2. Reagent
[0037] Fasudil hydrochloride hydrate was provided by Asahi Kasei
Pharma Corporation (Tokyo, Japan). Methamphetamine hydrochloride
(produced by Sumitomo Dainippon Pharma Co., Ltd., Osaka, Japan) and
(+)-MK-801 maleate (Sigma, St. Louis, Mo., USA) were purchased.
Measurement of Fasudil in Blood and Brain
2.3.1. Preparation of Blood Samples
[0038] The blood samples obtained were treated with heparin and
centrifuged (4.degree. C., 1000.times.g, 10 min). The supernatant
was collected and then centrifuged again (4.degree. C., 2000 rpm,
20 min). The supernatant was stored at -80.degree. C. as a plasma
sample. After the plasma sample was thawed at room temperature, 800
.mu.l of 1% formic-acid-methanol solution and 160 .mu.l of an
internal standard solution (125 ng/ml
1-(5-isoquinolinesulfonyl)piperazine, dihydrochloride, produced by
Sigma-Aldrich) were added to 200 l of the thawed plasma sample, and
the resulting mixture was centrifuged (4.degree. C., 1000.times.g,
5 min). The supernatant was collected and then subjected to
solid-phase extraction using Phree-SPE (Phenomenex, Torrance,
Calif., USA). The extract was evaporated to dryness at 40.degree.
C. The residue was re-dissolved with pure water and then
centrifuged (room temperature, 13000 rpm, 5 min). Using 20 .mu.l of
the collected supernatant, the fasudil content of the supernatant
was measured using a liquid chromatograph tandem mass spectrometer
(LC/MS/MS).
2.3.2. Preparation of Brain Samples
[0039] Brains removed from mice were washed with saline, and brain
regions except for the cerebellum were stored at -80.degree. C. 20
.mu.l of a 1% formic acid-methanol solution and 4 .mu.l of an
internal standard solution were added to 1 mg of each brain sample,
and the brain tissue was pulverized using an ultrasonic homogenizer
and centrifuged (4.degree. C., 10000 rpm, 5 min). The supernatant
was collected, and then the extract was evaporated to dryness at
40.degree. C. The residue was re-dissolved in pure water and then
centrifuged (room temperature, 13000 rpm, 5 min). Using 20 .mu.l of
the collected supernatant, the fasudil content of the supernatant
was measured using LC/MS/MS.
2.3.3. LC/MS/MS Measurement
[0040] An ACQUITY UPLC CSH C18 column (100 mm.times.2.1 mm, waters,
Milford, Mass., USA) was used as a separation column and heated to
35.degree. C. As mobile phases, solvent A (5 mM ammonium acetate
aqueous solution) and solvent B (methanol) were used. The
conditions were set to % B (5-80/90 sec) at a flow rate of 0.2
ml/min. Samples were ionized by ESI in positive ion mode, and
292.13 m/z.fwdarw.98.706 m/z, 292.13 m/z.fwdarw.98.703 m/z, and
292.13 m/z.fwdarw.69.5 m/z were detected as fasudil by the multiple
reaction monitoring measurement method. Based on the obtained peak
areas of fasudil and the internal standard substance, the peak area
ratio of fasudil to the internal standard substance was calculated.
The plasma fasudil concentration and the brain fasudil
concentration were calculated using calibration curves. The
calibration curve prepared by using a standard solution of fasudil
dissolved in fresh human frozen plasma showed a correlation
coefficient of 0.99 in the concentration range of 1 to 1000 ng/mL,
and a diurnal variation and a day-to-day variation of less than 5%.
The calibration curve prepared by using a fasudil standard solution
produced from a mouse brain homogenate solution showed a
correlation coefficient of 0.99 in the concentration range of 0.03
to 3 ng/brain tissue weight (mg), and a diurnal variation and a
day-to-day variation of less than 5%.
2.4. Behavioral Tests
2.4.1. Spontaneous Locomotor Activity
[0041] Mice were placed one by one in measurement cages (25
cm.times.30.times.cm.times.18 cm). Immediately after the placement,
spontaneous locomotor activity of mice was measured using a
measurement device equipped with infrared light (Brain Science
Idea, Osaka, Japan) (Ibi et al., 2010).
