U.S. patent application number 14/647931 was filed with the patent office on 2015-12-10 for therapeutic agent for disease based on inhibitory effect of macrophage migration inhibitory factor.
This patent application is currently assigned to TOYAMA CHEMICAL CO., LTD.. The applicant listed for this patent is TOYAMA CHEMICAL CO., LTD.. Invention is credited to Kimiko MORIMOTO, Keiichi TANAKA.
Application Number | 20150353519 14/647931 |
Document ID | / |
Family ID | 52346269 |
Filed Date | 2015-12-10 |
United States Patent
Application |
20150353519 |
Kind Code |
A1 |
TANAKA; Keiichi ; et
al. |
December 10, 2015 |
THERAPEUTIC AGENT FOR DISEASE BASED ON INHIBITORY EFFECT OF
MACROPHAGE MIGRATION INHIBITORY FACTOR
Abstract
A benzopyran derivative represented by general formula [1], or a
salt thereof, is useful in therapeutic or preventive treatment of
disease, wherein said therapeutic or preventive treatment is
effective because said benzopyran derivative, or salt thereof,
binds to MIF, has an MIF inhibitory effect, and inhibits MIF.
##STR00001## [In the formula, R.sup.1 represents an optionally
substituted C.sub.1-6 alkyl group, one of R.sup.2 and R.sup.3
represents a hydrogen atom, and the other of R.sup.2 and R.sup.3
represents a hydrogen atom, an optionally substituted amino group,
an optionally substituted acylamino group, an optionally
substituted carbamoyl group, or an optionally substituted aryl
group.]
Inventors: |
TANAKA; Keiichi; (Toyama,
JP) ; MORIMOTO; Kimiko; (Toyama, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TOYAMA CHEMICAL CO., LTD. |
Tokyo |
|
JP |
|
|
Assignee: |
TOYAMA CHEMICAL CO., LTD.
Shinjuku-ku, Tokyo
JP
|
Family ID: |
52346269 |
Appl. No.: |
14/647931 |
Filed: |
July 17, 2014 |
PCT Filed: |
July 17, 2014 |
PCT NO: |
PCT/JP14/69026 |
371 Date: |
May 28, 2015 |
Current U.S.
Class: |
549/402 ;
549/401; 549/403 |
Current CPC
Class: |
A61P 25/02 20180101;
C07D 311/30 20130101; A61P 43/00 20180101; A61K 31/352 20130101;
C07D 311/24 20130101; A61P 25/00 20180101; C07D 311/22 20130101;
A61P 25/04 20180101; A61P 29/00 20180101 |
International
Class: |
C07D 311/24 20060101
C07D311/24; C07D 311/30 20060101 C07D311/30; C07D 311/22 20060101
C07D311/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 18, 2013 |
JP |
2013-149690 |
Claims
1-8. (canceled)
9. A treatment agent for the relapsing-remitting or secondary
progressive multiple sclerosis at the time of relapse, comprising a
benzopyran derivative represented by the following general formula
or a salt thereof, ##STR00005## wherein R.sup.1 represents an
optionally substituted C.sub.1-6 alkyl group; one of R.sup.2 and
R.sup.3 represents a hydrogen atom; and the other of R.sup.2 and
R.sup.3 represents a hydrogen atom, an optionally substituted amino
group, an optionally substituted acylamino group, an optionally
substituted carbamoyl group or an optionally substituted aryl
group.
10. The treatment agent for the relapsing-remitting or secondary
progressive multiple sclerosis at the time of relapse according to
claim 9, wherein one of R.sup.2 and R.sup.3 represents a hydrogen
atom; and the other of R.sup.2 and R.sup.3 represents an optionally
substituted acylamino group.
11. The treatment agent for the relapsing-remitting or secondary
progressive multiple sclerosis at the time of relapse according to
claim 9, wherein the benzopyran derivative is
N-[7-(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]formamid-
e,
N-(3-amino-4-oxo-6-phenoxy-4H-1-benzopyran-7-yl)methanesulfonamide,
N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]acetami-
de, N-(4-oxo-6-phenoxy-4H-1-benzopyran-7-yl)methanesulfonamide,
7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-2-carboxamide,
N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-2-yl]acetami-
de,
7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-carboxamid-
e,
N-[7-[(ethylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]formam-
ide or
N-(4-oxo-6-phenoxy-2-phenyl-4H-1-benzopyran-7-yl)methanesulfonamide-
.
12. The treatment agent for the relapsing-remitting or secondary
progressive multiple sclerosis at the time of relapse according to
claim 9, wherein the benzopyran derivative is
N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]formami-
de.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of using a
benzopyran derivative having macrophage migration inhibitory factor
(hereinafter referred to as MIF) inhibitory activity or a salt
thereof for a therapeutic or preventive treatment of a nervous
system disease such as neuropathic pain or multiple sclerosis.
Furthermore, it relates to a pharmaceutical composition containing
a benzopyran derivative or a salt thereof useful for a therapeutic
or preventive treatment of a nervous system disease.
BACKGROUND ART
[0002] Neuropathic pain (hereinafter referred to as NP) is a kind
of chronic pain diseases caused by peripheral nerve and/or central
nerve disorder and functional disorder due to cancer or physical
injury. Such a pain has lost its original significance of alerting
tissue disorder but is no more than a pain. The quality of life
(QOL) of a patient is remarkably reduced due to such a pain.
[0003] The symptom of NP is, in addition to continuous spontaneous
pain, mainly allodynia that a tactile stimulus is felt as a drastic
pain. Such a pain is strongly resistant to non-steroidal
anti-inflammatory drugs (hereinafter referred to as NSAIDs) such as
ibuprofen, and is resistant also to morphine, that is, a narcotic
analgesic (Non Patent Document 1).
[0004] The pathologic physiology and the cause of NP have not been
completely elucidated yet, but the followings have been proved as a
result of recent fundamental research:
[0005] (1) NP is induced by a damage of peripheral and/or central
nerve.
[0006] (2) A variety of cytokines and chemokines are released from
damaged nerve cells.
[0007] (3) The released cytokines and the like cause remarkable
activation of microglia known as an immunocompetent cell for the
central nerve system.
