U.S. patent application number 16/891708 was filed with the patent office on 2020-09-17 for novel therapeutic uses of benzylideneguanidine derivatives for the treatment of proteopathies.
The applicant listed for this patent is InFlectis BioScience. Invention is credited to Philippe GUEDAT.
Application Number | 20200289436 16/891708 |
Document ID | / |
Family ID | 1000004870178 |
Filed Date | 2020-09-17 |
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United States Patent
Application |
20200289436 |
Kind Code |
A1 |
GUEDAT; Philippe |
September 17, 2020 |
NOVEL THERAPEUTIC USES OF BENZYLIDENEGUANIDINE DERIVATIVES FOR THE
TREATMENT OF PROTEOPATHIES
Abstract
The present invention relates to novel methods of using a
compound of formula (I), or a tautomer and/or a pharmaceutically
acceptable salt thereof, ##STR00001## in treating a disorder
associated with the PPP1R15A pathway and associated with protein
misfolding stress and in particular with accumulation of misfolded
proteins selected in the group of tauopathies, synucleinopathies,
polyglutamine and polyalanine diseases, leukodystrophies, cystic
fibrosis, multiple sclerosis, lysosomal storage disorders,
amyloidosis diseases, inflammation, metabolic disorders,
cardio-vascular disorders, osteoporosis, nervous system trauma,
ischemia.
Inventors: |
GUEDAT; Philippe;
(MONTENOIS, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
InFlectis BioScience |
NANTES |
|
FR |
|
|
Family ID: |
1000004870178 |
Appl. No.: |
16/891708 |
Filed: |
June 3, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15316785 |
Dec 6, 2016 |
10709677 |
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PCT/EP2015/065161 |
Jul 2, 2015 |
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16891708 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/155 20130101;
A61K 31/53 20130101; A61K 31/44 20130101 |
International
Class: |
A61K 31/155 20060101
A61K031/155; A61K 31/44 20060101 A61K031/44; A61K 31/53 20060101
A61K031/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 2, 2014 |
EP |
14306075.4 |
Claims
1. A method for treating a disorder selected from the group
consisting of an inflammatory condition, an infectious disease,
dystrophy, oculopharyngeal muscular dystrophy (OPMD), systemic
inflammatory response syndrome (SIRS), and sepsis comprising
administering to a patient in need thereof, a compound of formula
(I), or a tautomer or pharmaceutically acceptable salt thereof,
##STR00026## wherein: R.sub.1 is alkyl, O-alkyl, Cl, F or Br;
R.sub.2 is H or F; R.sub.3 is H or alkyl; R.sub.4 is H or
C(O)R.sub.6; R.sub.5 is H; or R.sub.4 and R.sub.5 are linked to
form a 5 to 6 membered saturated or unsaturated heterocyclic group
optionally comprising 1 or 2 heteroatoms such as N, in addition to
the N atoms to which R.sub.4 and R.sub.5 are bound, and where said
heterocyclic group is optionally substituted with one or more
R.sub.10 groups; R.sub.6 is selected from the group consisting of
R.sub.7, OR.sub.7 and NR.sub.8R.sub.9; R.sub.7, R.sub.8 and R.sub.9
are each independently selected from the group consisting of alkyl,
cycloalkyl, aralkyl, cycloalkenyl, heterocyclyl, and aryl, each of
which is optionally substituted with one or more R.sub.10 groups;
each R.sub.10 is independently selected from the group consisting
of halogen, OH, .dbd.O, CN, COO-alkyl, aralkyl, 50.sub.2-alkyl,
50.sub.2-aryl, COOH, CO-alkyl, CO-aryl, NH.sub.2, NH-alkyl,
N(alkyl).sub.2, CF.sub.3, alkyl, and alkoxy; X and Z are each
independently CR.sub.11, and Y is CR.sub.11 or N; and R.sub.11 is
H, alkyl or F.
2. A method for treating a disorder selected from the group
consisting of an inflammatory condition and an infectious disease
comprising administering to a patient in need thereof, a compound
of formula (I), or a tautomer or pharmaceutically acceptable salt
thereof, ##STR00027## wherein: R.sub.1 is alkyl, Cl, F or Br;
R.sub.2 is H or F; R.sub.3 is H or alkyl; R.sub.4 is H or
C(O)R.sub.6; R.sub.5 is H; or R.sub.4 and R.sub.5 are linked to
form a 5 to 6 membered saturated or unsaturated heterocyclic group
optionally comprising 1 or 2 heteroatoms such as N, in addition to
the N atoms to which R.sub.4 and R.sub.5 are bound, and where said
heterocyclic group is optionally substituted with one or more
R.sub.10 groups; R.sub.6 is selected from the group consisting of
R.sub.7, OR.sub.7 and NR.sub.8R.sub.9; R.sub.7, R.sub.8 and R.sub.9
are each independently selected from the group consisting of alkyl,
cycloalkyl, aralkyl, cycloalkenyl, heterocyclyl, and aryl, each of
which is optionally substituted with one or more R.sub.10 groups;
each R.sub.10 is independently selected from the group consisting
of halogen, OH, CN, COO-alkyl, aralkyl, SO.sub.2-alkyl, SO2-aryl,
COOH, CO-alkyl, CO-aryl, NH.sub.2, NH-alkyl, N(alkyl).sub.2,
CF.sub.3, alkyl, and alkoxy; X and Z are each independently
CR.sub.11, and Y is CR.sub.11 or N; and R.sub.11 is H, or F.
3. The method according to claim 1 wherein R.sub.1 is Cl, Br, Me,
or F.
4. The method according to claim 1 wherein R.sub.1 is Cl.
5. The method according to claim 1 wherein R.sub.2 is H.
6. The method according to claim 1 wherein Y is CR.sub.11.
7. The method according to claim 1 wherein R.sub.3 and R.sub.4 are
both H.
8. The method according to claim 1 wherein R.sub.3 is H and R.sub.4
is C(O)R.sub.6, with wherein R.sub.6 is Me or OMe.
9. The method according to claim 1 wherein in Formula (I): R.sub.1
is Cl, R.sub.2 is H, R.sub.3=R.sub.4=R.sub.5=H, X=Z=CH, Y is
CR.sub.11, and R.sub.11 is F.
10. The method according to claim 1 wherein said compound is
selected from the group consisting of the following compounds:
##STR00028## ##STR00029## or a tautomer or acceptable salt
thereof.
11. The method according to claim 1 where the compound is selected
from the group consisting of the following compounds: ##STR00030##
or a tautomer or acceptable salt thereof.
12. The method according to claim 1 where the inflammatory
condition is selected from the group consisting of lung infections,
Respiratory Distress Syndrome, bronchopulmonary dysplasia, colitis,
ulcerative colitis, Inflammatory Bowel Disease, diabetic
nephropathy, hemorrhagic shock, spondylo-arthropathies,
pancreatitis; inflammation-induced cancer, allergy, asthma,
hypercytokinemia, graft versus host disease (GVHD), and acute
respiratory distress syndrome (ARDS),
13. The method according to claim 1 where the infectious disease is
selected from the group consisting of influenza virus infection,
smallpox virus infection, herpes virus infection, severe acute
respiratory syndrome (SARS), chikungunya virus infection, West Nile
Virus infection, dengue virus infection, Japanese encephalitis
virus infection, yellow fever virus infection, and hepatitis C
virus infection.
14. The method according to claim 2 wherein R.sub.1 is Cl, Br, Me,
or F.
15. The method according to claim 2 wherein R.sub.1 is Cl.
16. The method according to claim 2 wherein R.sub.2 is H.
17. The method according to claim 2 wherein Y is CR.sub.11.
18. The method according to claim 2 wherein R.sub.3 and R.sub.4 are
both H.
19. The method according to claim 2 wherein R.sub.3 is H and
R.sub.4 is C(O)R.sub.6, and wherein R.sub.6 is Me or OMe.
20. The method according to claim 2 wherein in Formula (I): R.sub.1
is Cl, R.sub.2 is H, R.sub.3=R.sub.4=R.sub.5=H, X=Z=CH, Y is
CR.sub.11, and R.sub.11 is F.
21. The method according to claim 2 wherein said compound is
selected from the group consisting of the following compounds:
##STR00031## ##STR00032## or a tautomer or acceptable salt
thereof.
22. The method according to claim 2 where the compound is selected
from the group consisting of the following compounds: ##STR00033##
or a tautomer or acceptable salt thereof.
23. The method according to claim 2 where the inflammatory
condition is selected from the group consisting of lung infections,
Respiratory Distress Syndrome, bronchopulmonary dysplasia, colitis,
ulcerative colitis, Inflammatory Bowel Disease, diabetic
nephropathy, hemorrhagic shock, spondylo-arthropathies,
pancreatitis; inflammation-induced cancer, allergy, asthma,
hypercytokinemia, graft versus host disease (GVHD), and acute
respiratory distress syndrome (ARDS),
24. The method according to claim 2 where the infectious disease is
selected from the group consisting of influenza virus infection,
smallpox virus infection, herpes virus infection, severe acute
respiratory syndrome (SARS), chikungunya virus infection, West Nile
Virus infection, dengue virus infection, Japanese encephalitis
virus infection, yellow fever virus infection, and hepatitis C
virus infection.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/316,785, having a filing date of Dec. 6, 2016, which is a
371 application of International Patent Application
PCT/EP2015/065161, filed Jul. 2, 2015, which claims the benefit of
EP application 14306075.4, filed Jul. 2, 2014, all of said
applications incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compounds that have
potential therapeutic applications in treating disorders associated
with protein misfolding stress and in particular with an
accumulation of misfolded proteins. In particular, the invention
provides compounds that are capable of exhibiting a protective
effect against cytotoxic endoplasmic reticulum (ER) stress.
BACKGROUND TO THE INVENTION
[0003] The compound
2-(2,6-dichlorobenzylidene)hydrazinecarboximidamide, also referred
to as guanabenz, is an alpha agonist of the alpha-2 type that is
used as an antihypertensive drug.
##STR00002##
[0004] Various derivatives of guanabenz have also been reported.
For example, U.S. Pat. No. 3,982,020 (Sandoz, Inc.) discloses
substituted benzylidene hydrazines and their use as
hypoglycemic-antihyperglycemic agents, anti-obesity agents and
anti-inflammatory agents. US 2004/0068017 (Bausch & Lomb Inc.)
discloses substituted benzylidene hydrazines that are capable of
increasing the activity of gelatinase A in ocular cells. The
molecules have applications in the treatment of primary open angle
glaucoma. WO 2008/061647 (Acure Pharma AB) discloses the use of
N-(2-chloro-3,4,-dimethoxybenzylideneamino)guanidine as a VEGFR
inhibitor and its associated applications in the treatment or
prevention of undesired blood vessel formation during tumour growth
and/or inflammatory conditions. WO 2005/031000 (Acadia
Pharmaceuticals, Inc.) discloses substituted benzylidene hydrazines
and their use in treating acute pain and chronic neuropathic pain.
Finally, EP1908464 (CNRS) discloses guanabenz and chloroguanabenz
and their use in the treatment of polyglutamine expansion
associated diseases, including Huntington's disease.
[0005] More recently it has been reported that guanabenz has
therapeutic potential in a number of other areas. Guanabenz, was
recently noted to have anti-prion activity (D. Tribouillard-Tanvier
et al., 2008 PLoS One 3, e1981). It has been reported that its
activity in protecting against protein misfolding is surprisingly
much broader and includes attenuating accumulation of mutant
Huntingtin in cell-based assays (WO 2008/041133) and protection
against the lethal effects of expression of misfolding prone
Insulin Akita mutant in the endoplasmic reticulum (ER) of Min6 and
INS-1 pancreatic beta-cells (Tsaytler et al., Science 2011 Vol.
332, 1 pp 91-94). WO2014/138298 and Way et al. (2015 Nature
Communications 6:6532 DOI: 10.1038/ncomms7532) disclose guanabenz
ant its use in the treatment of demyelinating disorder, such as
multiple sclerosis.
[0006] Guanabenz has also been shown to promote survival of HeLa
cells exposed to otherwise cytotoxic ER-stress induced by the
N-glycosylation inhibitor tunicamycin, in a dose-dependent manner
(Tsaytler et al., Science 2011). Quantitative assessment of cell
viability revealed that guanabenz doubled the number of cells
surviving ER stress with a median effective concentration of
.about.0.4 .mu.M. Neither the .alpha.2-adrenergic receptor agonist
clonidine, nor the .alpha.2-adrenergic receptor antagonist efaroxan
protected cells from cytotoxic ER stress and efaroxan did not
interfere with guanabenz's protective effect (Tsaytler et al.,
Science 2011). These observations demonstrate that guanabenz
rescues cells from lethal ER stress by a mechanism independent of
the .alpha.2-adrenergic receptor. Guanabenz protects cells from
otherwise lethal accumulation of misfolded proteins by binding to a
regulatory subunit of protein phosphatase 1, PPP1R15A (GADD34),
selectively disrupting the stress-induced dephosphorylation of the
a subunit of translation initiation factor 2 (eIF2.alpha.).
Guanabenz sets the translation rates in stressed cells to a level
manageable by available chaperones, thereby restoring protein
homeostasis. It was reported that Guanabenz does not bind to the
constitutive PPP1R15B (CReP) and therefore does not inhibit
translation in non-stressed cells (Tsaytler et al., Science
2011).
[0007] Failure to maintain proteostasis in the ER by mounting an
adequate unfolded protein response (UPR) is recognized as a
contributing factor to many pathological conditions. Thus, the
molecules described here, which inhibit eIF2.alpha. phosphatase to
fine-tune protein synthesis, may be of therapeutic benefit to a
large number of diseases caused protein misfolding stress and in
particular with an accumulation of misfolded proteins.
[0008] The present invention seeks to provide alternative compounds
based on a guanabenz core structure that have potential therapeutic
applications in treating disorders associated with protein
misfolding stress and in particular with an accumulation of
misfolded proteins.
SUMMARY OF THE INVENTION
[0009] A first aspect of the invention relates to a compound of
formula (I), or a pharmaceutically acceptable salt thereof,
##STR00003##
wherein: R.sub.1 is alkyl, O-alkyl, Cl, F or Br;
R.sub.2 is H or F;
[0010] R.sub.3 is selected from H and alkyl; R.sub.4 is selected
from H and C(O)R.sub.6;
R.sub.5 is H;
[0011] or R.sub.4 and R.sub.5 are linked to form a 5 to 6 membered
saturated or unsaturated heterocyclic group optionally comprising 1
or 2 heteroatoms such as N, in addition to the N atoms to which
R.sub.4 and R.sub.5 are bound, and where said heterocyclic group is
optionally substituted with one or more R.sub.10 groups; R.sub.6 is
selected from R.sub.7, OR.sub.7 and NR.sub.8R.sub.9; R.sub.7,
R.sub.8 and R.sub.9 are each independently selected from alkyl,
cycloalkyl, aralkyl, cycloalkenyl, heterocyclyl and aryl, each of
which is optionally substituted with one or more R.sub.10 groups;
each R.sub.10 is independently selected from halogen, OH, .dbd.O,
CN, COO-alkyl, aralkyl, SO.sub.2-alkyl, SO.sub.2-aryl, COOH,
CO-alkyl, CO-aryl, NH.sub.2, NH-alkyl, N(alkyl).sub.2, CF.sub.3,
alkyl and alkoxy; X and Z are each independently CR.sub.11, and Y
is selected from CR.sub.11 and N; R.sub.11 is H, alklyl or F; for
use in treating a proteopathy and/or a disorder associated with
protein misfolding stress and in particular with an accumulation of
misfolded proteins.