2.4.2. Visual Discrimination Test
[0042] A touchscreen operant device (Phenosys, Berlin, Germany;
Brain Science Idea, Osaka, Japan) was used. The front surface of a
touch screen monitor (5.times.4 inches) installed inside the device
was equipped with an acrylic plate with two square holes, through
which mice could touch the touch screen monitor. The back surface
of the touch screen monitor was provided with a water supply nozzle
for providing milk as a reward. The mice were allowed to consume
food and water only for two hours a day for one week before the
start of the behavioral experiment, and food restriction was
continued throughout the training period. Mice were trained to
obtain food by touching a white square presented on the touch
screen. Mice that achieved a correct response rate of 75% or more
for two consecutive days were used in the subsequent visual
discrimination experiment. In the experiment, a fan-shaped pattern
and a ball-shaped pattern were presented simultaneously in the left
and right holes of the screen, and a reward was given when one
specific pattern was touched, whereas no reward was given when the
other pattern was touched. During the test period, the patterns
were presented randomly on both sides, and the combination of
pattern and reward was fixed. Patterns were presented thirty times
in one test. The correct response rate, the test completion rate,
the latency to correct response, and the latency to obtain the
reward were recorded (Wulaer et al., 2018).
2.4.3 Social Behavior Test
[0043] Before conducting the experiment, test mice were kept in
breeding cages one by one for 2 days. On the third day, mice that
had been normally group-reared in another breeding cage were placed
as invading mice into the cages of the test mice, and social
behaviors of the test mice, such as tracking and sniffing, were
measured for 5 minutes (Ibi et al., 2010).
2.5 Analysis Using iPS Cells
[0044] 2.5.1 Neuron Induction from iPS Cells
[0045] Dopaminergic neurons were induced from iPS cells of healthy
subjects and patients with ARHGAP10 mutation. The induction method
was performed according to a method already published (Transl
Psychiatry. 2018 Jul. 19; 8(1): 129; and patent application
PCT/JP2018/015304). To give an overview, iPS cells were cultured in
an iPS cell medium supplemented with SB431542 (3 .mu.M), CHIR99021
(3 .mu.M), and dorsomorphin (3 .mu.M) for 7 days (day 0 to day 7).
The cultured cells were then dispersed with TrypLE.TM. select
(Thermo Fisher Scientific Inc.) and passed through a cell strainer.
The resulting cells were float-cultured for 2 weeks in a
neurosphere medium (a medium obtained by preparing a DMEM/F12
medium with 1.times.N2 supplement, 0.6% glucose,
penicillin/streptomycin, and 5 mM HEPES (MHM medium) and adding to
this medium 1.times.B27 supplement, 20 ng/ml bFGF, 10 ng/ml human
LIF, 10 .mu.M Y27632, 3 .mu.M CHIR99021, 2 .mu.M SB431542, 100
ng/ml FGF8, and 1 .mu.M plumorphamine). Neurospheres were thereby
formed (day 7 to day 21). GF8 and plumorphamine were added from day
10. The neurospheres were collected on day 14, dispersed to form
single cells, and then float-cultured again to re-form neurospheres
(secondary neurospheres). On day 21, the neurospheres were seeded
onto a culture dish coated with Matrigel.TM. (BD) or with
poly-L-ornithine/laminin/fibronectin (poly-L-ornithine, laminin,
and fibronectin), and then cultured in a medium for dopamine
neurons (an MHM medium with B27 supplement, 10 .mu.M DAPT, 20 ng/ml
BDNF, 20 ng/ml GDNF, 0.2 mM ascorbic acid, 1 ng/ml TGF-.beta.3, and
0.5 mM dbcAMP) to induce differentiation into dopamine neutrons
(day 21 onward). All cultures were grown in a normal CO.sub.2
incubator (5% CO.sub.2; oxygen concentration: 18.5% to 19.5% (not
adjusted)).
2.5.2 Rho Kinase Inhibitors
[0046] Fasudil hydrochloride hydrate was provided by Asahi Kasei
Pharma (Tokyo, Japan). Y-27632 (produced by Wako Pure Chemical Co.,
Ltd.) and lipasudil (produced by Selec Chemicals, Inc.) were
purchased.