[0008] NP is treated for purposes of relieving the pain, increasing
the functional capacity of the patient, and improving his/her
activity. For these purposes, for example, administration of an
antidepressant, a narcotic analgesic or the like, a nerve block
treatment, and an acupuncture and moxibustion treatment are
performed. However, any excellent therapeutic method based on the
developing mechanism of NP has not been known, and an excellent
therapeutic method for NP is desired.
[0009] Multiple sclerosis (hereinafter referred to as MS) is a
disease that has a focus generated in a central nerve system such
as a brain or a spinal cord, and causes various neurological
symptoms (such as visual disturbance, dyskinesia, hypesthesia,
dysesthesia, pain, dysequilibrium, shivering, dysuria, sexual
dysfunction, fatigue, and emotional disorder). MS is divided,
depending on the progressive mode of a patient's condition, into a
"relapsing-remitting type" wherein relapse and remission are
repeated, and a "chronic progressive type" that the symptom is
gradually worsened. The chronic progressive type is further divided
into a "secondary progressive type" that the relapsing-remitting MS
subsequently shows chronic progression, and a "primary progressive
type" that obvious relapse does not occur but the symptom is
gradually worsened from the initial stage of the onset.
[0010] The cause of MS has not been elucidated yet. There is a
report about the cause of MS that T cells or macrophages infiltrate
into nerve tissues and attack the patient's own myelin covering
axon of nerve cells of the brain or the spinal cord, and as a
result, inflammation is caused in the myelin and hence
demyelination is caused, which leads to MS (Non Patent Document
2)
[0011] A therapeutic method for MS is divided into three
categories, that is, inhibition of inflammation in an acute period,
inhibition of relapse or progression, and relief of the
symptoms.
[0012] In a treatment in an acute period, glucocorticoid (a steroid
anti-inflammatory drug) is used to inhibit the inflammation of a
site where the myelin is damaged. MS is a disease difficult to
completely recover because relapse and remission repeatedly occur.
Various immunological treatments based on the pathogenetic
mechanism of MS have been studied (Non Patent Document 2), and it
is presumed that interferon .beta. and immunosuppressive agents are
effective. However, a sufficiently effective and safe therapeutic
method has not been established. In particular, an excellent
therapeutic method for MS at the time of relapse is desired.
[0013] MIF is a cytokine secreted from activated lymphocytes and
having various biological activities. It is known to exhibit
activities for, for example, an immune system, an endocrine system,
and proliferation and differentiation of cells. Particularly, MIF
plays a significant role in systemic inflammation and immune
response, and is a factor pertaining also to a delayed
hypersensitivity reaction for inhibiting random migration of
macrophages. Besides, MIF has dopachrome tautomerase activity (Non
Patent Document 3).
[0014] On the other hand, MIF is known to have homology to
glutathione S-transferase, to show detoxification, to be secreted
from adenohypophysis at the time of endotoxic shock, to be induced
by a low level of glucocorticoid, and to oppose its
immunosuppressive effect (Non Patent Document 4). In other words,
MIF inhibits the activity of glucocorticoid, antagonizes the
anti-inflammatory effect of endogenous or therapeutically
administered glucocorticoid, and works also as a cause or an
aggravating factor of an inflammatory disease and an inflammatory
state.
[0015] Besides, MIF is indispensable for activation of T cells, is
expressed in various cells, and is strongly expressed particularly
in the nerve system.
[0016] In the relation between MIF and diseases, for example, an
MIF inhibitor relieves an allodynia symptom of an animal model for
NP. On the other hand, a mouse model showing a stimulus sensitivity
reaction aggravated by stress can be produced by injecting
recombinant MIF to a normal mouse (Non Patent Document 5). Besides,
in an animal model for NP, specifically, in a model for the
allodynia induced by sciatic nerve ligation, MIF is highly
expressed in the ipsilateral dorsal horn of spinal cord, and
signaling molecules on the downstream side from MIF are activated
(Non Patent Document 6). Furthermore, in an MIF knockout mouse, the
allodynia induced by sciatic nerve ligation is eliminated (Non
Patent Documents 5 and 6). Accordingly, MIF is presumed to be
indispensable for expression of the symptoms of NP.
[0017] On the other hand, in an MS patient, the MIF concentration
in a cerebrospinal fluid is significantly increased at the time of
relapse, when compared to the time of remission (Non Patent
Document 7). Besides, experimental autoimmune encephalomyelitis
(hereinafter referred to as EAE) of a mouse, that is, a model
animal for MS, can be prevented for the relapse by knocking out MIF
genes (Non Patent Document 8). It is obvious from these facts that
MIF plays an extremely significant role in the formation of NP and
MS.
[0018] EAE, that is, the animal model for MS, includes a model for
reproducing primary onset of an acute period (monophasic) and a
model for reproducing chronic relapsing/remitting condition, and in
general, rats are used for the former and mice are used for the
latter to construct the model.
[0019] It is reported, for example, that cyclophosphamide of an
immunosuppressive agent inhibits the onset of acute EAE in rats but
is ineffective for relapsing type or chronic progressive type of
EAE in mice (Non Patent Document 9).
[0020] It is also reported that a rat or mouse anti-.alpha.4
integrin antibody equivalent to an MS therapeutic agent,
natalizumab, delayed the onset and reduced the severity of the
disease with respect to the acute EAE in rats, and inhibited the
EAE onset in mice by administration for preventing EAE, but the
symptoms were aggravated by therapeutic administration (Non Patent
Documents 10 and 11).
[0021] A benzopyran derivative exhibits an antiarthritic effect
(Patent Document 1), an inhibitory effect for production of
inflammatory cytokines, such as interleukin-.beta. and
interleukin-6, and an immunomodulatory effect (Non Patent Documents
12, 13 and 14), and is known to be useful for a treatment of
rheumatoid arthritis and other arthritis, and autoimmune diseases
(Patent Document 2). Besides, it is known to be effective for the
acute EAE in rats (Non Patent Document 15).
[0022] It is, however, not known at all that the benzopyran
derivative binds to MIF to inhibit its biological activities, as
mentioned above.
[0023] Besides, the effectiveness of the benzopyran derivative for
NP as mentioned above is not known at all, and the effectiveness
thereof for the relapsing-remitting or secondary progressive MS at
the time of relapse is also not known at all.
PRIOR ART DOCUMENTS
Patent Document
[0024] Patent Document 1: JP 02-049778 A
[0025] Patent Document 2: Pamphlet of International Publication No.