[0012] A second aspect of the invention relates to a compound of
formula (I), or a pharmaceutically acceptable salt thereof,
##STR00004##
wherein: R.sub.1 is alkyl, O-alkyl, Cl, F or Br;
R.sub.2 is H or F;
[0013] R.sub.3 is selected from H and alkyl; R.sub.4 is selected
from H and C(O)R.sub.6;
R.sub.5 is H;
[0014] or R.sub.4 and R.sub.5 are linked to form a 5 to 6 membered
saturated or unsaturated heterocyclic group optionally comprising 1
or 2 heteroatoms such as N, in addition to the N atoms to which
R.sub.4 and R.sub.5 are bound, and where said heterocyclic group is
optionally substituted with one or more R.sub.10 groups; R.sub.6 is
selected from R.sub.7, OR.sub.7 and NR.sub.8R.sub.9; R.sub.7,
R.sub.8 and R.sub.9 are each independently selected from alkyl,
cycloalkyl, aralkyl, cycloalkenyl, heterocyclyl and aryl, each of
which is optionally substituted with one or more R.sub.10 groups;
each R.sub.10 is independently selected from halogen, OH, .dbd.O,
CN, COO-alkyl, aralkyl, SO.sub.2-alkyl, SO.sub.2-aryl, COOH,
CO-alkyl, CO-aryl, NH.sub.2, NH-alkyl, N(alkyl).sub.2, CF.sub.3,
alkyl and alkoxy; X, Y and Z are each independently CR.sub.11.
R.sub.11 is H, alkyl or F; for use in treating a proteopathy and/or
a disorder associated with protein misfolding stress and in
particular with an accumulation of misfolded proteins.
[0015] A third aspect of the invention relates to a compound of
formula (I), or a pharmaceutically acceptable salt thereof,
##STR00005##
wherein:
R1 is Cl;
R2 is H;
[0016] R3 is selected from H and alkyl; R4 is selected from H and
C(O)R.sub.6;
R5 is H;
[0017] or R.sub.4 and R.sub.5 are linked to form a 5 to 6 membered
saturated or unsaturated heterocyclic group optionally comprising 1
or 2 heteroatoms such as N, in addition to the N atoms to which
R.sub.4 and R.sub.5 are bound, and where said heterocyclic group is
optionally substituted with one or more R.sub.10 groups; R6 is
selected from R7, OR7 and NR8R9; R7, R8 and R9 are each
independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclyl and aryl, each of which is optionally
substituted with one or more R10 groups; each R10 is independently
selected from halogen, OH, .dbd.O, CN, COO-alkyl, aralkyl,
SO2-alkyl, SO2-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl,
N(alkyl)2, CF3, alkyl and alkoxy; X, Y and Z are each independently
CR11; R11 is H, alkyl, or F; for use in treating a proteopathy
and/or a disorder associated with protein misfolding stress and in
particular with an accumulation of misfolded proteins.
[0018] A fourth aspect of the invention relates to a compound of
formula (I), or a pharmaceutically acceptable salt thereof,
##STR00006##
wherein: R1 is alkyl, O-alkyl, Cl, F or Br;
R2 is H or F;
[0019] R3 is selected from H and alkyl; R4 is selected from H and
C(O)R.sub.6;
R5 is H;
[0020] or R4 and R5 are linked to form a 5 to 6 membered saturated
or unsaturated heterocyclic group optionally comprising 1 or 2
heteroatoms such as N, in addition to the N atoms to which R.sub.4
and R.sub.5 are bound, and where said heterocyclic group is
optionally substituted with one or more R.sub.10 groups; R6 is
selected from R7, OR7 and NR8R9; R7, R8 and R9 are each
independently selected from alkyl, cycloalkyl, aralkyl,
cycloalkenyl, heterocyclyl and aryl, each of which is optionally
substituted with one or more R10 groups; each R10 is independently
selected from halogen, OH, .dbd.O, CN, COO-alkyl, aralkyl,
SO2-alkyl, SO2-aryl, COOH, CO-alkyl, CO-aryl, NH2, NH-alkyl,
N(alkyl)2, CF3, alkyl and alkoxy; X and Z are each independently
CR11, and Y is N; R11 is H, alkyl or F;
[0021] Previous studies have indicated that the aryl group must be
at least di-substituted in order for the compounds to exhibit
useful pharmacological activity (see for example, D.
Tribouillard-Tanvier et al., PLoS One 3, e1981 (2008) and
EP1908464A, CNRS). However, contrary to the results of previous
studies, the present Applicant has surprisingly found that
mono-substituted aryl derivatives are also active.
[0022] Moreover, compounds of formula (I) as defined above
advantageously exhibit no activity or low activity toward the
adrenergic .alpha.2A receptor relative to prior art compounds such
as Guanabenz. This loss in alpha-2 adrenergic activity renders the
compounds therapeutically useful in the treatment of proteopathies
and/or disorders associated with protein misfolding stress and in
particular with an accumulation of misfolded proteins. The absence
of alpha-2 adrenergic activity means that compounds of formula (I)
can be administered at a dosage suitable to treat the
aforementioned diseases, without any significant effect on blood
pressure.
[0023] A further aspect of the invention relates to pharmaceutical
compositions comprising a compound of formula (I) as described
above, admixed with a suitable pharmaceutically acceptable diluent,
excipient or carrier.
DETAILED DESCRIPTION
[0024] As used herein, the term "alkyl" includes both saturated
straight chain and branched alkyl groups. Preferably, the alkyl
group is a C.sub.1-20 alkyl group, more preferably a C.sub.1-15,
more preferably still a C.sub.1-12 alkyl group, more preferably
still, a C.sub.1-6 alkyl group, more preferably a C.sub.1-3 alkyl
group. Particularly preferred alkyl groups include, for example,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl,
pentyl and hexyl
[0025] As used herein, the term "cycloalkyl" refers to a cyclic
alkyl group. Preferably, the cycloalkyl group is a C.sub.3-12
cycloalkyl group.
[0026] As used herein, the term "alkenyl" refers to a group
containing one or more carbon-carbon double bonds, which may be
branched or unbranched. Preferably the alkenyl group is a
C.sub.2-20 alkenyl group, more preferably a C.sub.2-15 alkenyl
group, more preferably still a C.sub.2-12 alkenyl group, or
preferably a C.sub.2-6 alkenyl group, more preferably a C.sub.2-3
alkenyl group. The term "cyclic alkenyl" is to be construed
accordingly.
[0027] As used herein, the term "aryl" refers to a C.sub.6-12
aromatic group. Typical examples include phenyl and naphthyl
etc.
[0028] As used herein, the term "heterocycle" (also referred to
herein as "heterocyclyl" and "heterocyclic") refers to 4 to 12
membered, preferably 4 to 12 membered saturated, unsaturated or
partially unsaturated cyclic group containing one or more
heteroatoms selected from N, O and S, and which optionally further
contains one or more CO groups. The term "heterocycle" encompasses
both heteroaryl groups and heterocycloalkyl groups as defined
below.
[0029] As used herein, the term "heteroaryl" refers to a 4 to 12
membered aromatic, which comprises one or more heteroatoms.
Preferably, the heteroaryl group is a 4 to 12 membered aromatic
group comprising one or more heteroatoms selected from N, O and S.
Suitable heteroaryl groups include pyrrole, pyrazole, pyrimidine,
pyrazine, pyridine, quinoline, thiophene, 1,2,3-triazole,
1,2,4-triazole, thiazole, oxazole, iso-thiazole, iso-oxazole,
imidazole, furan and the like.
[0030] As used herein, the term "heterocycloalkyl" refers to a 4 to
12 membered cyclic aliphatic group which contains one or more
heteroatoms. Preferred heterocycloalkyl groups include piperidinyl,
pyrrolidinyl, piperazinyl, thiomorpholinyl and morpholinyl. More
preferably, the heterocycloalkyl group is selected from
N-piperidinyl, N-pyrrolidinyl, N-piperazinyl, N-thiomorpholinyl and
N-morpholinyl.
[0031] As used herein, the term "aralkyl" includes, but is not
limited to, a group having both aryl and alkyl functionalities. By
way of example, the term includes groups in which one of the
hydrogen atoms of the alkyl group is replaced by an aryl group,
e.g. a phenyl group. Typical aralkyl groups include benzyl,
phenethyl and the like.
[0032] In one preferred embodiment, R.sub.1 is Cl, Br, Me or F,
more preferably, Cl.
[0033] In one preferred embodiment, R.sub.2 is H.
[0034] In one preferred embodiment, Y is CR.sub.11.
[0035] In another preferred embodiment, Y is N.
[0036] In one preferred embodiment, R.sub.3 and R.sub.4 are both
H.
[0037] In one preferred embodiment, R.sub.3 is H and R.sub.4 is
C(O)R.sub.6.
[0038] In one preferred embodiment, R.sub.6 is alkyl or alkoxy,
more preferably, Me or OMe.
[0039] In one preferred embodiment, R.sub.4 and R.sub.5 are linked
to form a 5 to 6 membered saturated or unsaturated heterocyclic
group optionally comprising 1 or 2 heteroatoms such as N, in
addition to the N atoms to which R.sub.4 and R.sub.5 are bound, and
where said heterocyclic group is optionally substituted with one or
more R.sub.10 groups;
[0040] In one preferred embodiment, said compound is of formula
(Ia), or a pharmaceutically acceptable salt thereof,
##STR00007##
wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.10 are as defined
above.
[0041] In one especially preferred embodiment, the compound of
formula (I) is selected from the following:
##STR00008## ##STR00009##
and pharmaceutically acceptable salts thereof.
[0042] In a first preferred embodiment, the compound of formula (I)
is selected from Compound 1 i.e. 1-[[(2-chlorophenyl)
methylidene]amino]-guanidine and Compound 2, i.e.
1-[[(2-chlorophenyl) methylidene]amino]-guanidine acetate, as set
out above.
[0043] In a second preferred embodiment, the compound of formula
(I) is selected from Compound 8 as set out above.
[0044] In a third preferred embodiment, the compound of formula (I)
is selected from Compound 6 and Compound 7, as set out above.
Therapeutic Applications
[0045] The compounds of formula (I) have potential therapeutic
applications in treating proteopathies and/or disorders associated
with accumulation of misfolded and/or unfolded proteins. In
particular, compounds of formula (I) have a protective effect
against cytotoxic endoplasmic reticulum (ER) stress and age related
disorders.
[0046] Another aspect of the invention relates to the use of a
compound of formula (I) as defined above in the preparation of a
medicament for treating a disorder associated with protein
misfolding stress and in particular with an accumulation of
misfolded proteins.
[0047] Another aspect of the invention relates to the use of a
compound of formula (I) as defined above in the preparation of a
medicament for treating diseases where accumulation of misfolded
and/or unfolded proteins is involved in the mode of action (Brown
et al, 2012, Frontiers in Physiology, 3, Article 263).
[0048] Another aspect of the invention relates to the use of a
compound of formula (I) as defined above in the preparation of a
medicament for treating a proteopathy. The proteopathies refer to a
class of diseases in which certain proteins become structurally
abnormal, and thereby disrupt the function of cells, tissues and
organs of the body. Often the proteins fail to fold into their
normal conformation, and in this misfolded and/or unfolded state,
the proteins can become toxic in some way (a gain of toxic
function) or they can lose their normal function or they can have a
reduce biological activity. The proteopathies, also known as
proteinopathies, protein conformational disorders, or protein
misfolding diseases, include many diseases such diseases as
Alzheimer's disease, Parkinson's disease, prion disease, type 2
diabetes, amyloidosis, and a wide range of other disorders (see non
limiting examples below).
[0049] As used herein the terms "proteinopathies, proteopathies,
protein conformational disorders, protein misfolding diseases,
diseases associated with protein misfolding stress, diseases
associated with an accumulation of misfolded protein, diseases
associated with a cytotoxic ER stress, UPR related diseases
associated with have the same meaning and refer to diseases wherein
certain protein become structurally abnormal and thereby disrupt
the cellular homeostasis.
[0050] As used herein the terms "misfolded protein" and "unfolded
protein" has the same meaning and refer to protein that fail to
fold into their normal conformation.
[0051] As used herein the phrase "preparation of a medicament"
includes the use of one or more of the above described compounds
directly as the medicament in addition to its use in a screening
programme for further active agents or in any stage of the
manufacture of such a medicament.
[0052] Yet another aspect of the invention relates to a method of
treating a proteinopathy and/or a disorder associated with protein
misfolding stress and/or with a cytotoxic ER stress and in
particular with an accumulation of misfolded proteins in a subject
in need thereof, said method comprising administering a
therapeutically effective amount of a compound of formula (I) as
defined above to said subject.
[0053] The term "method" refers to manners, means, techniques and
procedures for accomplishing a given task including, but not
limited to, those manners, means, techniques and procedures either
known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0054] Herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
disease or disorder, substantially ameliorating clinical symptoms
of a disease or disorder or substantially preventing the appearance
of clinical symptoms of a disease or disorder.
[0055] As used herein, the term disease , disorder , conditions has
the same meaning. The disease is associated with an ER stress
response activity and/or is associated with protein misfolding
stress and in particular with an accumulation of misfolded
proteins.
[0056] The term "therapeutically effective amount" refers to that
amount of the compound being administered which will relieve to
some extent one or more of the symptoms of the disease or disorder
being treated.
[0057] In another embodiment, the invention relates to a compound
of formula (I) as defined above for use in treating UPR disorders.
The unfolded protein response (UPR) is a component of the cellular
defense system against misfolded proteins that adapts folding in
the endoplasmic reticulum (ER) to changing conditions. The UPR is
activated in response to an accumulation of unfolded or misfolded
proteins in the lumen of the endoplasmic reticulum. In this
scenario, the UPR has two primary aims: (i) to restore normal
function of the cell by halting protein translation, and (ii) to
activate the signaling pathways that lead to the increased
production of molecular chaperones involved in protein folding. If
these objectives are not achieved within a certain time frame, or
the disruption is prolonged, the UPR aims towards apoptosis.
Upstream components of the UPR are the ER-resident trans-membrane
proteins IRE1, ATF6, and PERK, which sense folding defects to
reprogram transcription and translation in a concerted manner and
restore proteostasis. Activated IRE1 and ATF6 increase the
transcription of genes involved in ER folding, such as those
encoding the chaperones BiP and GRP94. Activated PERK attenuates
global protein synthesis by phosphorylating the subunit of
translation initiation factor 2 (eIF2.alpha.) on Ser51 while
promoting translation of the transcription factor ATF4. The latter
controls expression of CHOP, another transcription factor, which in
turn promotes expression of PPP1R15A/GADD34. PPP1R15A, an effector
of a negative feedback loop that terminates UPR signaling, recruits
a catalytic subunit of protein phosphatase 1 (PP1c) to
dephosphorylate eIF2.alpha., allowing protein synthesis to resume.