2.5.3 Measurement of Neurite Length
[0047] Dopaminergic progenitor cells were seeded onto a culture
dish coated with poly-L-ornithine/laminin/fibronectin
(poly-L-ornithine, laminin, and fibronectin), and an Rho kinase
inhibitor was added simultaneously. For the control (=0 .mu.M),
water, which was used as a solvent of the inhibitor, was added. The
length of neurites after 72 hours was automatically measured with
IncuCyte NeuroTrack (registered trademark).
3. Results and Discussion
3.1 Plasma Fasudil and Brain Fasudil Concentrations
[0048] In accordance with the experimental schedule shown in FIG.
3, panel A, fasudil was intraperitoneally administered into C57BL/6
mice, and plasma and brain samples were collected. The plasma
fasudil concentration in C57BL/6 mice intraperitoneally injected
with 10 mg/kg fasudil reached the highest value of about 1600 ng/ml
5 minutes after the administration and then decreased (FIG. 3,
panel B). The blood fasudil concentrations 5, 10 and 20 minutes
after the administration were higher than the Ki value of fasudil
for Rho-kinase of 104 ng/ml. The brain fasudil concentration showed
a high value of about 0.2 ng/mg tissue during a period from 5 to 20
minutes after the administration, and then decreased to the Ki
value of fasudil for Rho kinase of 0.104 ng/mg tissue 40 minutes
after the administration of fasudil (FIG. 3, panel C). The plasma
fasudil and brain fasudil concentrations 5 minutes after the
administration of fasudil showed an increase in a dose-dependent
manner, and the plasma fasudil concentration was higher than the Ki
value of fasudil for Rho kinase at a dose of 3 mg/kg or more,
whereas the brain fasudil concentration was higher than the Ki
value of fasudil for Rho kinase at a dose of 10 mg/kg or more (FIG.
3, panels D and E). These results suggest that when fasudil is
intraperitoneally administered at a dose of 10 mg/kg or more,
fasudil is present in the brain at a concentration that
sufficiently inhibits Rho kinase during a period from 5 to 40
minutes after administration.
3.2 Effect of Fasudil on Spontaneous Locomotor Activity
[0049] Saline or fasudil (20 or 30 mg/kg) was intraperitoneally
administered to C57BL/6 mice, and spontaneous locomotor activity of
the mice started to be measured immediately after the
administration (FIG. 4, panel A). As compared with the
saline-treated mice, the mice treated with 30 mg/kg of fasudil had
a significant decrease in spontaneous locomotor activity
immediately after the administration (FIG. 4, panel B). On the
other hand, the mice treated with 20 mg/kg of fasudil showed the
same level of spontaneous locomotor activity as the saline-treated
mice (FIG. 4, panel C). These results suggest that administration
of more than 30 mg/kg of fasudil to mice impairs locomotor
function.
3.3. Meth-Induced Visual Discrimination Learning Impairment in
C57BL/6 Mice (Wild Type) and ARHGAP10 Mutant Mice and Ameliorating
Effect of Fasudil
[0050] A research group of the present inventors previously found
that C57BL/6 mice (wild type) to which meth was administered in an
amount of 1.0 mg/kg 30 minutes before the test had visual
discrimination learning impairment (FIG. 5, panel A). The present
inventors also found that in ARHGAP10 mutant mice, meth-induced
visual discrimination learning impairment was induced by a lower
dose of meth (0.3 mg/kg) (FIG. 3, panel A). Accordingly, the
present inventors investigated ameliorating effects of fasudil on
meth-induced visual discrimination learning impairment observed in
both mice (FIG. 5, panel B). The saline-treated C57BL/6 mice had a
correct response rate of about 90% in the visual discrimination
learning test, whereas the 1.0 mg/kg meth-treated mice had a
correct response rate of about 70%, thus indicating learning
impairment. The meth-induced visual discrimination learning
impairment observed in C57BL/6 mice was improved by administration
of fasudil (10 mg/kg or 20 mg/kg) to the mice (FIG. 5, panel C).
Fasudil (20 mg/kg) also significantly improved the meth-induced
visual discrimination learning impairment observed in ARHGAP10
mutant mice (FIG. 5, panel D). These results show that fasudil
ameliorates the cognitive dysfunction exhibited by genetic animal
models with ARHGAP10 genomic mutation and in pharmacological animal
models using meth.