WO 94/23714
Non Patent Document
[0026] Non Patent Document 1: Lancet, 1999, vol. 353, pp.
1959-1964
[0027] Non Patent Document 2: N. Engl. J. Med., 2000, vol. 343, pp.
938-952
[0028] Non Patent Document 3: Nat. Rev. Drug Discov., 2006, vol. 5,
pp. 399-410
[0029] Non Patent Document 4: Molecular Medicine, 1996, vol. 2, pp.
143-149
[0030] Non Patent Document 5: Exp. Neurol., 2012, vol. 236, pp.
351-362
[0031] Non Patent Document 6: Anesthesiology, 2011, vol. 114, pp.
643-659
[0032] Non Patent Document 7: J. Neurol. Sci., 2000, vol. 179, pp.
127-131
[0033] Non Patent Document 8: J. Immunol., 2005, vol. 175, pp.
5611-5614
[0034] Non Patent Document 9: Clin. Exp. Immunol., 2009, vol. 159,
pp.159-168
[0035] Non Patent Document 10: J. Pharmacol. Exp. Ther., 2003, vol.
305, pp. 1150-62
[0036] Non Patent Document 11: J. Clin. Invest., 2001, vol. 107,
pp. 995-1006
[0037] Non Patent Document 12: Chem. Pharm. Bull., 2000, vol. 48,
pp. 131-139
[0038] Non Patent Document 13: J. Pharamcobiodyn., 1992, vol. 15,
pp. 649-655
[0039] Non Patent Document 14: Int. J. Immunotherapy, 1993, vol. 9,
pp. 69-78
[0040] Non Patent Document 15: J. Neuroimmunol., 1998, vol. 89, pp.
35-42
SUMMARY OF INVENTION
Technical Problem
[0041] A medical product useful for a therapeutic or preventive
treatment of diseases such as NP and MS is desired, and a
pharmaceutical composition for inhibiting MIF, that is, a factor
significant as the cause of these diseases, is desired.
[0042] In particular, a pharmaceutical composition useful for a
therapeutic or preventive treatment of NP, relapsing MS and the
like is desired.
Solution to Problem
[0043] Under these circumstances, the present inventors found that
a benzopyran derivative represented by the following general
formula [1] or a salt thereof binds to MIF, exhibits an MIF
inhibitory effect, and hence is useful for a therapeutic or
preventive treatment of a disease for which the inhibition of MIF
is effective, resulting in accomplishing the present invention:
##STR00002##
[0044] wherein R.sup.1 represents an optionally substituted
C.sub.1-.sub.6 alkyl group; one of R.sup.2 and R.sup.3 represents a
hydrogen atom; and the other of R.sup.2 and R.sup.3 represents a
hydrogen atom, an optionally substituted amino group, an optionally
substituted acylamino group, an optionally substituted carbamoyl
group or an optionally substituted aryl group.
Advantageous Effects of Invention
[0045] A benzopyran derivative represented by general formula [1]
or a salt thereof exhibits an MIF inhibitory effect and is useful
for a therapeutic or preventive treatment of diseases for which the
inhibition of MIF is effective, such as NP and the
relapsing-remitting and secondary progressive MS at the time of
relapse.
DESCRIPTION OF EMBODIMENTS
[0046] The present invention will be described below in detail.
[0047] The terms as used herein have the following meanings unless
otherwise noted. A halogen atom means a fluorine atom, a chlorine
atom, a bromine atom or an iodine atom.
[0048] A C.sub.1-6 alkyl group means a linear or branched C.sub.1-6
alkyl group such as a methyl group, an ethyl group, a propyl group,
an isopropyl group, a butyl group, a sec-butyl group, an isobutyl
group, a tert-butyl group, a pentyl group, an isopentyl group and a
hexyl group.
[0049] A C.sub.1-6 alkoxy group means a linear or branched
C.sub.1-6 alkyloxy group such as a methoxy group, an ethoxy group,
a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy
group, a sec-butoxy group, a tert-butoxy group, a pentyloxy group
and a hexyloxy group.
[0050] A C.sub.2-12 alkanoyl group means a linear or branched
C.sub.2-12 alkanoyl group such as an acetyl group, a propionyl
group, a valeryl group, an isovaleryl group and a pivaloyl
group.
[0051] An aroyl group means a benzoyl group or a naphthoyl
group.
[0052] A heterocyclic carbonyl group means a nicotinoyl group, a
tenoyl group, a pyrrolizinocarbonyl group or a furoyl group.
[0053] An (.alpha.-substituted) aminoacetyl group means an
(.alpha.-substituted) aminoacetyl group which is derived from an
amino acid (such as glycine, alanine, valine, leucine, isoleucine,
serine, threonine, cysteine, methionine, aspartic acid, glutamic
acid, asparagine, glutamine, arginine, lysine, histidine,
hydroxylysine, phenylalanine, tyrosine, tryptophan, proline and
hydroxyproline) and which may have a protected N-terminal.
[0054] An acyl group means a formyl group, a succinyl group, a
glutaryl group, a maleoyl group, a phthaloyl group, a C.sub.2-12
alkanoyl group, an aroyl group, a heterocyclic carbonyl group or an
(.alpha.-substituted) aminoacetyl group.
[0055] An acylamino group means an amino group substituted with an
acyl group.
[0056] An aryl group means a phenyl group, a naphthyl group, an
indanyl group, an indenyl group, a tetrahydronaphthyl group or the
like.
[0057] The C.sub.1-6 alkyl group of R.sup.1 may be substituted with
one or more halogen atoms.
[0058] The amino group or the carbamoyl group of R.sup.2 and
R.sup.3 may be substituted with one or more C.sub.1-6 alkyl
groups.
[0059] The acylamino group of R.sup.2 and R.sup.3 may be
substituted with one or more halogen atoms.
[0060] The aryl group of R.sup.2 and R.sup.3 may be substituted
with one or more groups selected from a halogen atom, an amino
group, a hydroxyl group, a C.sub.1-6 alkyl group which may be
substituted with one or more halogen atoms, and a C.sub.1-6 alkoxy
group which may be substituted with one or more halogen atoms.
[0061] Preferable examples of the benzopyran derivative represented
by general formula [1] of the present invention include compounds
described below.