UPR failure contributes to many pathological conditions that might
be corrected by adequate boost of this adaptive response. Selective
inhibitors of the stressed-induced eIF2.alpha. phosphatase
PPP1R15A-PP1 delays eIF2.alpha. dephosphorylation and consequently
protein synthesis selectively in stressed cells, without affecting
protein synthesis in unstressed cells. This prolongs the beneficial
effects of the UPR. A transient reduction of protein synthesis is
beneficial to stressed cells because decreasing the flux of
proteins synthetized increases the availability of chaperones and
thus protects from misfolding stress (Tsaytler et al., Science
2011). Non-selective inhibitors of the 2 eIF2.alpha. phosphatases
might have undesirable effects, as persistent translation
inhibition is deleterious. Indeed, genetic ablation of both
PPP1R15A and PPP1R15B results in early embryonic lethality in mice
indicating that inhibition of the two eIF2.alpha. phosphatases
PPP1R15A-PP1 and PPP1R15B-PP1 is deleterious in an organismal
context. In contrast, genetic ablation of PPP1R15A has no harmful
consequence in mice (Harding et al., 2009, Proc Natl Acad Sci USA,
106, 1832-1837). Furthermore, specific inhibitors of PPP1R15A are
predicted to be inert in unstressed cells, as the PPP1R15A is not
expressed in absence of stress. Thus, selective PPP1R15A inhibitors
are predicted to be safe. Non-selective inhibitors of the two
eIF2.alpha. phosphatases may also be useful to treat protein
misfolding diseases, when used at doses that result in only a
partial inhibition of the phosphatases.
[0058] Cytoprotection against ER stress can be measured by a
suitable assay. For example, cytoprotection can be measured in HeLa
cells in which ER stress is elicited by the addition of media
containing tunicamycin, a mixture of homologous nucleoside
antibiotics that inhibits the UDP-HexNAc: polyprenol-P HexNAc-1-P
family of enzymes and is used to induce unfolded protein response.
Cell viability can be detected in the presence and absence of
inhibitor compounds after a set period of time, by measuring the
reduction of WST-8 into formazan using a standard cell viability
kit (such as Cell Viability Counting Kit-8 from Dojindo). The man
skilled in the art may use other class of tetrazolium compounds
such as MTT, MTS, XTT. Cytoprotection from ER stress is measured in
terms of the percentage increase in viable cells (relative to
control) after ER stress. Alternative cell viability assays may be
used such as luminogenic ATP assay. Further details of a suitable
assay are set forth in the accompanying Examples section.
[0059] In one preferred embodiment, the compound of formula (I) is
capable of prolonging the protective effect of the UPR relative to
the control (i.e. in the absence of inhibitor compound) by at least
10%, at least 20%, more preferably, at least 30%, even more
preferably, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, more preferably still, at least 90%.
[0060] The compounds of formula (I) are inhibitors of PPP1R15A-PP1
interaction which induce a protective effect. Preferably, the
compound exhibits a protective effect with EC.sub.50 of less than
about 10 M, even more preferably, less than about 5 .mu.M, more
preferably still, less than about 1 .mu.M. The compound should
preferably be devoid of alpha2 adrenergic activity. Thus, in one
preferred embodiment the compound does not exhibit any activity in
a functional alpha-2-adrenergic assay.
[0061] Certain compounds of formula (I) selectively inhibit
PPP1R15A-PP1, and thus prolong the protective effect of the UPR,
thereby rescuing cells from protein misfolding stress. Inhibitors
of PPP1R15A-PP1 described in the present invention therefore have
therapeutic applications in the treatment of a variety of diseases
associated with protein misfolding stress and in particular with an
accumulation of misfolded proteins and/or proteinopathies.
[0062] In one embodiment, the compound of formula (I) is capable of
inhibiting PPP1R15A and PPP1R15B. In highly preferred embodiment,
the compound of formula (I) is capable of selectively inhibiting
PPP1R15A over PPP1R15B.
[0063] In one embodiment, the invention relates to a compound of
formula (I) as defined above for use in treating a disorder
associated with the eIF2.alpha. phosphorylation pathway where
accumulation of misfolded proteins is involved in the mode of
action. Preferably, the disorder is a PPP1R15A-related disease.
Examples of such disorders include protein misfolding diseases
and/or proteinopathies.
[0064] In another embodiment, the invention relates to a compound
of formula (I) as defined above for use in treating a disorder
caused by, associated with or accompanied by eIF2.alpha.
phosphorylation and/or PPP1R15A activity where accumulation of
misfolded proteins is involved in the mode of action.
[0065] As used herein, "PPP1R15A related disease or disorder"
refers to a disease or disorder characterized by abnormal PPP1R15A
activity where accumulation of misfolded proteins is involved in
the mode of action. Abnormal activity refers to: (i) PPP1R15A
expression in cells which normally do not express PPP1R15A; (ii)
increased PPP1R15A expression; or, (iii) increased PPP1R15A
activity.
[0066] In another embodiment, the invention relates to a method of
treating a mammal having a disease state alleviated by the
inhibition of PP1R15A, where accumulation of misfolded proteins is
involved in the mode of action, wherein the method comprises
administering to a mammal a therapeutically effective amount of a
compound of formula (I) as defined above.
[0067] In another embodiment, the invention relates to a PPP1R15A
inhibitor of formula (I) or a pharmaceutical acceptable salt
thereof for the use in treating disorders associated with protein
misfolding stress and in particular with an accumulation of
misfolded proteins and/or UPR disorders, wherein said compound has
no or reduced adrenergic alpha 2 agonist activity in comparison
with Guanabenz.
[0068] In another embodiment, the invention relates to a PPP1R15A
inhibitor of formula (I) or a pharmaceutical acceptable salt
thereof for the use in treating disorders associated with protein
misfolding stress and in particular with an accumulation of
misfolded proteins and/or UPR disorders, wherein said compound does
not inhibit protein translation in non-stressed cells expressing
PPP1R15B.
[0069] In another embodiment, the invention relates to a method of
treating a disorder characterized by ER stress response activity
with an accumulation of misfolded proteins, the method comprising
administering to a patient a therapeutically effective amount of at
least one compound of formula (I) wherein said compound modulates
ER stress response.
[0070] In another embodiment, the invention relates to PPP1R15A
inhibitor of formula (I) or a pharmaceutical acceptable salt
thereof for the use in treating proteopathies and/or disorders
associated with protein misfolding stress and in particular with an
accumulation of misfolded proteins and/or UPR disorders, wherein
said compound has a selectivity towards PPP1R15A-PP1
holophosphatase, having but no or reduced activity towards
PPP1R15B-PP1 holophosphatase, and wherein the ratio (activity
towards PPP1R15A-PP1 holophosphatase/activity towards PPP1R15B-PP1)
for said compound is at least equal or superior to the ratio
(activity towards PPP1R15A-PP1 holophosphatase/activity towards
PPP1R15B-PP1) for Guanabenz.
[0071] In another embodiment, the invention relates to a PPP1R15A
inhibitor of formula (I) or a pharmaceutical acceptable salt
thereof for the use in treating proteopathies and/or disorders
associated with protein misfolding stress and in particular with an
accumulation of misfolded proteins and/or UPR disorders: [0072]
wherein said compound has an activity towards PPP1R15A-PP1
holophosphatase but no or reduced activity towards PPP1R15B-PP1
holophosphatase, and; [0073] wherein the ratio (activity towards
PPP1R15A-PP1 holophosphatase/activity towards PPP1R15B-PP1) for
said compound is at least equal or superior to the ratio (activity
towards PPP1R15A-PP1 holophosphatase/activity towards PPP1R15B-PP1)
for Guanabenz; and [0074] wherein said compound has no or reduced
adrenergic alpha 2 agonist activity in comparison with
Guanabenz.
[0075] The disease or disorders according to the invention is:
[0076] (i) associated with an ER stress response activity; and/or
[0077] (ii) protein misfolding stress and in particular with an
accumulation of misfolded and/or unfolded proteins; and/or [0078]
(iii) an UPR disorder; and/or [0079] (iv) PPP1R15A related disease;
and/or [0080] (v) A proteopathy.
[0081] Non limiting examples of disease according to the invention
include, but are not limited to:
[0082] Neurodegenerative diseases such as tauopathies (such as
Alzheimer's disease among others), synucleinopathies (such as
Parkinson disease among others), Huntington disease and related
polyglutamine diseases, polyalanine diseases (such as
oculo-pharyngeal muscular dystrophy), prion diseases (also named
transmissible spongiform encephalopathies), demyelination disorders
such as Charcot-Marie Tooth diseases (also named hereditary motor
and sensory neuropathy), leukodystrophies, amyotrophic lateral
sclerosis (also referred to as motor neurone disease and as Lou
Gehrig's disease), seipinopathies and multiple sclerosis.
[0083] Examples of tauopathies include, but are not limited to
Alzheimer's disease, progressive supranuclear palsy, corticobasal
degeneration, frontotemporal lobar degeneration or frontotemporal
dementia (FTD) (Pick's disease). FTD is a neurodegenerative disease
characterized by progressive neuronal loss predominantly involving
the frontal and/or temporal lobes; second only to Alzheimer's
disease (AD) in prevalence, FTD accounts for 20% of young onset
dementia cases. The involvement of UPR in tauopathies is well
documented (see Stoveken 2013, The Journal of Neuroscience
33(36):14285-14287). Without to be bound by a theory, it is
anticipated that compounds of the invention which are PPP1R15A
inhibitors will ameliorate disease manifestations of tauopathies.
According to a preferred embodiment, the invention relates to a
PPP1R15A inhibitor of formula (I) or a pharmaceutical acceptable
salt thereof for the use in treating Alzheimer disease. According
to a another preferred embodiment, the invention relates to a
PPP1R15A inhibitor of formula (I) or a pharmaceutical acceptable
salt thereof for the use in treating a disease selected among
frontotemporal dementia (FTD), supranuclear palsy and corticobasal
degeneration, preferably FTD.
[0084] Examples of synucleinopathies include, but are not limited
to Parkinson's disease, dementia with Lewy bodies, pure autonomic
failure, and multiple system atrophy. Recently, Colla et al. (J. of
Neuroscience 2012 Vol. 32 N.sup.o 10 pp 3306-3320) demonstrated
that Salubrinal a small molecule that increases the phosphorylation
of eIF2 alpha by inhibiting the PPP1R15A mediated dephosphorylation
of eIF2.alpha. (Boyce et al. 2005 Science Vol. 307 pp 935-939),
significantly attenuates disease manifestations in two animal
models of alpha-synucleinopathy. Without to be bound by a theory,
it is anticipated that compounds of the invention which are
PPP1R15A inhibitors will ameliorate disease manifestations of
alpha-syncleinopathies. According to a preferred embodiment, the
invention relates to a PPP1R15A inhibitor of formula (I) or a
pharmaceutical acceptable salt thereof for the use in treating
alpha-syncleinopathies. According to a preferred embodiment, the
invention relates to a PPP1R15A inhibitor of formula (I) or a
pharmaceutical acceptable salt thereof for the use in treating
Parkinson's disease.
[0085] Examples of polyglutamine diseases include but are not
limited to Spinobulbar muscular atrophy (or Kennedy disease),
Huntington disease, Dentatorubral-pallidoluysian atrophy,
Spinocerebellar ataxia type 1, Spinocerebellar ataxia type 2,
Spinocerebellar ataxia type 3 (or Machado-Joseph disease),
Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7 and
Spinocerebellar ataxia type 17. Guanabenz is able to attenuate the
accumulation of mutant Huntingtin in cell-based assays
(WO2008/041133). This finding is unexpected since mutant huntingtin
is either cytosolic or nuclear. However, there is evidence that
mutant huntingtin metabolism has previously been connected to the
ER stress response (Nishitoh et al., 2002, Genes Dev, 16, 1345-55;
Rousseau et al., 2004, Proc Natl Acad Sci USA, 101, 9648-53;
Duennwald and Lindquist, 2008, Genes Dev, 22, 3308-19). The
findings that guanabenz protects cells from cytotoxic ER stress and
reduces mutant huntingtin accumulation further supports the idea
that there may be aspects of the ER stress response that impact on
mutant huntingtin accumulation. However, Guanabenz is not useful
for the treatment of human protein misfolding diseases due to its
hypotensive activity. In contrast, the Guanabenz derivative
PPP1R15A inhibitors devoid of alpha2 adrenergic activity of the
invention could be useful to treat polyglutamine diseases and more
specifically selected in the group of Huntington disease,
Spinobulbar muscular atrophy (or Kennedy disease),
Dentatorubral-pallidoluysian atrophy, Spinocerebellar ataxia type
1, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3 (or
Machado-Joseph disease), Spinocerebellar ataxia type 6,
Spinocerebellar ataxia type 7 and Spinocerebellar ataxia type 17.
According to a preferred embodiment, the invention relates to a
PPP1R15A inhibitor of formula (I) or a pharmaceutical acceptable
salt thereof for the use in treating polyglutamine disease.
According to a preferred embodiment, the invention relates to a
PPP1R15A inhibitor of formula (I) or a pharmaceutical acceptable
salt thereof for the use in treating Huntington disease.
[0086] Example of polyalanine diseases include oculo-pharyngeal
muscular dystrophy which is caused by poly-alanine tract in poly(A)
binding protein nuclear 1 (PABPN1). Barbezier et al. (2011, EMBO
Vol. 3 pp 35-49) demonstrated that Guanabenz reduces aggregation in
oculopharyngeal muscular atrophy. According to a preferred
embodiment, the invention relates to a PPP1R15A inhibitor of
formula (I) or a pharmaceutical acceptable salt thereof for the use
in treating polyalanine disease. According to a preferred
embodiment, the invention relates to a PPP1R15A inhibitor of
formula (I) or a pharmaceutical acceptable salt thereof for the use
in treating oculopharyngeal muscular atrophy.
[0087] Examples of prion diseases of humans include but are not
limited to classic Creutzfeldt-Jakob disease, new variant
Creutzfeldt-Jakob disease (nvCJD, a human disorder related to
Bovine spongiform encephalopathy), Gerstmann-Strassler-Scheinker
syndrome, fatal familial insomnia and kuru. Guanabenz reduces the
symptoms of prion infected mice (D. Tribouillard-Tanvier et al.,
2008 PLoS One 3, e1981). However, Guanabenz is not useful for the
treatment of human protein misfolding diseases due to its
hypotensive activity. In contrast, the Guanabenz derivative
PPP1R15A inhibitors devoid of alpha2 adrenergic activity of the
invention could be useful to treat prion diseases. According to a
preferred embodiment, the invention relates to a PPP1R15A inhibitor
of formula (I) or a pharmaceutical acceptable salt thereof for the
use in treating a disease selected in the group of
Creutzfeldt-Jakob disease, new variant Creutzfeldt-Jakob disease,
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia
and kuru. Demyelination disorders are characterized by a loss of
oligodendrocytes in the central nervous system or Schwann cells in
the peripheral nervous system. The phenomenon associated with a
demyelination disorder is characterized by a decrease in myelinated
axons in the central nervous system or peripheral nervous system.