3.4. Inhibitory Effect of Fasdil on Hyperactivityin MK-801-Treated
Mice
[0051] Saline or fasudil (10 or 20 mg/kg) was intraperitoneally
administered to C57BL/6 mice. After 5 minutes, MK-801 (0.3 mg/kg)
was intraperitoneally administered. The amount of locomotor
activity started to be measured immediately after the
administration (FIG. 6, panel A). MK-801-induced hyperactivity was
significantly inhibited in mice treated with fasudil (20 mg/kg)
(FIG. 6, panel B). Accordingly, fasudil can be thought to inhibit
psychomotor excitation in animal models using MK-801.
Ameliorating Effect of Fasudil on Social Behavior Disorder in
MK-801-Treated Mice
[0052] Saline or MK-801 (0.1 mg/kg) was intraperitoneally
administered to C57BL/6 mice 30 minutes before the start of the
experiment. Five minutes before the start of the experiment, saline
or fasudil (5 or 10 mg/kg) was intraperitoneally administered, and
social behavior was measured. (FIG. 7, panel A). The time of social
behavior disorder was significantly reduced in the MK-801-treated
mice, as compared with the control mice (FIG. 7, panel B). The
impaired social behavior observed in MK-801-treated mice returned
to the same level as the social behavior in control mice by
administration of fasudil (10 mg/kg) (FIG. 7, panel B). These
results show that fasudil has an ameliorating effect on social
behavior disorders present in MK-801-treated animal models.
3.6. Ameliorating Effect of Fasudil on Visual Discrimination
Learning Impairment in MK-801-Treated Mice
[0053] Saline or MK-801 (0.1 mg/kg) was intraperitoneally
administered to C57BL/6 mice 30 minutes before the start of the
experiment. 5 minutes before the start of the experiment, saline or
fasudil (3, 10, or 20 mg/kg) was intraperitoneally administered to
the C57BL/6 mice, and a visual discrimination test was performed
(FIG. 8, panel A). The saline-treated C57BL/6 mice had a correct
response rate of 90% in the visual discrimination learning test,
whereas the MK-801-treated mice had a correct response rate of
about 70%, thus indicating learning impairment (FIG. 8, panel B).
The MK-801-induced visual discrimination learning impairment
observed in the C57BL/6 mice returned to the control level by
administration of fasudil (20 mg/kg) (FIG. 8, panel B). In this
test, none of the groups showed significant changes in correct
response rate, test completion rate, latency to correct response,
or latency to obtain the reward (FIG. 8, panels C to E).
Accordingly, fasudil was shown to improve cognitive dysfunction
exhibited by the MK-801 animal model without producing adverse
effects on attention or motivation.
3.7. Rho Kinase Inhibitor Addition Experiment Using Human iPS
Cells
[0054] Dopaminergic neurons were induced from iPS cells derived
from healthy subjects and patients with ARHGAP10 mutation. The
effects of Rho kinase inhibitors (fasudil, repasudil, Y-27632) were
examined. The length of neurites was used as an index of
evaluation. The length of neurites was automatically detected and
measured with IncuCyte (registered trademark) NeuroTrack (produced
by Essen Bioscience). FIG. 9 shows examples of neurite
detection.
[0055] The length of neurites (mm) per mm.sup.2 was calculated for
each field of view, and analysis was performed for 18 fields of
view in each group. The length of neurites in neurons of the
ARHGAP10 patients was shorter than in the healthy subjects.
However, the addition of each Rho kinase inhibitor tended to
improve the length of neurites in a concentration-dependent manner
(FIG. 10; the graph shows mean.+-.SD).
4. Summary
[0056] Table 1 shows the results obtained in this study. These
results clearly show that the Rho kinase inhibitor fasudil has an
ameliorating effect on abnormal behaviors observed in the gene
mutation models with a patient-type ARHGAP10 mutation
(deletion/SNV), dopamine hypothesis models, and glutamate
hypothesis models. Also observed were improving effects
(improvement in protrusion length) of three types of Rho kinase
inhibitors, including fasudil, on neurons derived from patient iPS
cells.