[0062] A compound in which R.sup.1 is an optionally substituted
C.sub.1-6 alkyl group; one of R.sup.2 and R.sup.3 is a hydrogen
atom; and the other of R.sup.2 and R.sup.3 is an optionally
substituted acylamino group is preferred.
[0063] Specifically,
N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]formami-
de,
N-(3-amino-4-oxo-6-phenoxy-4H-1-benzopyran-7-yl)methanesulfonamide,
N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]acetami-
de, N-(4-oxo-6-phenoxy-4H-1-benzopyran-7-yl)methanesulfonamide,
7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-2-carboxamide,
N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-2-yl]acetami-
de,
7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-carboxamid-
e,
N-[7-[(ethylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]formam-
ide, and
N-(4-oxo-6-phenoxy-2-phenyl-4H-1-benzopyran-7-yl)methanesulfonami-
de are preferred, and
N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]formami-
de is more preferred.
[0064] The benzopyran derivative of general formula [1] used in the
present invention is produced by combining the publicly
acknowledged methods, and can be produced by, for example, a method
described in Patent Document 1.
[0065] Examples of the salt of the benzopyran derivative of general
formula [1] include a salt with an alkali metal such as sodium or
potassium; a salt with an alkali earth metal such as calcium and
magnesium; an ammonium salt; and a salt with a nitrogen-containing
organic base such as trimethylamine, triethylamine, tributylamine,
pyridine, N,N-dimethylaniline, N-methylpiperidine,
N-methylmorpholine, diethylamine, dicyclohexylamine, procaine,
dibenzylamine, N-benzyl-.beta.-phenethylamine, 1-efenamine and
N,N'-dibenzylethylenediamine.
[0066] Among the aforementioned salts, pharmacologically acceptable
salts are preferred.
[0067] MIF has high homology to a bacterial tautomerase, and
catalyzes a dopachrome tautomerase reaction (Molecular Medicine,
1996, vol. 2, pp. 143-149). Therefore, the biological activity of
MIF can be evaluated by using a tautomerase reaction with a
dopachrome as a substrate.
[0068] The benzopyran derivative of general formula [1] or the salt
thereof of the present invention has an effect to inhibit the MIF
tautomerase activity (namely, an MIF inhibitory effect), and a drug
containing the benzopyran derivative of general formula [1] or the
salt thereof is useful for a therapeutic or preventive treatment of
diseases for which the MIF inhibition is effective.
[0069] Examples of the disease for which the MIF inhibition is
effective include NP and the relapsing-remitting and secondary
progressive MS at the time of relapse, and preferably include
NP.
[0070] Examples of NP include fibromyalgia, postherpetic pain,
diabetic neuropathy, post-spinal cord injury pain, postapoplectic
pain, chronic pain, complex regional pain syndrome, backache for
which NSAIDs are insufficiently effective, sciatica, pelvic pain,
trigeminal neuralgia, osteoarthritis pain for which NSAIDs are
insufficiently effective, deafferentation pain syndrome, pain due
to myositis, pain due to fasciitis, and pain due to seronegative
arthritis. Examples of the deafferentation pain syndrome include
thalamic pain, pain due to MS, pain after avulsion injury, phantom
limb pain, and postoperative pain syndrome. Examples of the pain
due to seronegative arthritis include pain due to axial joint
disorder, pain due to ankylosing spondylitis, pain due to
sacroiliac joint disorder, and pain due to seronegative
spondylitis.
[0071] Preferable examples include fibromyalgia, postherpetic pain,
diabetic neuropathy, backache for which NSAIDs are insufficiently
effective, osteoarthritis pain for which NSAIDs are insufficiently
effective, pain due to myositis, pain due to fasciitis, and pain
due to seronegative arthritis, and more preferable examples include
postherpetic pain, diabetic neuropathy, backache for which NSAIDs
are insufficiently effective, osteoarthritis pain for which NSAIDs
are insufficiently effective, pain due to myositis, pain due to
fasciitis, and pain due to seronegative arthritis.
[0072] The relapsing-remitting MS has a characteristic that it is
ameliorated over several weeks or several months after a
neurological symptom having acutely appeared reaches the fastigium,
and then it relapses to reproduce or aggravate the neurological
symptom. A remission period follows the relapse. A relapse occurred
every several months or years and a slow or gradual remission are
repeated.
[0073] The secondary progressive MS has a characteristic that a
remission state of a patient having initially developed the
relapsing-remitting MS is gradually aggravated while the relapse
and the remission are repeated.
[0074] Examples of the symptoms of the relapsing-remitting MS and
secondary progressive MS at the time of relapse include reduced
vision, motor paralysis, sensory disturbance, multiple vision,
dysuria and dysarthria.
[0075] The compound of the present invention can be formed into
pharmaceutical formulations such as an oral preparation (including
a tablet, a capsule, a powder, a granule, a fine granule, a pill, a
suspension, an emulsion, a liquid and a syrup), an injection and an
eye drop by mixing with various pharmaceutical additives such as an
excipient, a binder, a disintegrant, a disintegration inhibitor, an
anti-caking agent, a lubricant, a carrier, a solvent, an expander,
a tonicity adjusting agent, a solubilizing agent, an emulsifier, a
suspending agent, a thickener, a coating agent, an absorption
enhancer, a gelling enhancer, a coagulation accelerator, a light
stabilizer, a preservative, a desiccating agent, an emulsion
stabilizer, a suspension stabilizer, a dispersion stabilizer, a
coloring inhibitor, an oxygen absorber, an antioxidant, a taste
masking agent, an odor masking agent, a coloring agent, a foaming
agent, an antifoaming agent, a soothing agent, an antistatic agent,
a diluent, a pH buffer, and a pH adjustor.
[0076] The above-described various formulations are formulated by
usual methods.