Non-limiting exemples of misfolded proteins of a myelinating cell
(including oligodendrocyte and Schwann cell) is selected from the
group consisting of CC1, myelin basic protein (MBP), ceramide
galactosyltransferase (CGT), myelin associated glycoprotein (MAG),
myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte-myelin
glycoprotein (OMG), cyclic nucleotide phosphodiesterase (CNP),
myelin protein zero (MPZ), peripheral myelin protein 22 (PMP22),
Connexin 32 (Cx32), protein 2 (P2), galactocerebroside (GaIC),
sulfatide and proteolipid protein (PLP). MPZ, PMP22, Cx32 and P2
are preferred misfolded proteins for Schwann cells. PLP, MBP, MAG
are preferred misfolded proteins for oligodendrocytes.
[0088] In certain embodiments, the demyelination disorder is
selected from the group consisting of Charcot-Marie Tooth (CMT)
diseases. CMT refer to a group of hereditary neuropathy disorders
characterized by a chronic motor and sensory polyneuropathy.
Different types of CMT were identified such as CMT1, CMT2, CMT4,
CMTX and Dejerine-Sottas disease. CMT subtypes may be further
subdivided primarily on molecular genetic findings. For examples
CMT1 is subdivided in CMT1A, 1B, 10, 1D, 1E, 1F/2E. Over a 100
mutations in the gene encoding myelin protein zero (P0), a
single-pass transmembrane protein, which is the major protein
produced by myelinating Schwann cells causes Charcot-Marie-Tooth
neuropathy (D'Antonio et al., 2009, J Neurosci Res, 87, 3241-9).
The mutations are dominantly inherited and cause the disease
through a gain of toxic function (D'Antonio et al., 2009, J
Neurosci Res, 87, 3241-9). Deletion of serine 63 from PO (P0S63del)
causes Charcot-Marie-Tooth 1B neuropathy in humans and a similar
demyelinating neuropathy in transgenic mice. The mutant protein
accumulates in the ER and induces the UPR (D'Antonio et al., 2009,
J Neurosci Res, 87, 3241-9). Genetic ablation of CHOP, a
pro-apoptotic gene in the UPR restores motor function in
Charcot-Marie-Tooth mice (Pennuto et al., 2008, Neuron, 57,
393-405). The finding that PPP1R15A inhibition in cells nearly
abolishes CHOP expression in ER-stressed cells indicates that
genetic or pharmacological inhibition of PPP1R15A should reduce
motor dysfunction in Charcot-Marie-Tooth mice. Recently, D'Antonio
et al. (2013 J. Exp. Med Vol. pp 1-18) demonstrated that P0S63del
mice treated with salubrinal, regained almost normal motor capacity
in rotarod analysis and was accompanied by a rescue of
morphological and electro-physiological abnormalities. Accumulation
of the of CMT-related mutant in the ER proteins is not unique to
P0S63del; at least five other PO mutants have been identified that
are retained in the ER and elicit an UPR (Pennuto et al., 2008
Neuron Vol. 57 pp 393-405; Saporta et al., 2012 Brain Vol. 135 pp
2032-2047). In addition, protein misfolding and accumulation of
misfolded protein in the ER have been implicated in the
pathogenesis of other CMT neuropathies as a result of mutations in
PMP22 and Cx32 (Colby et al., 2000 Neurobiol. Disease Vol. 7 pp
561-573; Kleopa et al., 2002 J. Neurosci. Res. Vol. 68 pp 522-534;
Yum et al., 2002 Neurobiol. Dis. Vol. 11 pp 43-52). However,
Salubrinal is toxic and cannot be used to treat human patients
D'Antonio et al. (2013 J. Exp. Med Vol. pp 1-18). In contrast, the
PPP1R15A inhibitors of formula (I) are predicted to be safe and
could be useful for the treatment of CMTs, preferably CMT-1A and
1B. According to a preferred embodiment, the invention relates to a
PPP1R15A inhibitor of formula (I) or a pharmaceutical acceptable
salt thereof for the use in treating CMT, more preferably CMT-1 and
Dejerine-Sottas disease. According to a preferred embodiment, the
invention relates to a PPP1R15A inhibitor of formula (I) or a
pharmaceutical acceptable salt thereof for the use in treating CMT
associated with an accumulation of misfolded protein in the ER.
According to a preferred embodiment, the invention relates to a
PPP1R15A inhibitor of formula (I) or a pharmaceutical acceptable
salt thereof for the use in treating CMT-1A. According to a
preferred embodiment, the invention relates to a PPP1R15A inhibitor
of formula (I) or a pharmaceutical acceptable salt thereof for the
use in treating CMT-1B. According to a preferred embodiment, the
invention relates to a PPP1R15A inhibitor of formula (I) or a
pharmaceutical acceptable salt thereof for the use in treating
CMT-1E.
[0089] In another embodiment, the compound of formula (I) is for
use in treating CMT, more preferably for use in treating CMT-1, in
association with at least one compound selected in the group of
D-Sorbitol, baclofen, pilocarpine, naltrexone, methimazole,
mifepristone, ketoprofene and salts thereof. According to another
embodiment, the invention relates to guanabenz or salubrinal (i.e.
PPP1R15A inhibitors) or a pharmaceutical acceptable salt thereof
for the use in treating CMT, preferably CMT-1, in association with
at least one compound selected in the group of D-Sorbitol,
baclofen, pilocarpine, naltrexone, methimazole, mifepristone,
ketoprofene and salts thereof. The compounds are combined for a
grouped or separate administration, simultaneously or
sequentially.
[0090] The invention relates to composition comprising a PPP1R15A
inhibitor selected in the group of compound of formula (I),
guanabenz and salubrinal or a pharmaceutical acceptable salt
thereof, and at least one marketed compound and salts thereof, for
use in the treatment of neurodegenerative diseases, preferably CMT,
more preferably CMT-1. The dosage of compounds in the composition
shall lie within the range of doses not above the usually
prescribed doses for long term maintenance treatment or proven to
be safe on phase 3 clinical trial; the most preferred dosage of
compounds in the combination shall corresponds to amounts for 1% up
to 10% of those usually prescribes for long term maintenance
treatment.
[0091] Thus, the invention relates to composition comprising a
PPP1R15A inhibitor selected in the group of compound of formula
(I), guanabenz and salubrinal or a pharmaceutical acceptable salt
thereof, and a compound increasing the expression of PM P22
protein, selected in the group of D-Sorbitol, baclofen,
pilocarpine, naltrexone, methimazole, mifepristone, ketoprofene and
salts thereof, for use in the treatment of CMT, preferably CMT-1,
more preferably CMT-1A.
[0092] In other embodiments, the demyelination disorder is selected
from the group consisting of leukodystrophies. Examples of
leukodystrophies include but are not limited to
adrenoleukodystrophy (ALD), Alexander disease, Canavan disease,
Krabbe disease, Metachromatic Leukodystrophy (MLD),
Pelizaeus-Merzbacher disease (PMD), childhood ataxia with central
nervous system hypomyelination (also known as vanishing white
matter disease), CAMFAK syndrome, Refsum Disease, Cockayne
Syndrome, Ver der Knapp Syndrome, Zellweger Syndrome,
Guillain-Barre Syndrome (GBS), chronic inflammatory demyelinating
polyneuropathy (CIDP), multifocual motor neuropathy (MMN) and
progressive supernuclear palsy, progressive Multifocal
Leuko-encephalopathy (PML), Encephalomyelitis, Central Pontine
Myelolysis (CPM), Anti-MAG Disease, among others. Gow et al.
(Neuron, 2002 Vol. 36, 585-596) demonstrated that the unfolded
protein response is activated in PMD, and show that this pathway is
duplication of, the PLP1 gene.
[0093] According to a preferred embodiment, the invention relates
to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof for the use in treating leukodystrophies,
and preferably Pelizaeus-Merzbacher disease (PMD).
[0094] Amyotrophic lateral sclerosis (ALS) is referred to as motor
neurone disease and as Lou Gehrig's disease. It is now well
recognized that protein misfolding plays a central role in both
familial and sporadic ALS (Matus et al. 2013 Int. J. Cell Biol.
ID674751 http://dx.doi.org/10.1155/2013/674751). Saxena et al.
(Nature Neuroscience 2009 Vol. 12 pp 627-636) demonstrated that
Salubrinal extends the life span of a G93A-SOD1 transgenic mouse
model of motor neuron disease. More recently, Jiang et al.
(Neuroscience 2014) demonstrated that Guanabenz delays the onset of
disease symptoms, extends lifespan, improves motor performance and
attenuates motor neuron loss in the SOD1 G93A mouse model of ALS.
Das et al. (2015 Science 388, 239-242) demonstrated that a
guanabenz derivative prevents the motor, morphological and
molecular defects of ALS in mutant G93A SOD1 mice. According to a
preferred embodiment, the invention relates to a PPP1R15A inhibitor
of formula (I) or a pharmaceutical acceptable salt thereof for the
use in treating familial and sporadic forms of ALS.
[0095] Examples of seipinopathies include, but are not limited to
Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2)-related
motor disease, congenital generalized lipodystrophy (CGL), Silver
syndrome, distal hereditary motor neuropathy type V (dHMN-V). The
expression of mutant forms of seipin in cultured cells activates
the unfolded protein response (UPR) pathway and induces ER
stress-mediated cell death (Ito & Suzuki, 2009 Brain 132:
87-15). According to a preferred embodiment, the invention relates
to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof for the use in treating seipinopathy.
[0096] In another embodiment, the demyelination disorder referred
therein is multiple sclerosis and related disease such as
Schilder's disease. According to a preferred embodiment, the
invention relates to a PPP1R15A inhibitor of formula (I) or a
pharmaceutical acceptable salt thereof for the use in treating
multiple sclerosis.
Cystic fibrosis (CF)
[0097] Norez et al. (2008 Eur. J. Pharmacol. Vol. 592 pp 33-40)
demonstrated that Guanabenz activates Ca.sup.2+ dependent chloride
currents in cystic fibrosis human airway epithelial cells. Without
to be bound by a theory, it is anticipated that compounds of the
invention which are guanabenz derivative PPP1R15A inhibitors will
ameliorate disease manifestations of cystic fibrosis. According to
a preferred embodiment, the invention relates to a PPP1R15A
inhibitor of formula (I) or a pharmaceutical acceptable salt
thereof for the use in treating cystic fibrosis.
Retinal Diseases.
[0098] Recently published literature has provided evidences that
the UPR is involved in the development of retinal degeneration:
inherited retinal degeneration such as retinal ciliopathies &
retinitis pigmentosa, macular degeneration, retinopathy of
premarurity, light-induced retinal degeneration, retinal
detachment, diabetic retinopathy and glaucoma (for review Gorbatyuk
et Gorbatyuk 2013 Molecular Vision Vol. 19 pp 1985-1998; Jing et
al., 2012, Exp Diabetes Res, 2012, 589589). Retinal ciliopathies
are a group of rare genetic disorders originating from a defect in
the primary cilium of photoreceptors thus inducing retinitis
pigmentosa. This defect has been reported to induce an ER stress
due to protein accumulation in the inner segment of the
photoreceptor which in turn induces the UPR (WO2013/124484).
Retinal degeneration is a very common feature in ciliopathies that
can be observed either in isolated retinitis pigmentosa such as
Leber's congenital amaurosis or X-linked retinitis pigmentosa, or
also in syndromic conditions like the Bardet-Biedl Syndrome (BBS),
the Alstrom syndrome (ALMS) or the Usher syndrome. The retinal
ciliopathy is selected from the group consisting of Bardet-Biedl
syndrome, Senior-Loken syndrome, Joubert syndrome, Salidono-Mainzer
syndrome, Sensenbrenner syndrome, Jeune syndrome, Meckel-Gruder
syndrome, Alstrom syndrome, MORM syndrome. In one preferred
embodiment, the compound of formula (I) is for use in treating
retinal diseases, more preferably, inherited retinal degeneration
such as retinal ciliopathies & retinitis pigmentosa, macular
degeneration, retinopathy of premarurity, light-induced retinal
degeneration, retinal detachment, diabetic retinopathy and
glaucoma.
[0099] According to a preferred embodiment, the invention relates
to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof for the use in treating retinitis
pigmentosa. According to a preferred embodiment, the invention
relates to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof for the use in treating Leber's congenital
amaurosis. According to another preferred embodiment, the invention
relates to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof for the use in treating Bardet-Biedl
syndrome. According to another preferred embodiment, the invention
relates to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof for the use in treating Alstrom syndrome.
According to another preferred embodiment, the invention relates to
a PPP1R15A inhibitor of formula (I) or a pharmaceutical acceptable
salt thereof for the use in treating Husher syndrome.
[0100] In preferred embodiment, the compound of formula (I) is for
use in treating retinal diseases, more preferably for use in
treating diseases selected in the group of inherited retinal
degeneration such as retinal ciliopathies, retinitis pigmentosa,
macular degeneration, retinopathy of premarurity, light-induced
retinal degeneration, retinal detachment, diabetic retinopathy and
glaucoma in association with a compound increasing the expression
and/or the activity of BIP protein, such as Valproic acid or a
derivative thereof, trichostatin A, lithium,
1-(3,4-dihydroxy-penyl)-2-thiocyanate-ethanone and exendin-4. Thus,
the invention relates to composition comprising a PPP1R15A
inhibitor of formula (I) or a pharmaceutical acceptable salt
thereof and a compound increasing the expression and/or the
activity of BIP protein, preferably Valproic acid, for use in the
treatment of diseases selected in the group of inherited retinal
degeneration such as retinal ciliopathies, retinitis pigmentosa,
macular degeneration, retinopathy of premarurity, light-induced
retinal degeneration, retinal detachment, diabetic retinopathy and
glaucoma.
[0101] In preferred embodiment, the compound of formula (I), is for
use in treating retinal diseases, more preferably for use in
treating diseases selected in the group of inherited retinal
degeneration such as retinal ciliopathies, retinitis pigmentosa,
macular degeneration, retinopathy of premarurity, light-induced
retinal degeneration, retinal detachment, diabetic retinopathy and
glaucoma in association with a gene therapy vectors, Non limiting
examples of gene therapy vectors include lentivirus, adenovirus,
and adeno-associated vectors (AAVs); these vectors are effective in
delivering genes of interest to the retina and retinal pigment
epithelium for ocular gene therapy. It is anticipated that in an
ocular gene therapy of inherited retinal degeneration associated
with an accumulation of mutated misfolded proteins, protein
accumulation in the endoplasmic reticulum will remain present while
a normal protein is expressed from the gene therapy vector. It
remains the need to decrease the protein accumulation/load in the
cell, preferably in the ER with PPP1 R15A inhibitors. The invention
also relates to composition comprising PPP1 R15A inhibitor selected
in the group of compound of formula (I), guanabenz and salubrinal
or a pharmaceutical acceptable salt thereof, in combination with
ocular gene therapy.