[0057] The above suggests that the Rho kinase inhibitors can be
useful as novel therapeutic agents based on the pathology of
schizophrenia. Further, in view of the fact that the same genome
mutation (especially a neurodevelopment-related gene) is a risk for
not only schizophrenia, but also autism spectrum disorder and
bipolar disorder, and that existing antipsychotic drugs for
schizophrenia are also used for the treatment of autism spectrum
disorder, bipolar disorder, and depression, Rho kinase inhibitors
are expected to be useful as therapeutic agents not only for
schizophrenia but also for autism spectrum disorder, bipolar
disorder, and depression, which are associated with
neurodevelopment.
TABLE-US-00001 TABLE 1 Antipsychotic effect of Fasudil Fasudil dose
(mg/kg, intraperitoneal administration) Animal model Behavioral
test 3 5 10 20 30 Patient-type ARHGAP Meth-induced Improved N.D.
Improved Improved N.D. mutant mice (0.3 mg/kg) (genetic mutation
visual discrimination impairment model) Meth-treated mice
Meth-induced Improvement N.D. Improved Improved N.D. (dopamine
hypothesis (1.0 mg/kg) tendency model) visual discrimination
impairment MK-801 treated mice Spontaneous locomotor activity N.D.
N.D. N.D. .+-. Decreased (glutamate hypothesis MK-801-induced
hypermobility N.D. N.D. .+-. Improved N.D. model) MK-801-induced
N.D. .+-. Improved .+-. N.D. social behavior disorder
MK-801-induced (1.0 mg/kg) .+-. N.D. Tendency of Improved N.D.
visual discrimination disorder improvement N.D. : No data .+-.: No
change
REFERENCES
[0058] Cadinu D. et al., NMDA receptor antagonist rodent models for
cognition in schizophrenia and identification of novel drug
treatments, an update. Neuropharmacology pii:
S0028-3908(17)30584-1, 2017. [0059] Howes O., McCutcheon R, Stone
J., Glutamate and dopamine in schizophrenia: an update for the 21st
century. J Psychopharmacol 29:97-115, 2015. [0060] Ibi D. et al.,
Combined effect of neonatal immune activation and mutant DISC1 on
phenotypic changes in adulthood. Behav Brain Res 206:32-37, 2010.
[0061] Javitt D. C. et al., Recent advances in the phencyclidine
model of schizophrenia. Am J Psychiatry 148: 1301-1308, 1991.
[0062] Krystal J. H. et al., Subanesthetic effects of the
noncompetitive NMDA antagonist, ketamine, in humans.
Psychotomimetic, perceptual, cognitive, and neuroendocrine
responses. Arch Gen Psychiatry 51:199-214, 1994. [0063] Kushima I.
et al., High-resolution copy number variation analysis of
schizophrenia in Japan. Mol Psychiatry 22:430-440, 2017. [0064]
Lieberman J. A., Kane J. M., Alvir J., Provocative tests with
psychostimulant drugs in schizophrenia. Psychopharmacology
91:415-33, 1987. [0065] Neill J. C. et al., Animal models of
cognitive dysfunction and negative symptoms of schizophrenia: Focus
on NMDA receptor antagonism. Pharmacol Ther 128:419-432, 2010.
[0066] Tamminga C. A. et al., Glutamate pharmacology and the
treatment of schizophrenia: current status and future directions.
Int Clin Psychopharmacol 10:29-37, 1995. [0067] Volkow N. D. et
al., Neuropsychiatric disorders: investigation of schizophrenia and
substance abuse. Semin Nucl Med 22:254-267, 1992. [0068] Wulaer B.
et al., Repetitive and compulsive-like behaviors lead to cognitive
dysfunction in Disc1.sup..DELTA.2-3/.DELTA.2-3 mice. Genes Brain
Behav April 10: e12478, 2018.
INDUSTRIAL APPLICABILITY
[0069] The antipsychotic drug of the present invention can be used
as a pathology-based treatment agent for various mental disorders
with abnormal neurodevelopment as a pathological condition. That
is, since the present invention can be applied to the treatment of
schizophrenia as well as the treatment of autism spectrum disorder,
bipolar disorder, depression, etc., its utility value and clinical
significance is extremely great.
[0070] The present invention is not limited in any way to the above
explanation of the embodiments or the examples of the present
invention. Various modifications easily conceivable by a person
skilled in the art without departing from the scope of the claims
also fall within the scope of the present invention. The entire
contents of the literature, laid-open patent publications, patent
gazettes, and the like specified in the present specification are
incorporated herein by reference.
* * * * *