[0077] An oral solid formulation such as a tablet, a powder and a
granule may be formulated by a usual method using a pharmaceutical
additive of, for example, an excipient such as lactose, saccharose,
sodium chloride, glucose, starch, calcium carbonate, kaolin,
crystalline cellulose, anhydrous dibasic calcium phosphate, partly
pregelatinized starch, corn starch and alginic acid; a binder such
as a simple syrup, a glucose solution, a starch solution, a gelatin
solution, polyvinyl alcohol, polyvinyl ether, polyvinyl
pyrrolidone, carboxymethyl cellulose, shellac, methyl cellulose,
ethyl cellulose, sodium alginate, acacia, hydroxypropyl methyl
cellulose and hydroxypropyl cellulose; a disintegrant such as dry
starch, alginic acid, powdered agar, starch, cross-linked polyvinyl
pyrrolidone, cross-linked carboxymethyl cellulose sodium,
carboxymethyl cellulose potassium and sodium starch glycolate; a
disintegration inhibitor such as stearyl alcohol, stearic acid,
cacao butter and hydrogenated oil; an anti-caking agent such as
aluminum silicate, calcium hydrogen phosphate, magnesium oxide,
talc and silicic anhydride; a lubricant such as carnauba wax, light
anhydrous silicic acid, aluminum silicate, magnesium silicate, a
hydrogenated oil, a hydrogenated vegetable oil derivative, sesame
oil, white beeswax, titanium oxide, dried aluminum hydroxide gel,
stearic acid, calcium stearate, magnesium stearate, talc, calcium
hydrogen phosphate, sodium lauryl sulfate and polyethylene glycol;
an absorption enhancer such as a quaternary ammonium salt, sodium
lauryl sulfate, urea and an enzyme; and a carrier such as starch,
lactose, kaolin, bentonite, silicic anhydride, hydrated silicon
dioxide, magnesium aluminometasilicate and colloidal silicic
acid.
[0078] The tablet may be formed, if necessary, into a general
coated tablet, such as a sugar coated tablet, a gelatin coated
tablet, a gastric soluble coated tablet, an enteric coated tablet
and a water-soluble film coated tablet.
[0079] The capsule is prepared by filling a hard gelatin capsule, a
soft capsule and the like with any of the aforementioned various
pharmaceutical additives.
[0080] Alternatively, a pharmaceutical additive such as a solvent,
an expander, a tonicity adjusting agent, a solubilizing agent, an
emulsifier, a suspending agent and a thickener may be used for
preparation by a usual method to obtain an aqueous or oil
suspension, a solution, a syrup and an elixir.
[0081] The injection may be prepared by a usual method using a
pharmaceutical additive of, for example, a diluent such as water,
ethyl alcohol, macrogol, propylene glycol, citric acid, acetic
acid, phosphoric acid, lactic acid, sodium lactate, sulfuric acid
and sodium hydroxide; a pH buffer and a pH adjuster such as sodium
citrate, sodium acetate and sodium phosphate; an emulsion
stabilizer, a suspension stabilizer and a dispersion stabilizer
such as sodium pyrosulfite, ethylenediaminetetraacetic acid,
thioglycolic acid and thiolactic acid; a tonicity adjusting agent
such as common salt, glucose, mannitol and glycerin; a solubilizing
agent such as carboxymethyl cellulose sodium, propylene glycol,
sodium benzoate, benzyl benzoate, urethane, ethanolamine and
glycerin; a soothing agent such as calcium gluconate,
chlorobutanol, glucose and benzyl alcohol; and a local
anesthetic.
[0082] The eye drop may be prepared by a usual method by
appropriately mixing with a preservative such as chlorobutanol,
sodium dehydroacetate, benzalkonium chloride, cetylpyridinium
chloride, phenethyl alcohol, methyl paraoxybenzoate and
benzethonium chloride; a pH buffer and a pH adjustor such as borax,
boric acid and potassium dihydrogenphosphate; a thickener such as
methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose,
hydroxypropyl methyl cellulose, polyvinyl alcohol, carboxymethyl
cellulose sodium and chondroitin sulfate; a solubilizing agent such
as polysorbate 80 and polyoxyethylene hardened castor oil 60; an
emulsion stabilizer, a suspension stabilizer and a dispersion
stabilizer such as sodium edetate and sodium hydrogen sulfite; and
a tonicity adjusting agent such as sodium chloride, potassium
chloride and glycerin.
[0083] An administration method of the formulation is not
especially limited, and is appropriately determined in accordance
with the form of the formulation, the age, sex and other conditions
of a patient, and the degree of the symptom of the patient.
[0084] A dose of an active ingredient of the present formulation is
appropriately selected in accordance with the usage, the age and
sex of a patient, the form of a disease and the other conditions,
and generally, it may be administered at a dose of 0.1 to 500 mg,
preferably 10 to 200 mg per day once or dividedly several times a
day for an adult.
EXAMPLES
[0085] Next, the present invention will be described with reference
to test examples, and it is noted that the present invention is not
limited to these examples.
[0086] Abbreviations used in the respective test examples have the
following meanings.
[0087] MES: 2-(N-morpholino)ethanesulfonic acid
[0088] DMF: N,N-dimethylformamide
[0089] EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
[0090] EDTA: ethylenediaminetetraacetic acid
[0091] NHS: N-hydroxysuccinimide
[0092] PLP: Proteolipid protein
[0093] TBST: Tween 20-containing Tris-buffered saline
[0094] The following compounds were used as test substances.
TABLE-US-00001 TABLE 1 ##STR00003## Compound R.sup.1 R.sup.2
R.sup.3 Compound A CH.sub.3 NHCHO H Compound B CH.sub.3 NH.sub.2 H
Compound C CH.sub.3 NHCOCH.sub.3 H Compound D CH.sub.3 H H Compound
E CH.sub.3 H CONH.sub.2 Compound F CH.sub.3 H NHCOCH.sub.3 Compound
G CH.sub.3 CONH.sub.2 H Compound H CH.sub.3CH.sub.2 NHCHO H
Compound I CH.sub.3 H ##STR00004##
[0095] Test Example 1 (Confirmation of the binding of Compound A
and MIF)
[0096] As a test substance, Compound A
(N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]formam-
ide) was used.
(1) Preparation of Cell Lysate
[0097] THP-1 cells were cultured for about 6 hours in PRMI 1640
medium containing 1% fetal bovine serum and 50 .mu.mol/L
2-mercaptoethanol. Then, lipopolysaccharide (E. coli 0127:B8, Sigma
Aldrich) was added to the culture plate at a final concentration of
1 .mu.g/mL, and the cells were cultured for about 30 minutes. The
cells were harvested and washed with phosphate buffered saline, and
mixed with about 2-fold volume of cell lysis buffer (20 mmol/L
Tris, 150 mmol/L sodium chloride, 1 mmol/L magnesium chloride, 0.1%
NP-40, 1 mmol/L dithiothreitol, 0.1% Triton X-100, pH 7.4). The
resultant mixture was placed on ice with occasional stirring for
about 30 minutes, and was centrifuged (20000.times.g, 4.degree. C.,
8 minutes). The separated and gained supernatant was used as cell
lysate. Protein concentration of the cell lysate was measured with
BCA protein assay reagent (Thermo Fisher Scientific K.K.) in
accordance with its manual.