Lysosomal Storage Diseases;
[0102] Lysosomal storage diseases are a group of approximately 50
rare inherited metabolic disorders that result from defects in
lysosomal function. The lysosomal dysfunction is usually the
consequence of deficiency of a single enzyme required for the
metabolism of lipids, glycoproteins or so-called
mucopolysaccharides. Examples of lysosomal storage diseases which
can be treated with by PPP1 R15A inhibitors of formula (I))
described herein include, but are not limited to, Activator
Deficiency/GM2 gangliosidosis, alpha-mannosidosis,
aspartylglucosaminuria, cholesteryl ester storage disease,
cystinosis, Danon disease, Fabry disease, Farber disease,
Niemann-Pick disease, fucosidosis, galactosialidosis, Gaucher
disease (Types I, II, II), GM1 gangliosidosis (infantile, late
infantile/juvenile, adult/chronic), I-cell disease/Mucolipidosis,
Infantile free sialic acid storage disease/ISSD, Juvenile
hexosaminidase A deficiency, Krabbe disease (infantile onset, late
onset), lysosomal acid lipase deficiency (early onset/late onset),
metachromatic leukodystrophy, mucopolysaccharidoses disorders (such
as Pseudo-Hurler polydystrophy/mucolipidosis IIIA,
mucopolysaccharidosis I (MPS I) Hurler syndrome, MPS I Scheie
syndrome, MPS I Hurler-Scheie syndrome, MPS II Hunter syndrome,
Sanfilippo syndrome Type A (MPS IIIA), Sanfilippo syndrome Type B
(MPS IIIB), Sanfilippo syndrome Type C (MPS IIIC), Sanfilippo
syndrome Type D (MPS IIID), Morquio Type A/MPS IVA, Morquio Type
B/MPS IVB, MPS IX hyaluronidase deficiency, MPS VI Maroteaux-Lamy,
MPS VII Sly syndrome, mucopolylipidosis 1/sialidosis, mucolipidosis
IIIC, mucolipidosis type IV(multiple sulfatase deficiency,
Niemann-Pick disease (Types A, B, C), CLN6 disease (atypical late
infantile, late onset variant, early juvenile),
Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease, Finnish Variant
late infantile CLN5, Jansky-Bielschosky disease/late infantile
CLN2/TPP1 disease, Kufs/Adult-onset NCL/CLN4 disease, Northern
epilepsy/variant late infantile CLN8, Santavuori-Haltia/infantile
CLN1/PPT disease, beta-mannosidosis, Pompe disease/glycogen storage
disease type II, pycnodysostosis, Sandhoff disease/GM2
gangliosidosis (adult onset, infantile onset, juvenile onset),
Schindler disease, Sall disease/sialic acid storage disease,
Tay-Sachs/GM2 gangliosidosis, and Wolman disease. According to
another preferred embodiment, the invention relates to a PPP1R15A
inhibitor of formula (I) or a pharmaceutical acceptable salt
thereof for the use in treating lysosomal storage diseases which
are the consequence of deficiency of at least one single enzyme
required for the metabolism of lipids, glycoproteins or so-called
mucopolysaccharides and wherein said enzyme is misfolded in the
endoplasmic reticulum (ER). According to a preferred embodiment,
the lysosomal storage disease is Gaucher disease.
Amyloidosis Diseases:
[0103] Amyloidosis is a non-specific term that refers to a number
of different diseases collectively called amyloidosis. Amyloids are
proteins whose secondary structure change, causing the proteins to
fold in a characteristic form, the beta-pleated sheet. When the
normally soluble proteins fold to become amyloids, they become
insoluble, deposit and accumulate in organs or tissues, disrupting
normal function. Different types of amyloidosis have different
signs and symptoms depending on where and in which organs the
amyloid proteins aggregate. Example of amyloidosis diseases
includes, but are not limited to, AL, AH, ALH amyloidosis (amyloid
derived from light-chain, heavy-chain, heavy and light chain
antibodies respectively), AA amyloidosis (amyloid derived from
derived from serum A protein), ATTR amyloidosis (amyloid derived
from transthyrethin), primary systemic amyloidosis, secondary
systemic amyloidosis, senile systemic amyloidosis, familial amyloid
polyneuropathy 1, hereditary cerebral amyloid angiopathy,
hemodialysis-related amyloidosis, familial amyloid polyneuropathy
III, Finnish hereditary systemic amyloidosis, atrial amyloidosis,
hereditary non-neuropathic systemic amyloidosis,
injection-localized amyloidosis, hereditary renal amyloidosis and
Alzheimer disease among others. According to another preferred
embodiment, the amyloid is Amyloid beta (A.beta. or Abeta) and the
invention relates to a PPP1R15A inhibitor of formula (I) or (II) or
a pharmaceutical acceptable salt thereof for the use in treating
Alzheimer disease.
[0104] According to another preferred embodiment, the amyloid is
HLA-B27 (Colbert et al. 2009 Prion Vol. 3 (1) pp 15-16) and the
invention relates to a PPP1R15A inhibitor of formula (I) or a
pharmaceutical acceptable salt thereof for the use in treating
spondylo-arthropathies, more preferably ankylosing spondylitis.
Inflammation
[0105] PPP1R15A represents a promising target to control
inflammation by blocking the release of inflammatory cytokines and
other secreted molecular mediators leading to pathogenic
conditions. Non-limiting examples of diseases or conditions having
inflammation associated therewith which can be treated with by
PPP1R15A inhibitors of formula (I) described herein include, but
are not limited to infection-related or non-infectious inflammatory
conditions in the lung (i.e., sepsis, lung infections, Respiratory
Distress Syndrome, bronchopulmonary dysplasia, etc.);
infection-related or non-infectious inflammatory conditions in
other organs such as colitis, ulcerative colitis, Inflammatory
Bowel Disease, diabetic nephropathy, hemorrhagic shock,
spondylo-arthropathies, pancreatitis; inflammation-induced cancer
(i.e., cancer progression in patients with colitis or Inflammatory
Bowel Disease); and the like.
[0106] Examples of such pathogenic inflammatory conditions include
auto-immune diseases, hereditary diseases, chronic diseases and
infectious diseases such as allergy, asthma, hypercytokinemia
including graft versus host disease (GVHD), acute respiratory
distress syndrome (ARDS), sepsis, systemic inflammatory response
syndrome (SIRS) (see WO2011/061340). Preferably, infectious disease
is selected from influenza virus infection, smallpox virus
infection, herpes virus infection, severe acute respiratory
syndrome (SARS), chikungunya virus infection, West Nile Virus
infection, dengue virus infection, Japanese encephalitis virus
infection, yellow fever virus infection, and hepatitis C virus
infection.
[0107] Preferably auto-immune disease is selected from Sjogren's
syndrome, systemic lupus erythematosus, psoriasis, dermatitis
herpetiformis, vitiligo, mycosis fungoides, allergic contact
dermatitis, atopic dermatitis, lichen planus, Pityriasis
lichenoides et varioliforms acuta (PLEVA), arthritis, catastrophic
antiphospholipid syndrome.
[0108] According to another preferred embodiment, the invention
relates to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof for the use in treating a disease selected
in the group of colitis, ulcerative colitis, Inflammatory Bowel
Disease, pancreatitis, sepsis.
[0109] According to another preferred embodiment, the invention
relates to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof, for the use in treating pancreatitis.
[0110] According to another preferred embodiment, the invention
relates to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof, for the use in treating sepsis.
[0111] According to another preferred embodiment, the invention
relates to a PPP1R15A inhibitor of formula (I) or a pharmaceutical
acceptable salt thereof for the use in treating
spondylo-arthropathies, more preferably ankylosing spondylitis.
[0112] Metabolic and/or cardio-vascular disorders, such adiposity,
hyper-lipidemia, familial hyper-cholesterolemia, obesity,
atherosclerosis, hypertension, heart diseases, cardiac ischaemia,
stroke, myocardial infraction, trans-aortic constriction, vascular
stroke and diabetes and related disorders include hyperglycemia,
impaired glucose tolerance, hyper-insulinemia (pre-diabetes),
insulin hypersensitivity type I and II diabetes, insulin
resistance, Wolcott-Rallison Syndrome among others.
[0113] In one preferred embodiment, the compound of formula (I) is
for use in treating atherosclerosis. In second preferred
embodiment, the compound of formula (I) is for use in treating a
disease selected in the group of hypertension, heart diseases,
cardiac ischaemia, stroke, myocardial infraction, trans-aortic
constriction or vascular stroke. In one preferred embodiment, the
compound of formula (I) is for use in treating cardiac ischemia. In
another preferred embodiment, the compound of formula (I) is for
use in treating a disease selected in the group of hyperglycemia,
impaired glucose tolerance, hyper-insulinemia (pre-diabetes),
insulin hypersensitivity type I and II, insulin resistance and
Wolcott-Rallison Syndrome. In another preferred embodiment, the
compound of formula (I) is for use in treating pre-diabetes or
diabetes, more preferably type 2 diabetes.
Osteoporosis:
[0114] Yokota et al. (BMC Musculoskeletal disorders 2013, 14, 197)
and He et al. (Cellular Signaling 2013, 25 552-560) demonstrated
that Salubrinal (Boyce et al. 2005) efficiently block osteoporosis
in mice model and stimulates bone formation. However, Salubrinal is
toxic and cannot be used to treat human patients. In contrast, the
PPP1R15A inhibitors of formula (I) are predicted to be safe and
could be useful for the treatment of osteoporosis. In one preferred
embodiment, the compound of formula (I) is for use in treating
osteoporosis.
Nervous System Trauma
[0115] Ohri et al. (Neurobiology of disease, 2013 Vol. 58 pp 29-37)
demonstrated that Salubrinal significantly improved hindlimb
locomotion which corresponds with an improved white matter sparing
and a decreased oligodendrocytes apoptosis, thus improving
functional recovery after spinal cord injury.
[0116] The PPP1R15A inhibitors of formula (I) of the invention are
predicted to be safe and could be useful to reduce the
oligodendrocyte loss after traumatic spinal cord injury and for the
prophylactic and/or therapeutic treatment of spinal cord injury. In
one preferred embodiment, the compound of formula (I) is for the
prophylactic and/or therapeutic treatment of spinal cord
injury.
Ischemia, Cerebral Ischemia, Sleep Apnoea
[0117] The present invention provides methods of using PPP1R15A
inhibitors of formula (I) of the invention to prevent and/or treat
tissue damage resulting from cell damage or death due to necrosis
or apoptosis. Example of neural tissue damage include ischemia and
reperfusion injury, such as cerebral ischemic stroke and head
trauma. In one preferred embodiment, the compound of formula (I) is
for the prophylactic and/or therapeutic treatment of cerebral
ischemia, such as cerebral ischemic stroke and head trauma.
Aging
[0118] Aging is associated with the degeneration of cells, tissues,
and organs, resulting in diseases such as cancer, cardiovascular
failure, obesity, type 2 diabetes mellitus, non-alcoholic fatty
liver, and neurodegenerative diseases, as well as the decline of
most measures of physiological performance.
[0119] In biology, senescence is the state or process of aging.
Cellular senescence is a phenomenon where isolated cells
demonstrate a limited ability to divide in culture (the Hayflick
Limit, discovered by Leonard Hayflick in 1961), while organismal
senescence is the ageing of organisms. Organismal senescence is
characterised by the declining ability to respond to stress,
increasing homeostatic imbalance and the increased risk of disease;
in particular, the UPR is impaired with age (Naidoo et al., 2008, J
Neurosci, 28, 6539-48). Thus, prolonging the beneficial effect of
the UPR by inhibition of eIF2.alpha. phosphatase could ameliorate
age-related disorders. Therefore, the PPP1R15A inhibitors of
formula (I) of the invention are predicted to be safe and could be
useful to prevent and/or treat diseases or disorders relating to
lifespan or proliferative capacity of cells, and diseases or
disease conditions induced or exacerbated by cellular senescence in
an animal, more specifically humans.
[0120] According to a particular embodiment, the present invention
concerns one compound selected from:
##STR00010## ##STR00011##
[0121] For use in the treatment and/or prevention of one or more
diseases selected from selected in the group of cystic fibrosis,
lysosomal storage disease, amyloidosis diseases, cancers,
inflammation preferably sepsis, colitis and pancreatitis, metabolic
disorders, diabetes, cardio-vascular disorders, osteoporosis,
central nervous system trauma, ischemia, retinal diseases,
seipinopathies, neurodegenerative diseases, preferably Alzheimer's
disease, Parkinson's disease, Amyotrophic Lateral Sclerosis,
Huntington's disease, polyglutamine and polyalanine diseases,
Charcot-Marie-Tooth diseases, leukodystrophies and multiple
sclerosis.
[0122] According to an embodiment, the present invention also
concerns one compound selected from the above compounds 1 to 11, as
well as the pharmaceutical compositions comprising the same.
Pharmaceutical Compositions
[0123] For use according to the present invention, the compounds or
physiologically acceptable salts, esters or other physiologically
functional derivatives thereof, described herein, may be presented
as a pharmaceutical formulation, comprising the compounds or
physiologically acceptable salt, ester or other physiologically
functional derivative thereof, together with one or more
pharmaceutically acceptable carriers therefore and optionally other
therapeutic and/or prophylactic ingredients. The carrier(s) must be
acceptable in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof. The pharmaceutical compositions may be for human or animal
usage in human and veterinary medicine.
[0124] Examples of such suitable excipients for the various
different forms of pharmaceutical compositions described herein may
be found in the "Handbook of Pharmaceutical Excipients, 2.sup.nd
Edition, (1994), Edited by A Wade and P J Weller.
[0125] Acceptable carriers or diluents for therapeutic use are well
known in the pharmaceutical art, and are described, for example, in
Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R.
Gennaro edit. 1985).
[0126] Examples of suitable carriers include lactose, starch,
glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol
and the like. Examples of suitable diluents include ethanol,
glycerol and water.
[0127] The choice of pharmaceutical carrier, excipient or diluent
can be selected with regard to the intended route of administration
and standard pharmaceutical practice. The pharmaceutical
compositions may comprise as, or in addition to, the carrier,
excipient or diluent any suitable binder(s), lubricant(s),
suspending agent(s), coating agent(s), solubilising agent(s),
buffer(s), flavouring agent(s), surface active agent(s),
thickener(s), preservative(s) (including anti-oxidants) and the
like, and substances included for the purpose of rendering the
formulation isotonic with the blood of the intended recipient.
[0128] Examples of suitable binders include starch, gelatin,
natural sugars such as glucose, anhydrous lactose, free-flow
lactose, beta-lactose, corn sweeteners, natural and synthetic gums,
such as acacia, tragacanth or sodium alginate, carboxymethyl
cellulose and polyethylene glycol.
[0129] Examples of suitable lubricants include sodium oleate,
sodium stearate, magnesium stearate, sodium benzoate, sodium
acetate, sodium chloride and the like.
[0130] Preservatives, stabilizers, dyes and even flavoring agents
may be provided in the pharmaceutical composition. Examples of
preservatives include sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid. Antioxidants and suspending agents may be
also used.
[0131] Pharmaceutical formulations include those suitable for oral,
topical (including dermal, buccal, ocular and sublingual), rectal
or parenteral (including subcutaneous, intradermal, intramuscular
and intravenous), nasal, intra-ocularly and pulmonary
administration e.g., by inhalation. The formulation may, where
appropriate, be conveniently presented in discrete dosage units and
may be prepared by any of the methods well known in the art of
pharmacy. All methods include the step of bringing into association
an active compound with liquid carriers or finely divided solid
carriers or both and then, if necessary, shaping the product into
the desired formulation.
[0132] Pharmaceutical formulations suitable for oral administration
wherein the carrier is a solid are most preferably presented as
unit dose formulations such as boluses, capsules or tablets each
containing a predetermined amount of active compound. A tablet may
be made by compression or moulding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared by
compressing in a suitable machine an active compound in a
free-flowing form such as a powder or granules optionally mixed
with a binder, lubricant, inert diluent, lubricating agent,
surface-active agent or dispersing agent. Moulded tablets may be
made by moulding an active compound with an inert liquid diluent.
Tablets may be optionally coated and, if uncoated, may optionally
be scored. Capsules may be prepared by filling an active compound,
either alone or in admixture with one or more accessory
ingredients, into the capsule shells and then sealing them in the
usual manner. Cachets are analogous to capsules wherein an active
compound together with any accessory ingredient(s) is sealed in a
rice paper envelope. An active compound may also be formulated as
dispersible granules, which may for example be suspended in water
before administration, or sprinkled on food. The granules may be
packaged, e.g., in a sachet. Formulations suitable for oral
administration wherein the carrier is a liquid may be presented as
a solution or a suspension in an aqueous or non-aqueous liquid, or
as an oil-in-water liquid emulsion.
[0133] Formulations for oral administration include controlled
release dosage forms, e.g., tablets wherein an active compound is
formulated in an appropriate release-controlling matrix, or is
coated with a suitable release-controlling film. Such formulations
may be particularly convenient for prophylactic use.
[0134] Pharmaceutical formulations suitable for rectal
administration wherein the carrier is a solid are most preferably
presented as unit dose suppositories. Suitable carriers include
cocoa butter and other materials commonly used in the art. The
suppositories may be conveniently formed by admixture of an active
compound with the softened or melted carrier(s) followed by
chilling and shaping in moulds.
[0135] Pharmaceutical formulations suitable for parenteral
administration include sterile solutions or suspensions of an
active compound in aqueous or oleaginous vehicles.
[0136] Pharmaceutical formulations of the invention are suitable
for ophthalmic administration, in particular for intra-ocular,
intra-vitreal, topical ocular or peri-ocular administration, more
preferably for topical ocular or intra-vitreal administration.
[0137] Injectible preparations may be adapted for bolus injection
or continuous infusion. Such preparations are conveniently
presented in unit dose or multi-dose containers which are sealed
after introduction of the formulation until required for use.
Alternatively, an active compound may be in powder form which is
constituted with a suitable vehicle, such as sterile, pyrogen-free
water, before use.
[0138] An active compound may also be formulated as long-acting
depot preparations, which may be administered by intramuscular
injection or by implantation, e.g., subcutaneously or
intramuscularly. Depot preparations may include, for example,
suitable polymeric or hydrophobic materials, or ion-exchange
resins. Such long-acting formulations are particularly convenient
for prophylactic use.
[0139] Formulations suitable for pulmonary administration via the
buccal cavity are presented such that particles containing an
active compound and desirably having a diameter in the range of 0.5
to 7 microns are delivered in the bronchial tree of the recipient.
As one possibility such formulations are in the form of finely
comminuted powders which may conveniently be presented either in a
pierceable capsule, suitably of, for example, gelatin, for use in
an inhalation device, or alternatively as a self-propelling
formulation comprising an active compound, a suitable liquid or
gaseous propellant and optionally other ingredients such as a
surfactant and/or a solid diluent. Suitable liquid propellants
include propane and the chlorofluorocarbons, and suitable gaseous
propellants include carbon dioxide. Self-propelling formulations
may also be employed wherein an active compound is dispensed in the
form of droplets of solution or suspension.
[0140] Such self-propelling formulations are analogous to those
known in the art and may be prepared by established procedures.
Suitably they are presented in a container provided with either a
manually-operable or automatically functioning valve having the
desired spray characteristics; advantageously the valve is of a
metered type delivering a fixed volume, for example, 25 to 100
microlitres, upon each operation thereof.
[0141] As a further possibility an active compound may be in the
form of a solution or suspension for use in an atomizer or
nebuliser whereby an accelerated airstream or ultrasonic agitation
is employed to produce a fine droplet mist for inhalation.
[0142] Formulations suitable for nasal administration include
preparations generally similar to those described above for
pulmonary administration. When dispensed such formulations should
desirably have a particle diameter in the range 10 to 200 microns
to enable retention in the nasal cavity; this may be achieved by,
as appropriate, use of a powder of a suitable particle size or
choice of an appropriate valve. Other suitable formulations include
coarse powders having a particle diameter in the range 20 to 500
microns, for administration by rapid inhalation through the nasal
passage from a container held close up to the nose, and nasal drops
comprising 0.2 to 5% w/v of an active compound in aqueous or oily
solution or suspension.
[0143] Pharmaceutically acceptable carriers are well known to those
skilled in the art and include, but are not limited to, 0.1 M and
preferably 0.05 M phosphate buffer or 0.8% saline. Additionally,
such pharmaceutically acceptable carriers may be aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Preservatives and other additives
may also be present, such as, for example, antimicrobials,
anti-oxydants, chelating agents, inert gases and the like.
[0144] Formulations suitable for topical formulation may be
provided for example as gels, creams or ointments. Such
preparations may be applied e.g. to a wound or ulcer either
directly spread upon the surface of the wound or ulcer or carried
on a suitable support such as a bandage, gauze, mesh or the like
which may be applied to and over the area to be treated.
[0145] Liquid or powder formulations may also be provided which can
be sprayed or sprinkled directly onto the site to be treated, e.g.
a wound or ulcer. Alternatively, a carrier such as a bandage,
gauze, mesh or the like can be sprayed or sprinkle with the
formulation and then applied to the site to be treated.
[0146] According to a further aspect of the invention, there is
provided a process for the preparation of a pharmaceutical or
veterinary composition as described above, the process comprising
bringing the active compound(s) into association with the carrier,
for example by admixture.
[0147] In general, the formulations are prepared by uniformly and
intimately bringing into association the active agent with liquid
carriers or finely divided solid carriers or both, and then if
necessary shaping the product. The invention extends to methods for
preparing a pharmaceutical composition comprising bringing a
compound of general formula (I) in conjunction or association with
a pharmaceutically or veterinarily acceptable carrier or
vehicle.
Salts/Esters
[0148] The compounds of the invention can be present as salts or
esters, in particular pharmaceutically and veterinarily acceptable
salts or esters.
[0149] Pharmaceutically acceptable salts of the compounds of the
invention include suitable acid addition or base salts thereof. A
review of suitable pharmaceutical salts may be found in Berge et
al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example
with strong inorganic acids such as mineral acids, e.g. hydrohalic
acids such as hydrochloride, hydrobromide and hydroiodide, sulfuric
acid, phosphoric acid sulphate, bisulphate, hemisulphate,
thiocyanate, persulphate and sulphonic acids; with strong organic
carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon
atoms which are unsubstituted or substituted (e.g., by halogen),
such as acetic acid; with saturated or unsaturated dicarboxylic
acids, for example oxalic, malonic, succinic, maleic, fumaric,
phthalic or tetraphthalic; with hydroxycarboxylic acids, for
example ascorbic, glycolic, lactic, malic, tartaric or citric acid;
with aminoacids, for example aspartic or glutamic acid; with
benzoic acid; or with organic sulfonic acids, such as
(C.sub.1-C.sub.4)-alkyl- or aryl-sulfonic acids which are
unsubstituted or substituted (for example, by a halogen) such as
methane- or p-toluene sulfonic acid. Salts which are not
pharmaceutically or veterinarily acceptable may still be valuable
as intermediates.
[0150] Preferred salts include, for example, acetate,
trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate,
malate, pantothenate, adipate, alginate, aspartate, benzoate,
butyrate, digluconate, cyclopentanate, glucoheptanate,
glycerophosphate, oxalate, heptanoate, hexanoate, fumarate,
nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate,
pivalate, proprionate, tartrate, lactobionate, pivolate,
camphorate, undecanoate and succinate, organic sulphonic acids such
as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate,
camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate,
p-chlorobenzenesulphonate and p-toluenesulphonate; and inorganic
acids such as hydrochloride, hydrobromide, hydroiodide, sulphate,
bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and
sulphonic acids. According to a preferred embodiment the salt is
acetate.
[0151] Esters are formed either using organic acids or
alcohols/hydroxides, depending on the functional group being
esterified. Organic acids include carboxylic acids, such as
alkanecarboxylic acids of 1 to 12 carbon atoms which are
unsubstituted or substituted (e.g., by halogen), such as acetic
acid; with saturated or unsaturated dicarboxylic acid, for example
oxalic, malonic, succinic, maleic, fumaric, phthalic or
tetraphthalic; with hydroxycarboxylic acids, for example ascorbic,
glycolic, lactic, malic, tartaric or citric acid; with aminoacids,
for example aspartic or glutamic acid; with benzoic acid; or with
organic sulfonic acids, such as (C.sub.1-C.sub.4)-alkyl- or
aryl-sulfonic acids which are unsubstituted or substituted (for
example, by a halogen) such as methane- or p-toluene sulfonic acid.
Suitable hydroxides include inorganic hydroxides, such as sodium
hydroxide, potassium hydroxide, calcium hydroxide, aluminium
hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms
which may be unsubstituted or substituted, e.g. by a halogen).
Enantiomers/Tautomers
[0152] In all aspects of the present invention previously
discussed, the invention includes, where appropriate all
enantiomers, diastereoisomers and tautomers of the compounds of the
invention. The person skilled in the art will recognise compounds
that possess optical properties (one or more chiral carbon atoms)
or tautomeric characteristics. The corresponding enantiomers and/or
tautomers may be isolated/prepared by methods known in the art.
Enantiomers are characterised by the absolute configuration of
their chiral centres and described by the R- and S-sequencing rules
of Cahn, Ingold and Prelog. Such conventions are well known in the
art (e.g. see `Advanced Organic Chemistry`, 3.sup.rd edition, ed.
March, J., John Wiley and Sons, New York, 1985).
[0153] Compounds of formula (I) thus also include the tautomer
forms of formula:
##STR00012##
[0154] As an illustrative example, a tautomer form of example 1
is:
##STR00013##
[0155] Compounds of the invention containing a chiral centre may be
used as a racemic mixture, an enantiomerically enriched mixture, or
the racemic mixture may be separated using well-known techniques
and an individual enantiomer may be used alone.
Stereo and Geometric Isomers
[0156] Some of the compounds of the invention may exist as
stereoisomers and/or geometric isomers--e.g. they may possess one
or more asymmetric and/or geometric centres and so may exist in two
or more stereoisomeric and/or geometric forms. The present
invention contemplates the use of all the individual stereoisomers
and geometric isomers of those inhibitor agents, and mixtures
thereof. The terms used in the claims encompass these forms,
provided said forms retain the appropriate functional activity
(though not necessarily to the same degree).
[0157] The present invention also includes all suitable isotopic
variations of the agent or a pharmaceutically acceptable salt
thereof. An isotopic variation of an agent of the present invention
or a pharmaceutically acceptable salt thereof is defined as one in
which at least one atom is replaced by an atom having the same
atomic number but an atomic mass different from the atomic mass
usually found in nature. Examples of isotopes that can be
incorporated into the agent and pharmaceutically acceptable salts
thereof include isotopes of hydrogen, carbon, nitrogen, oxygen,
phosphorus, sulfur, fluorine and chlorine such as .sup.2H, .sup.3H,
.sup.13O, .sup.14O, .sup.15N, .sup.17O, .sup.18O, .sup.31P,
.sup.32P, .sup.35S, .sup.18F and .sup.36Cl, respectively. Certain
isotopic variations of the agent and pharmaceutically acceptable
salts thereof, for example, those in which a radioactive isotope
such as .sup.3H or .sup.14C is incorporated, are useful in drug
and/or substrate tissue distribution studies. Tritiated, i.e.,
.sup.3H, and carbon-14, i.e., .sup.14C, isotopes are particularly
preferred for their ease of preparation and detectability. Further,
substitution with isotopes such as deuterium, i.e., .sup.2H, may
afford certain therapeutic advantages resulting from greater
metabolic stability, for example, increased in vivo half-life or
reduced dosage requirements and hence may be preferred in some
circumstances. For example, the invention includes compounds of
general formula (I) where any hydrogen atom has been replaced by a
deuterium atom. Isotopic variations of the agent of the present
invention and pharmaceutically acceptable salts thereof of this
invention can generally be prepared by conventional procedures
using appropriate isotopic variations of suitable reagents.
Prodrugs
[0158] The invention further includes the compounds of the present
invention in prodrug form, i.e. covalently bonded compounds which
release the active parent drug according to general formula (I) in
vivo. Such prodrugs are generally compounds of the invention
wherein one or more appropriate groups have been modified such that
the modification may be reversed upon administration to a human or
mammalian subject. Reversion is usually performed by an enzyme
naturally present in such subject, though it is possible for a
second agent to be administered together with such a prodrug in
order to perform the reversion in vivo. Examples of such
modifications include ester (for example, any of those described
above), wherein the reversion may be carried out be an esterase
etc. Other such systems will be well known to those skilled in the
art.
Solvates
[0159] The present invention also includes solvate forms of the
compounds of the present invention. The terms used in the claims
encompass these forms.
Polymorphs
[0160] The invention further relates to the compounds of the
present invention in their various crystalline forms, polymorphic
forms and (an)hydrous forms. It is well established within the
pharmaceutical industry that chemical compounds may be isolated in
any of such forms by slightly varying the method of purification
and or isolation form the solvents used in the synthetic
preparation of such compounds.
Administration
[0161] The pharmaceutical compositions of the present invention may
be adapted for rectal, nasal, intrabronchial, topical (including
buccal, sublingual and ophthalmic administration, in particular for
intra-ocular, intra-vitreal, topical ocular or peri-ocular
administration), vaginal or parenteral (including subcutaneous,
intramuscular, intravenous, intraarterial and intradermal),
intraperitoneal or intrathecal administration. Preferably the
formulation is an orally administered formulation. The formulations
may conveniently be presented in unit dosage form, i.e., in the
form of discrete portions containing a unit dose, or a multiple or
sub-unit of a unit dose. By way of example, the formulations may be
in the form of tablets and sustained release capsules, and may be
prepared by any method well known in the art of pharmacy.
[0162] Formulations for oral administration in the present
invention may be presented as: discrete units such as capsules,
gellules, drops, cachets, pills or tablets each containing a
predetermined amount of the active agent; as a powder or granules;
as a solution, emulsion or a suspension of the active agent in an
aqueous liquid or a non-aqueous liquid; or as an oil-in-water
liquid emulsion or a water-in-oil liquid emulsion; or as a bolus
etc. Preferably, these compositions contain from 1 to 250 mg, more
preferably from 10-100 mg, and more preferably from 1-100 mg, of
active ingredient per dose.
[0163] For compositions for oral administration (e.g. tablets and
capsules), the term "acceptable carrier" includes vehicles such as
common excipients e.g. binding agents, for example syrup, acacia,
gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone),
methylcellulose, ethylcellulose, sodium carboxymethylcellulose,
hydroxypropylmethylcellulose, sucrose and starch; fillers and
carriers, for example corn starch, gelatin, lactose, sucrose,
microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate,
sodium chloride and alginic acid; and lubricants such as magnesium
stearate, sodium stearate and other metallic stearates, glycerol
stearate stearic acid, silicone fluid, talc waxes, oils and
colloidal silica. Flavouring agents such as peppermint, oil of
wintergreen, cherry flavouring and the like can also be used. It
may be desirable to add a colouring agent to make the dosage form
readily identifiable. Tablets may also be coated by methods well
known in the art.