(2) Synthesis of
4-amino-N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl-
]butanamide hydrochloride
[0098] To a solution of 500 mg of
N-(3-amino-4-oxo-6-phenoxy-4H-1-benzopyran-7-yl)methanesulfonamide
in 5.0 mL of DMF, 293 mg of 4-(tert-butoxycarbonylamino)butyric
acid and 304 mg of EDC were added, followed by stirring at room
temperature for 1 hour and 30 minutes. To the thus obtained
reaction mixture, 111 mg of EDC was added, followed by stirring at
room temperature for 3 hours and 30 minutes. Ethyl acetate and 10%
citric acid aqueous solution were added to the resulting reaction
mixture, the resultant was stirred at room temperature for 30
minutes, and a solid was filtered. To the thus obtained solid, DMF,
ethyl acetate and 10% citric acid aqueous solution were added,
followed by stirring at room temperature for 1 hour. Then, the
solid was filtered to obtain 397 mg of
tert-butyl[4-[[7-[(methylsulfonyl)amino]-4-oxo-6-phonoxy-4H-1-benzopyran--
3-yl]amino]-4-oxobutyl]carbamate in the form of a pale yellow
solid. 300 mg of the thus obtained tert-butyl
[4-[[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl]amino-
]-4-oxobutyl]carbamate was suspended in 3.0 mL of methylene
chloride, 0.60 mL of trifluoroacetic acid was added thereto under
ice cooling, the resultant was stirred at room temperature for 40
minutes, and then the solvent was distilled off under reduced
pressure. To the thus obtained residue, 3 mL of ethyl acetate and
0.25 mL of 4 mol/L hydrogen chloride/ethyl acetate solution were
added, and the solvent was distilled off under reduced pressure. To
the thus obtained residue, 5.0 mL of ethyl acetate and 0.50 mL of 4
mol/L hydrogen chloride/ethyl acetate solution were added, followed
by stirring at room temperature for 2 hours. Then, a solid was
filtered to obtain 232 mg of
4-amino-N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl-
]butanamide hydrochloride as a pale yellow solid.
(3) Preparation of Beads
[0099] Immobilization of
4-amino-N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl-
]butanamide hydrochloride on Dynabeads M-270 Carboxylic Acid (Life
Technologies Corporation) was performed by a general method.
[0100] Briefly, after NHS esterifying COOH terminals of about 30 mg
of the beads (Dynabeads M-270 Carboxylic Acid, Life Technologies
Corporation), 0.010 mL of DMF, 0.90 mL of 0.01 mol/L
4-amino-N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl-
]butanamide hydrochloride/DMF solution, and 0.090 mL of 1 mol/L
N,N-diisopropylethylamine/DMF solution were added thereto, and the
resultant was shook at room temperature for 70 minutes. The beads
were washed with 0.5 mL of DMF twice, and then, 0.94 mL of DMF and
0.060 mL of 2-aminoethanol were added thereto, followed by shaking
at room temperature for 2 hours. The beads were washed with 0.5 mL
of DMF twice, and then washed with 1 mL of 0.05 mol/L phosphate
buffer (pH 6) four times, and thus, beads to which Compound A was
bound via a linker were obtained (hereinafter referred to as the
compound beads). Besides, beads obtained by a similar reaction
without adding
4-amino-N-[7-[(methylsulfonyl)amino]-4-oxo-6-phenoxy-4H-1-benzopyran-3-yl-
]butanamide hydrochloride were used as control beads. Each type of
these beads was suspended in 1 mL of 0.05 mol/L phosphate buffer
(pH 6) to be stored in a refrigerator until use. Just before use, a
part of the beads was separated and washed with cell lysis buffer
three times.
[0101] (4) Reaction Between Cell Extract and Beads
[0102] 0.1 mL of cell lysate (2 mg/mL of protein) and about 0.9 mg
of the compound beads or control beads were thoroughly mixed
overnight at 4.degree. C. Supernatants were separated from the
beads by using a magnet, and were collected as Flow-through
fraction. Besides, the beads were rinsed with cell lysis buffer,
and then were reacted with 40 .mu.L of cell lysis buffer containing
0.5 mmol/L of Compound A at 4.degree. C. for about 8 hours with
vigorous stirring. Supernatants were separated from the beads by
using a magnet, and were collected as Compound
[0103] A-eluate. Moreover, the beads were lightly rinsed with cell
lysis buffer, were mixed with 15 .mu.L of SDS-PAGE sample buffer
(2ME+) (Wako Pure Chemical Industries, Ltd.), which had been
diluted to 4 times, and were heated at about 95.degree. C. for 5
minutes. Thereafter, supernatants separated from the beads by using
a magnet were collected as Heat-eluted fraction.
(5) Detection of MIF by Western Blotting
[0104] Western blotting was carried out by a general method.
[0105] Both of a part of the Flow-through fraction and a part of
the Compound A-eluate obtained as described above in (4) were
respectively mixed with the SDS-PAGE sample buffer (2ME+), and were
heated. These heat-treated samples and the Heat-eluted fraction
were electrophoresed on SDS-PAGE gel (SuperSep Ace 15%, Wako Pure
Chemical Industries, Ltd.) at 30 to 50 mA for about 80 minutes, and
then were electrophoretically transferred to a PVDF membrane
(Hybond-P, GE Healthcare Japan Corporation) at 100 mA for about 60
minutes. The protein-transferred membrane was immersed and gently
shaken in TBST solution (10 mmol/L Tris, 100 mmol/L sodium
chloride, 0.1% Tween-20, pH 7.5) containing 5% skim milk at room
temperature for about 1 hour. Thereafter, the membrane was immersed
in TBST solution containing 1/50000 volume of anti-MIF antibody
(Abcam Plc.) and 5% skim milk, and was reacted overnight at
4.degree. C. with gentle mixing. After lightly rinsing the membrane
with TBST solution three times, the resultant membrane was immersed
in TBST solution containing 1/5000 volume of HRP-modified anti-goat
IgG antibody (Santa Cruz Biotechnology Inc.) and 5% skim milk, and
was reacted at room temperature for 1 hour with gentle mixing.