[0164] A tablet may be made by compression or moulding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active agent in a
free flowing form such as a powder or granules, optionally mixed
with a binder, lubricant, inert diluent, preservative,
surface-active or dispersing agent. Moulded tablets may be made by
moulding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets may be
optionally be coated or scored and may be formulated so as to
provide slow or controlled release of the active agent.
[0165] Other formulations suitable for oral administration include
lozenges comprising the active agent in a flavoured base, usually
sucrose and acacia or tragacanth; pastilles comprising the active
agent in an inert base such as gelatin and glycerin, or sucrose and
acacia; and mouthwashes comprising the active agent in a suitable
liquid carrier.
[0166] Other forms of administration comprise solutions or
emulsions which may be injected intravenously, intraarterially,
intrathecally, subcutaneously, intradermally, intraperitoneally,
intra-ocularly, topical, peri-ocularly or intramuscularly, and
which are prepared from sterile or sterilisable solutions.
[0167] The pharmaceutical compositions of the present invention may
also be in form of suppositories, pessaries, suspensions,
emulsions, lotions, ointments, creams, gels, sprays, solutions or
dusting powders.
[0168] An alternative means of transdermal administration is by use
of a skin patch. For example, the active ingredient can be
incorporated into a cream consisting of an aqueous emulsion of
polyethylene glycols or liquid paraffin. The active ingredient can
also be incorporated, at a concentration of between 1 and 10% by
weight, into an ointment consisting of a white wax or white soft
paraffin base together with such stabilisers and preservatives as
may be required.
Dosage
[0169] A person of ordinary skill in the art can easily determine
an appropriate dose of one of the instant compositions to
administer to a subject without undue experimentation. Typically, a
physician will determine the actual dosage which will be most
suitable for an individual patient and it will depend on a variety
of factors including the activity of the specific compound
employed, the metabolic stability and length of action of that
compound, the age, body weight, general health, sex, diet, mode and
time of administration, rate of excretion, drug combination, the
severity of the particular condition, and the individual undergoing
therapy. The dosages disclosed herein are exemplary of the average
case. There can of course be individual instances where higher or
lower dosage ranges are merited, and such are within the scope of
this invention.
[0170] In accordance with this invention, an effective amount of a
compound of general formula (I) may be administered to target a
particular condition or disease. Of course, this dosage amount will
further be modified according to the type of administration of the
compound. For example, to achieve an "effective amount" for acute
therapy, parenteral administration of a compound of general formula
(I) is preferred. An intravenous infusion of the compound in 5%
dextrose in water or normal saline, or a similar formulation with
suitable excipients, is most effective, although an intramuscular
bolus injection is also useful. Typically, the parenteral dose will
be about 0.01 to about 100 mg/kg; preferably between 0.1 and 20
mg/kg, in a manner to maintain the concentration of drug in the
plasma at an effective concentration The compounds may be
administered one to four times daily at a level to achieve a total
daily dose of about 0.4 to about 400 mg/kg/day. The precise amount
of an inventive compound which is therapeutically effective, and
the route by which such compound is best administered, is readily
determined by one of ordinary skill in the art by comparing the
blood level of the agent to the concentration required to have a
therapeutic effect.
[0171] The compounds of this invention may also be administered
orally to the patient, in a manner such that the concentration of
drug is sufficient to achieve one or more of the therapeutic
indications disclosed herein. Typically, a pharmaceutical
composition containing the compound is administered at an oral dose
of between about 0.1 to about 50 mg/kg in a manner consistent with
the condition of the patient. Preferably the oral dose would be
about 0.1 to about 20 mg/kg. No unacceptable toxicological effects
are expected when compounds of the present invention are
administered in accordance with the present invention. The
compounds of this invention, which may have good bioavailability,
may be tested in one of several biological assays to determine the
concentration of a compound which is required to have a given
pharmacological effect.
Combinations
[0172] In a particularly preferred embodiment, the one or more
compounds of the invention are administered in combination with one
or more other active agents, for example, existing drugs available
on the market. In such cases, the compounds of the invention may be
administered consecutively, simultaneously or sequentially with the
one or more other active agents.
[0173] Drugs in general are more effective when used in
combination. In particular, combination therapy is desirable in
order to avoid an overlap of major toxicities, mechanism of action
and resistance mechanism(s). Furthermore, it is also desirable to
administer most drugs at their maximum tolerated doses with minimum
time intervals between such doses. The major advantages of
combining drugs are that it may promote additive or possible
synergistic effects through biochemical interactions and also may
decrease the emergence of resistance.
[0174] Beneficial combinations may be suggested by studying the
inhibitory activity of the test compounds with agents known or
suspected of being valuable in the treatment of a particular
disorder. This procedure can also be used to determine the order of
administration of the agents, i.e. before, simultaneously, or after
delivery. Such scheduling may be a feature of all the active agents
identified herein.
[0175] According to preferred embodiment, the invention relates to
a pharmaceutical composition comprising a PPP1R15A inhibitor of
formula (I) or a pharmaceutical acceptable salt thereof, and a
compound increasing the expression and/or the activity of protein
BiP and a pharmaceutically acceptable carrier and/or excipient (see
WO2013/124484). Preferably, the compound increasing the expression
and/or activity of protein BiP is selected from the group
consisting of valproic acid or a derivative thereof, trichostatin
A, lithium, I-(3,4-dihydroxy-phenyl)-2-thiocyanate-ethanone, and
exendin-4. According to a preferred embodiment the protein BiP is
valproic acid or a derivative thereof such as 2-ene-valproic
acid.
[0176] According to a preferred embodiment, the invention relates
to a pharmaceutical composition comprising a PPP1R15A inhibitor of
formula (I) or a pharmaceutical acceptable salt thereof, and a
compound increasing the expression and/or the activity of protein
BiP and a pharmaceutically acceptable carrier and/or excipient, to
treat a disorder associated with the PPP1R15A pathway and
associated with protein misfolding stress and in particular with
accumulation of misfolded proteins. Preferably, the disease is
selected in the group of tauopathies, synucleinopathies,
polyglutamine and polyalanine diseases, leukodystrophies,
charcot-marie-tooth diseases, seipinopathies, cystic fibrosis,
multiple sclerosis, lysosomal storage disorders, amyloidosis
diseases, retinal diseases, inflammation, metabolic disorders,
cardio-vascular disorders, osteoporosis, nervous system trauma,
ischemia.
Assay
[0177] A further aspect of the invention relates to the use of a
compound as described above in an assay for identifying further
candidate compounds capable of inhibiting PPP1R15A-PP1.
[0178] Preferably, the assay is a competitive binding assay. More
preferably, the competitive binding assay comprises contacting a
compound of the invention with PPP1R15A-PP1 and a candidate
compound and detecting any change in the interaction between the
compound according to the invention and the PPP1R15A-PP1.
[0179] Preferably, the candidate compound is generated by
conventional SAR modification of a compound of the invention. As
used herein, the term "conventional SAR modification" refers to
standard methods known in the art for varying a given compound by
way of chemical derivatisation.
[0180] Thus, in one aspect, the identified compound may act as a
model (for example, a template) for the development of other
compounds. The compounds employed in such a test may be free in
solution, affixed to a solid support, borne on a cell surface, or
located intracellularly. The abolition of activity or the formation
of binding complexes between the compound and the agent being
tested may be measured.
[0181] The assay of the present invention may be a screen, whereby
a number of agents are tested. In one aspect, the assay method of
the present invention is a high through-put screen.
[0182] This invention also contemplates the use of competitive drug
screening assays in which neutralising antibodies capable of
binding a compound specifically compete with a test compound for
binding to a compound.
[0183] Another technique for screening provides for high throughput
screening (HTS) of agents having suitable binding affinity to the
substances and is based upon the method described in detail in WO
84/03564.
[0184] It is expected that the assay methods of the present
invention will be suitable for both small and large-scale screening
of test compounds as well as in quantitative assays.
[0185] Preferably, the competitive binding assay comprises
contacting a compound of the invention with PPP1R15A-PP1 in the
presence of a known substrate of PPP1R15A-PP1 and detecting any
change in the interaction between said PPP1R15A-PP1 and said known
substrate.
[0186] A further aspect of the invention provides a method of
detecting the binding of a ligand to PPP1R15A-PP1, said method
comprising the steps of: [0187] (i) contacting a ligand with
PPP1R15A-PP1 in the presence of a known substrate; [0188] (ii)
detecting any change in the interaction between PPP1R15A-PP1 and
said known substrate; and wherein said ligand is a compound of the
invention.
[0189] One aspect of the invention relates to a process comprising
the steps of: [0190] (a) performing an assay method described
hereinabove; [0191] (b) identifying one or more ligands capable of
binding to a ligand binding domain; and [0192] (c) preparing a
quantity of said one or more ligands.
[0193] Another aspect of the invention provides a process
comprising the steps of: [0194] (a) performing an assay method
described hereinabove; [0195] (b) identifying one or more ligands
capable of binding to a ligand binding domain; and [0196] (c)
preparing a pharmaceutical composition comprising said one or more
ligands.
[0197] Another aspect of the invention provides a process
comprising the steps of: [0198] (a) performing an assay method
described hereinabove; [0199] (b) identifying one or more ligands
capable of binding to a ligand binding domain; [0200] (c) modifying
said one or more ligands capable of binding to a ligand binding
domain; [0201] (d) performing the assay method described
hereinabove; [0202] (e) optionally preparing a pharmaceutical
composition comprising said one or more ligands.
[0203] The invention also relates to a ligand identified by the
method described hereinabove.
[0204] Yet another aspect of the invention relates to a
pharmaceutical composition comprising a ligand identified by the
method described hereinabove.
[0205] Another aspect of the invention relates to the use of a
ligand identified by the method described hereinabove in the
preparation of a pharmaceutical composition for use in the
treatment of a disorder associated with accumulation of misfolded
proteins as defined above.
[0206] The above methods may be used to screen for a ligand useful
as an inhibitor of PPP1R15A-PP1.
[0207] Compounds of general formula (I) are useful both as
laboratory tools and as therapeutic agents. In the laboratory
certain compounds of the invention are useful in establishing
whether a known or newly discovered target contributes a critical
or at least significant biochemical function during the
establishment or progression of a disease state, a process commonly
referred to as `target validation`.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
[0208] The present invention is further described with reference to
the following figures wherein:
[0209] FIG. 1 shows dose dependent protection of Hela cells by
compound 1 of the invention from ER stress induced by 6 hour
exposure to tunicamycin.
[0210] FIG. 2 shows dose dependent protection of interferon-gamma
injured rat oligodendrocytes by compound 1 of the invention.
[0211] FIG. 3 shows dose dependent protection of rotenone injured
primary mesencephalic rat neurons by compound 2 of the
invention.
[0212] FIG. 4 shows dose dependent protection of amyloid-beta 1-42
injured primary cortical rat neurons by compound 2 of the
invention.
[0213] FIG. 5 shows the ability of compound 2 at different
concentration and regimen to prevent motor defects of ALS in mutant
SOD1 mice.
IFB-B: Compound 2 is administered orally. BID: administration twice
a day QD: administration one a day
[0214] FIG. 6 shows the ability of compound 2 at different
concentrations and regimen to prevent motor defects of CMT-1A in
PMP-22 in transgenic (TG) rat over-expressing PMP-22. Compound 2 is
administered orally once a day.
[0215] FIG. 7 shows the ability of compound 1 at 5 microM to
prevent the accumulation of T181P mutated DM20 protein in Human
293T cell.
[0216] FIG. 8 shows the ability of compounds 6 to prevent cell
death associated with the accumulation of misfold prone Insulin
Akita expressed in Min6 cells.
[0217] FIG. 9 shows the ability of compound 1 and compound 6 at
different concentrations to prevent Min6 insulinoma cell death
associated with accumulation of misfolded protein induced by 6 hour
exposure to tunicamycin.
[0218] FIG. 10 shows the ability of compound 1 and compound 6 at
different concentrations to prevent INS1 insulinoma cell death
associated with accumulation of misfolded protein induced by 6 hour
exposure to tunicamycin.
[0219] FIG. 11 shows the ability of compound 2 to protect
photoreceptors against apoptosis and preserves light detection in
BBS12-/- mice. Electroretinogram (ERG). Tabulated mean of the
percentage different between BBS12-/- eye with compound 2 (2.5
microM) in association with Valproic acid (0.2 mM) or compound 2
(2.5 microM) alone versus the vehicle-treated eye (PBS). Positive
translates an increase in ERG response and negative translates a
decrease in ERG compare to the PBS treated eye. n=10-14 per
group.
[0220] FIG. 12 shows the ability of compound 2 to decrease protein
load in the Endoplasmic Reticulum in BBS12-/- mice. Transmission
Electronic Microscopy (TEM) endoplasmic reticulum (ER) of BBS12-/-
photoreceptors in response to the administration of Compound 2 (2.5
microM) in combination with Valproic acid (VPA) (0.2 mM) or PBS.
Dilatation of the ER cisternaes is observed when PBS only is
injected (left) whereas Compound 2 in combination with VPA are able
to decrease this dilatation (right) after one single intra-vitreal
injection.
[0221] FIG. 13 shows the ability of compounds 1, 6 and 8 (at 25
microM) to prevent type-I Interferon production by mouse embryonic
fibroblasts lipofected with poly I:C.
[0222] FIG. 14 shows the ability of Compound 2 to protect neonatal
rat cardiomyocytes against hypoxia-induced apoptosis. The graph
shows the percentage of apoptotic cells measured by FACS analysis.
Cardiomyocytes were exposed to hypoxia (0.3% O.sub.2) for 36 h in
the absence (0 .mu.M) or in the presence of indicated
concentrations of Compound 2 (n=3).
[0223] The present invention is further described with reference to
the following non-limiting examples.
EXAMPLES
1--Materials & Methods
[0224] Compound 3 was purchased from Chembridge ref: 5173161
Compound 4 was purchased from Chemdiv ref: 0589-0012 Compound 5 was
purchased from Chemdiv ref: 1683-6502
1.1--Preparation of the Compounds According to the Present
Invention
[0225] The reactants and commercials compounds were purchased from
Acros Organics, Sigma-Aldrich. The compounds according to the
present invention can be prepared according to the following
general procedure:
[0226] General procedure A:
##STR00014##
[0227] To a solution of benzaldehyde (1 eq.) in ethanol (300 ml)
was sequentially added Aminoguanidine hydrochloride (1 eq.) and
sodium acetate (1 eq.) at 25.degree. C. The resulting reaction
mixture was heated at 80.degree. C. for next .about.6 hours.
Reaction completion was monitored on TLC using
dichloromethane/methanol (8/2) as mobile phase. After completion of
reaction, the reaction mixture was allowed to cool down to
25.degree. C. and dumped in the saturated solution of NaHCO.sub.3
(700 ml). The resulting precipitate were filtered off under vacuum
and washed with water (100 ml). The resulting solid material was
titurated with diethylether (2.times.25 ml) and dried under vacuum
to provide the desired substituted aminoguanidine derivative.
[0228] The following compounds were prepared according general
procedure A:
Compound 1: 2-(2-chlorobenzylidene)hydrazinecarboximidamide
[0229] Prepared following general procedure A from
2-chlorobenzaldehyde (10 g) to give 11.1 g of desired compound
(yield: 79.6%). .sup.1H-NMR (DMSO-d.sub.6): .delta. (ppm) 5.66 (s,
2H); 6.05 (s broad, 2H); 7.27 (m, 2H); 7.40 (m, 1H); 8.14 (dd, 1H);
8.27 (s, 1H); MS (ESI+): m/z=197.2 [M+H].sup.+.