After lightly rinsing the membrane with TBST solution three times,
MIF was detected by using ECL Prime reagent (GE Healthcare Japan
Corporation) in accordance with its manual.
[0106] FIG. 1 shows a photograph of the membrane on which binding
reactants of MIF and the antibody are detected by
chemiluminescence.
[0107] Fractions electrophoresed in respective lanes of the gel
were as follows:
[0108] Lane 1: Flow-through fraction of the compound beads
[0109] Lane 2: Compound A-eluate of the compound beads
[0110] Lane 3: Heat-eluted fraction of the compound beads
[0111] Lane 4: recombinant MIF (Abeam Plc.)
[0112] Lane 5: Flow-through fraction of the control beads
[0113] Lane 6: Compound A-eluate of the control beads
[0114] Lane 7: Heat-eluted fraction of the control beads
[0115] The Compound A-eluate of the compound beads contains
proteins that have binding capacity to the compound beads and is
relieved from the compound beads in the presence of an excessive
amount of Compound A. A band detected in the Compound A-eluate
(i.e., Lane 2) was proved to be MIF because it was bound to the
anti-MIF antibody and was in a position corresponding to the same
molecular weight as recombinant MIF.
[0116] In other words, it was revealed that MIF has binding
capacity to the compound beads.
[0117] On the other hand, both of the Compound A-eluate of the
control beads and the Heat-eluted fraction of the control beads
contain proteins having binding capacity to the control beads.
Since MIF was not detected in these fractions (namely, Lanes 6 and
7), it was shown that MIF has no binding capacity to the control
beads.
[0118] Accordingly, it was proved that MIF specifically binds to
Compound A.
Test Example 2
Confirmation of Inhibition of MIF Activity
[0119] Compounds A to I were used as test substances.
[0120] The inhibitory effect of test substances against tautomerase
activity of MIF was evaluated by referring to a method of Healy et
al. (Cancer Epidemiology Biomarkers and Prevention, 2011, vol. 20,
pp. 1516-1523).
[0121] In this method, tautomeric reaction from
L-3,4-dihydroxyphenylalanine methyl ester (L-dopachrome methyl
ester, colored) to 5,6-hydroxyindole-2-carboxylic acid methyl ester
(no color) is measured as a change in absorbance at 475 nm.
[0122] As an enzyme source, recombinant MIF manufactured by Abcam
Plc. or MIF produced and purified by referring to a method of
Lubetsky et al. (The Journal of Biological Chemistry, 2002, vol.
277, pp. 24976-24982) was used. A purification method for MIF is
described below.
[0123] The pET15b vector (Merck) into which the full length gene
sequence of MIF had been inserted was transfected E. coli BL21 Star
(DE3) strain (Life Technologies Corporation). The E. coli was
cultured until the culture medium exhibited absorbance (at 600 nm)
of 0.5 to 0.8, isopropyl-.beta.-thiogalactopyranoside (Wako Pure
Chemical Industries, Ltd.) was added thereto to a final
concentration of 0.1 mmol/L, and the protein expression was induced
for 4 hours. The E. coli were resuspended in buffer (pH 7.5)
containing 20 mmol/L Tris (Wako Pure Chemical Industries, Ltd.), 20
mmol/L sodium chloride (Wako Pure Chemical Industries, Ltd.) and 1
mmol/L dithiothreitol (Wako Pure Chemical Industries, Ltd.), and
the resultant was subjected to ultrasonic lysis and centrifugation
at 15000 rpm for 10 minutes. The thus obtained supernatant was
filtered using a 0.20 .mu.m filter, and was allowed to pass through
HiTrap Q HP and HiTrap SP HP columns (GE Healthcare Japan
Corporation), so as to separate flow-through fractions each of 5
mL. Each of 10 .mu.L of the separated fractions was subjected to
the electrophoresis using 5 to 20% polyacrylamide gel (Wako Pure
Chemical Industries, Ltd.), and all proteins were stained with
Coomassie brilliant blue reagent (Bio-Rad Laboratories, Inc.). On
the basis of results thus obtained, a fraction containing a large
amount of MIF and containing least amount of other proteins was
selected as purified MIF. The concentration of MIF protein was
measured by using BCA protein assay reagent (Thermo Fisher
Scientific K. K.).
[0124] Inhibitory effect of each test substance against MIF
tautomerase activity was measured as follows.
[0125] A final concentration of 10 to 50 nmol/L of MIF and a final
concentration of 30 .mu.mol/L of each test substance or 0.5%
dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.) as a
control were added to buffer (pH 6.2) containing 50 mmol/L Bis-Tris
(Dojindo Laboratories) and 1 mmol/L EDTA (Dojindo Laboratories),
and reaction was performed at room temperature for 15 minutes to
give a reaction solution 1.
[0126] On the other hand, 1/20 volume of 12 mmol/L
L-3,4-dihydroxyphenylalanine methyl ester (Sigma Aldrich) and 1/20
volume of 24 mmol/L sodium periodate (Wako Pure Chemical
Industries, Ltd.) were added to buffer having the same composition
as that used for obtaining the reaction solution 1, to give a
reaction solution 2.
[0127] Next, the reaction solution 1 and the reaction solution 2
were mixed, and the temporal change of absorbance at 475 nm of the
obtained mixture was immediately measured.
[0128] The difference of the absorbencies between the at about 1
minute after the measurement start and at about 5 minutes after was
obtained. Assuming that the absorbance change of the control is
100%, an inhibiting rate of the tautomerase reaction in presence
of-each test substance was calculated.
[0129] The results are shown in Table 2. In Table 2, the
tautomerase reaction inhibiting rate is shown as follows. "-"; less
than 50%, "+"; 50% or more and less than 75%, "++"; 75% or
more.
TABLE-US-00002 TABLE 2 Inhibition Rate of Test Substance
Tautomerase Reaction Compound A + Compound B + Compound C +
Compound D + Compound E ++ Compound F ++ Compound G + Compound H ++
Compound I ++
[0130] All of the test substances inhibited the tautomerase
activity of MIF.
[0131] The above-described results reveal that the compound of
general formula [1] or the salt thereof show the MIF inhibitory
effect and is useful as an MIF inhibitor.
Test Example 3
Effect of Compound A on Chronic Constriction Nerve Injury Model
Rat
[0132] This test was performed by referring to a method of Bennett
et al. (Pain, 1988, vol. 33, pp. 87-107).
[0133] Compound A was used as a test substance, and celecoxib, one
of NSAIDs, was used as a reference substance. The compound A was
administered at a dose of 30 mg/kg (Compound A group). The
celecoxib was administered at a dose of 30 mg/kg (celecoxib group).
To a control group, a 0.5% methyl cellulose aqueous solution used
as a vehicle was administered.
[0134] Under anesthetic with Somnopentyl (manufactured by Kyoritsu
Seiyaku Corporation, about 52 mg/kg, intraperitoneal
administration), nerve constriction operation was performed on the
left sciatic nerves of Sprague-Dawley male rats (7 weeks old).
Briefly, a left femoral region of each rat was dissected, the
sciatic nerve was detached from tissues around, and the nerve was
loosely constricted with a 4-0 silk suture (manufactured by Eticon
Inc., surgical silk) for having narrow parts in four positions at
intervals of about 1 mm. The muscular layer and the skin were
respectively sutured, and the operation region was disinfected. The
vehicle, the test substance or the reference substance was orally
administered once a day continuously for 14 days from the 16th day
after the operation.
[0135] From the start of the administration, pain sense (mechanical
allodynia) of the footpad of left hind paw was evaluated by von
Frey test. Briefly, von Frey filaments respectively having various
flexibilities (Semmes-Weinstein Von Frey Anesthesiometer
(manufactured by Danmic Global, LCC)) were vertically pressed
against the footpad of the left hind paw successively in order from
one having a lightest elasticity, and the elastic force of the
filament against which the rat showed a withdrawal response was
determined as a pain threshold value.
[0136] The evaluation of the pain sense was expressed as an average
of pain threshold values. The results are shown in Table 3.
TABLE-US-00003 TABLE 3 Average of pain thresholds (g) Number of
15th day after 30th day after Test group animals operation
operation Control group 8 2.7 4.2 Compound A group 8 2.4 13.5
Celecoxib group 7 2.5 3.1
[0137] The pain threshold value of the Compound A group was
increased to 13.5 g 14 days after the start of the administration
(on the 30th day after the operation), and thus, Compound A
inhibited the symptom of the mechanical allodynia as compared with
that in the control group.
[0138] On the other hand, the administration of celecoxib (30
mg/kg), NSAID, did not suppress the mechanical allodynia
symptom.
[0139] The above-described results reveal that Compound A
suppresses the symptom of the mechanical allodynia by a mechanism
different from that of NSAIDs.
Test Example 4
Effect on Chronic Relapsing EAE Model of Mouse
[0140] Compound A was selected as a test substance, and
salazosulfapyridine (hereinafter referred to as SASP) was selected
as a comparative control agent. Compound A was administered at a
dose of 30 mg/kg (Compound A group). SASP was administered at a
dose of 300 mg/kg (SASP group). In control group and normal group
(no induction treatment group), 0.5% methyl cellulose aqueous
solution used as vehicle of the administration liquid was
administered.
[0141] The chronic relapsing EAE was induced in SJL/J female mice
by immunization with partial PLP peptide. Briefly, an emulsion was
prepared by mixing equivalent volumes of phosphate buffer saline
containing 1 mg/mL of the peptide corresponding to residues 139-151
of the PLP and Freund's incomplete adjuvant containing 4 mg/mL of
killed M. Tuberculosis H37Ra. The emulsion was intradermically
injected (50 .mu.g of PLP per mouse) into four positions on the
back for immunization, and additionally, on the day of the
immunization and two days after, pertussis toxin was
intraperitoneally injected in each amount of 150 ng per mouse,
twice in total. The vehicle, the test substance or the reference
article was orally administered continuously for 44 days once daily
from the day of the immunization.
[0142] In the present experiment, paralysis was developed to reach
a peak on the 14th to 16th day after the immunization, and the
symptom remitted once, but relapsed to reach a peak on the 38th
day.
[0143] The symptom was evaluated in accordance with a report of
Weaver et al. (FASEB Journal, 2005, vol. 19, p. 1668). Briefly,
paralysis of all of the four limbs and the tail was evaluated by
scoring in 4 ranks of scores of 0 to 3 and in 3 ranks of scores of
0 to 2, respectively, and the sum of the scores was determined as
EAE score (the maximum score 14).
[0144] Incidence rates (the number of mice having developed the
disease/the number of used mice) and the average of EAE score of
each group were measured at the period of initial onset (on the
15th day after the immunization) and at the period of relapse (on
the 38th day after the immunization). The results are shown in
Table 4.
TABLE-US-00004 TABLE 4 Number Period of initial onset Period of
relapse of Incidence EAE Incidence EAE Test group animals rate
score rate score Normal group 2 0/2 0.0 0/2 0.0 Control group 8 6/8
6.8 6/8 1.9 Compound A group 8 1/8 1.4 2/8 0.3 SASP group 8 7/7 5.5
6/6 2.0 (1 animal (2 animals died) died)
[0145] In the Compound A group, the incidence rate was low both at
the period of initial onset and at the period of relapse, and the
EAE score was obviously low too.
[0146] On the other hand, in the SASP group, neither the incidence
rate nor the EAE score was lowered.
[0147] The above-described results reveal that Compound A
suppressed the occurrence of paralysis of the chronic relapsing
EAE. The effects of Compound A are obviously different from that of
SASP, even though both of these drugs were categorized into
immunomodulator.
[0148] The above-described results reveal that the compound of
general formula [1] or the salt thereof is useful as an MIF
inhibitor, and is also useful for a therapeutic or preventive
treatment of a disease for which the inhibition of MIF is
effective, such as NP and a relapse period of the
relapsing-remitting and secondary progressive MS.
BRIEF DESCRIPTION OF DRAWING
[0149] FIG. 1 is a photograph of a membrane on which reactants
resulting from binding between MIF and the antibody are detected by
chemiluminescence.
INDUSTRIAL APPLICABILITY
[0150] A benzopyran derivative represented by general formula [1]
or a salt thereof binds to MIF, exhibits an MIF inhibitory effect,
and is useful for a therapeutic or preventive treatment of a
disease for which the inhibition of MIF is effective.
* * * * *