Compound 2: 2-(2-chlorobenzylidene)hydrazinecarboximidamide
acetate
[0230] To a suspension of 2-chlorobenzaldehyde (30.0 g) and
Aminoguanidine bicarbonate (29.0 g) in Methanol (450 ml) was added
Acetic acid (30 ml) at 25.degree. C. The reaction mixture was
stirred at 70.degree. C. for 30 minutes. Reaction completion was
monitored on TLC using Dichloromethane/Methanol (8/2) as mobile
phase. After completion of reaction, the reaction mixture was
allowed to cool down to 25.degree. C. and concentrated under
vacuum. The residue was suspended in methanol (250 ml) and
insoluble material was removed by flirtation. The resulting
filtrate was concentrated under vacuum and the above mentioned
process (suspension in methanol+filtration) was repeated for three
more times. Then, the solid material was triturated with diethyl
ether (3.times.100 ml) and dried under vacuum to provide 46.0 g of
2-(2-chlorobenzylidene)hydrazinecarboximidamide acetate Salt
(yield: 84.2%) LC-MS: m/z=197.2 (M+H). .sup.1H-NMR (DMSO-d.sub.6):
.delta. (ppm) 1.81 (s, 3H), 7.12 (m, 4H); 7.34 (m, 2H); 7.46 (m,
1H); 8.22 (m, 1H); 8.36 (s, 1H); LC-MS: m/z=197.2 [M+H].sup.+.
Compound 6:
2-[(3-chloropyridin-4-yl)methylidene]hydrazinecarboximidamide
[0231] Prepared following general procedure A from
2-chlorobenzaldehyde (0.5 g) to give 0.16 g of desired compound
(yield: 23%). .sup.1H-NMR (DMSO-d.sub.6): .delta. (ppm) 6.00 (s
broad, 2H); 6.32 (s broad, 2H); 8.10 (d, 1H); 8.14 (s, 1H); 8.35
(dd, 1H); 8.52 (s, 1H); MS (ESI+): m/z=198.0 [M+H].sup.+.
Compound 7:
2-[(3-chloropyridin-4-yl)methylidene]hydrazinecarboximidamide
acetate
[0232] To a suspension of 3-chloroisonicotinaldehyde (2.0 g) and
aminoguanidine bicarbonate (2.12 g) in methanol (28 ml) was added
acetic acid (2 ml) at 25.degree. C. The reaction mixture was
stirred at 70.degree. C. for .about.2 hours. Reaction completion
was monitored on TLC using Dichloromethane/Methanol (8/2) as mobile
phase. After completion of reaction, the crude mixture were allowed
to cool down to 25.degree. C. and concentrated under vacuum. The
solid material was triturated with methanol:diethyl ether (9:1)
(4.times.50 ml) and dried under vacuum to 2.0 g of
2-[(3-chloropyridin-4-yl)methylidene]hydrazinecarboximidamide
acetate salt (yield: 55.1%). .sup.1H-NMR (DMSO-d.sub.6): .delta.
(ppm) 6.01 (brs, 2H); 6.48 (m, 4H); 8.12 (d, 1H); 8.16 (s, 1H);
8.38 (dd, 1H); 8.54 (s, 1H); MS (ESI+): m/z=198.1 [M+H].sup.+.
Compound 8:
2-(2-chloro-6-fluorobenzylidene)hydrazinecarboximidamide
acetate
[0233] To a suspension of 2-chloro-6-fluorobenzaldehyde (1.5 g) and
aminoguanidine bicarbonate (1.29 g) in methanol (22 ml) was added
acetic acid (1.5 ml) at 25.degree. C. The reaction mixture was
stirred at 70.degree. C. for .about.1 hour. Reaction completion was
monitored on TLC using Dichloromethane/Methanol (8/2) as mobile
phase. After completion of reaction, the mixture was allowed to
cool down to 25.degree. C. and concentrated under vacuum. The
resulting solid material was triturated with methanol:diethyl ether
(9:1) (3.times.50 ml) and dried under vacuum to give 2.2 g
2-(2-chloro-6-fluorobenzylidene)hydrazinecarboximidamide acetate
Salt (yield: 84.8%). .sup.1H-NMR (DMSO-d.sub.6): .delta. (ppm) 1.89
(s, 3H), 6.13 (s broad, 4H); 7.24 (m, 1H); 7.33 (m, 2H) 8.17 (s,
1H); MS (ESI+): m/z=215.1 [M+H].sup.+.
Compound 9:
2-(2-chloro-4-methylbenzylidene)hydrazinecarboximidamide
[0234] Prepared following general procedure A from
2-chloro-4-methylbenzaldehyde (0.2 g) to give 255 mg of desired
compound (yield: 93.8%). .sup.1H-NMR (DMSO-d.sub.6): .delta. (ppm)
2.29 (s, 3H); 5.60 (s broad, 2H); 6.00 (s broad, 2H); 7.10 (d, 2H);
7.27 (s, 1H); 8.02 (d, 1H); 8.24 (s, 1H); MS (ESI+): m/z=210.9
[M+H].sup.+.
Compound 10:
2-(2-chloro-5-methylbenzylidene)hydrazinecarboximidamide
[0235] Prepared following general procedure A from
2-chloro-5-methylbenzaldehyde (0.2 g) to give 156 mg of desired
compound (yield: 57.4%). .sup.1H-NMR (DMSO-d.sub.6): .delta. (ppm)
2.30 (s, 3H); 5.64 (s broad, 2H); 6.06 (s broad, 2H); 7.07 (d, 2H);
7.27 (d, 1H); 7.97 (s, 1H); 8.24 (s, 1H); MS (ESI+): m/z=210.9
[M+H].sup.+.
Compound 11:
2-(2-chloro-3-methylbenzylidene)hydrazinecarboximidamide
[0236] Prepared following general procedure A from
2-chloro-3-methylbenzaldehyde (0.2 g) to give 226 mg of desired
compound (yield: 83.1%). .sup.1H-NMR (DMSO-d.sub.6): .delta. (ppm)
2.17 (s, 3H); 5.64 (s broad, 2H); 6.03 (s broad, 2H); 7.18 (t, 2H);
7.24 (d, 1H); 7.99 (s, 1H); 8.37 (s, 1H); MS (ESI+): m/z=210.9
[M+H].sup.+.
[0237] Selected compounds according to the invention are set forth
in Table 1 below:
TABLE-US-00001 Compound Number Structure Chemical Name Compound 1
##STR00015## 2-(2- chlorobenzylidene) hydrazinecarboximidamide
Compound 2 ##STR00016## 2-(2- chlorobenzylidene)
hydrazinecarboximidamide acetate Compound 3 ##STR00017## 2-(2-
bromobenzylidene) hydrazinecarboximidamide Compound 4 ##STR00018##
2-(2- methoxybenzylidene) hydrazinecarboximidamide Compound 5
##STR00019## 3-{[(2- chlorobenzylidene)amino]methyl}-
6-methyl-1,2,4-triazin-5(4H)-one Compound 6 ##STR00020##
2-[(3-chloropyridin-4- yl)methylidene] hydrazinecarboximidamide
Compound 7 ##STR00021## 2-[(3-chloropyridin-4- yl)methylidene]
hydrazinecarboximidamide acetate Compound 8 ##STR00022##
2-(2-chloro-6- fluorobenzylidene) hydrazinecarboximidamide acetate
Compound 9 ##STR00023## 2-(2-chloro-4- methylbenzylidene)
hydrazinecarboximidamide Compound 10 ##STR00024## 2-(2-chloro-5-
methylbenzylidene) hydrazinecarboximidamide Compound 11
##STR00025## 2-(2-chloro-3- methylbenzylidene)
hydrazinecarboximidamide
[0238] In some of the experiments below, the salt of these
compounds may be used.
1.2--Mammalian Cell Culture, Constructs and Transfection
[0239] HeLa Cells were cultured in Eagle's Minimum Essential Medium
(EMEM) supplemented with Glutamine, Sodium Pyruvate, Non-Essential
Amino Acids, Penicillin and Streptomycin (Lonza) containing 10%
Foetal Bovine Serum (FBS) (Biowest).
[0240] 293T cells were cultured in Dubelcco's Modified Eagle's
Media (DMEM) supplemented with penicillin, streptomycin, glutamine
(Lonza) and 10% of fetal bovine serum (FBS) (Biowest). Min6 cells
were cultured in DMEM supplemented with penicillin, streptomycin,
glutamine, sodium pyruvate, 50 .mu.M .beta.-Mercaptoethanol and 15%
Foetal Bovine Serum (FBS) (Biowest).
[0241] INS1 cells were cultured in RPMI supplemented with
penicillin, streptomycin, glutamine, sodium pyruvate (Lonza), 50
.mu.M .beta.-ME and 10% of fetal bovine serum (FBS) (Biowest).
[0242] Each cell line was maintained at 37.degree. C. in 5%
CO.sub.2 atmosphere.
[0243] Human open reading frame (ORF) sequences for PLP1, DM20 and
Insulin were obtained from Life Technologies (Invitrogen)
(IOH41689, IOH5252 and IOH7334 respectively). Construct cloning
into the expression plasmid pDEST26 (Invitrogen) was performed by
Gateway.RTM. LR Clonase.TM. II Enzyme Mix (Invitrogen). ORF
mutations were carried out using the QuikChange Lightning
Site-Directed Mutagenesis Kit (Stratagene) (T181P mutation for PLP1
and DM20 ORFs, Akita (C96Y) for Insulin ORF).
[0244] Gene expression into mammalian cells was carried out by
nucleofection, using the Amaxa.TM. 4 D-Nucleofector.TM. System
(Lonza) or by transfection using Lipofectamine (Life
technologies).
1.3--Cytoprotection from ER Stress
[0245] This assay is described in Tsaytler et al. (Science
2011).
[0246] HeLa Cells were cultured in Eagle's Minimum Essential Medium
(EMEM) supplemented with Glutamine, Sodium Pyruvate, Non-Essential
Amino Acids, Penicillin and Streptomycin containing 10% Foetal
Bovine Serum (FBS), at 37.degree. C. in 5% CO.sub.2 atmosphere.
Cells were plated in 96 well plates at a density of 17,000 cells/mL
the day before the treatment. ER stress was elicited by addition of
5 .mu.g/mL tunicamycin (Sigma-Aldrich) together with the
phosphatases inhibitors (0.5-10 .mu.M). Media were changed 6 h
later with fresh media and the cytoprotection was maintained by the
addition of the phosphatases inhibitors (0.5-10 .mu.M). Cell
viability was assessed by measuring the reduction of WST-8 into
formazan using Cell Counting Kit-8 (Sigma) according to the
supplier's recommendation, 48 h or 72 h after tunicamycin
treatment.
[0247] Cytoprotection from ER stress is measured in terms of
cytoprotective potency effect compared to the reference compound
Guanabenz (Tsaytler et al., Science 2011) after ER stress: [0248]
`-` no cytoprotective effect; [0249] `+` lower cytoprotective
effect compared to Guanabenz; [0250] `++` similar cytoprotective
effect compared to Guanabenz.
[0251] Table 1 summarizes the results of cytoprotective effect of
different compounds of the invention, compared to guanabenz, after
the stress induced by a 6 hour exposure of tunicamycin.
1.4--Assessment of Translation Rates in Unstressed Cells
[0252] HeLa cells (100,000 cells/ml) were plated in 6-well plates
24 h before each experiment and were either left untreated or
treated with compounds (50 .mu.M) for 2.5, 5 and 9 h. Culture
medium was replaced by methionine-free DMEM medium (Invitrogen) 30
min before compounds addition. One hour before each time point, 50
.mu.M of Click-iT.RTM. AHA (L-azidohomoalanine) (Invitrogen) was
added to the culture medium in order to label newly synthesized
proteins. At the end of each time point, cells were washed with
ice-cold PBS and harvested by Trypsine dissociation (Lonza), then
lysed in a 50 mM Tris-HCl buffer containing 1% of SDS (Sigma) and
protease and phosphatase inhibitors (Sigma). Protein samples were
coupled to alkyne biotin (Invitrogen) using Click-iT.RTM. Protein
Reaction Buffer Kit (Invitrogen). Samples were denatured at
70.degree. C. for 10 min, resolved on ECL 4-20% precasted gels (GE
Healthcare) and transferred to nitrocellulose membranes (GE
Healthcare). Alkyne biotin coupled to Click-iT.RTM. AHA
incorporated to newly synthesized proteins was detected using
streptavidin-HRP (Gentex). Revelation was performed by incubation
of ECL Prime (GE Healthcare) and read by chemoluminiscence using
Fusion Solo 3S (Vilber Lourmat).
1.5--Assessment of Translation Rates in Stressed Cells
[0253] Treatments were performed as for measuring translation in
unstressed cells, except that Tunicamycin (5 .mu.g/ml) was added
together with the compounds.
1.6--Functional GPCR Assay for Adrenergic .alpha.2A Receptor
(CellKey Detection Method)
[0254] The agonist activity of compounds was evaluated on CHO cells
endogenously expressing human alpha2A receptor and was determined
by measuring their effects on impedance modulation using the
CellKey detection method.
[0255] Cells were seeded onto 96-well plate at density of
6.times.10.sup.4 cells/well in HBSS buffer (Invitrogen)+20 mM HEPES
(Invitrogen) with 0.1% BSA and are allowed to equilibrate for 60
min at 28.degree. C. before the start of the experiment. Plates
were placed onto the system and measurements were made at a
temperature of 28.degree. C. Solutions were added simultaneously to
all 96 wells using an integrated fluidics system: HBSS (basal
control), reference agonist at 100 nM (stimulated control),
reference agonist (EC.sub.50 determination) or the test compounds.
Impedance measurements are monitored for 10 minutes after ligand
addition. The standard reference agonist is epinephrine, which is
tested in each experiment at several concentrations to generate a
concentration-response curve from which its EC.sub.50 value is
calculated.
[0256] Dose-response data from test compounds were analysed with
Hill software using non-linear regression analysis of the
concentration-response curves generated with mean replicate values
using Hill equation curve fitting. Results are presented table 1,
compounds with EC50>33.3 .mu.M are considered to have no
significant alpha-2 adrenergic activity.
1.7--In Vitro Multiple Sclerosis Disease Model: Interferon-Gamma
Injured Rat Oligodendrocytes Co-Cultured with Neurons Culture of
Oligodendrocyte Co-Cultured with Neurons
[0257] Neurons/OPC were cultured as previously describes by Yang et
al. (2005 J Neurosci Methods; 149(1) pp 50-6) with modifications.
Briefly, the full brain (without cerebellum) obtained from 17-day
old rat embryos (Wistar, Janvier labs) were removed. The full
brains were treated for 20 min at 37.degree. C. with a trypsin-EDTA
(Pan Biotech) solution at a final concentration of 0.05% trypsin
and 0.02% EDTA. The dissociation was stopped by addition of
Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/liter of
glucose (Pan Biotech), containing DNAse I grade II (final
concentration 0.5 mg/ml; Pan Biotech, Batch: h140508) and 10% fetal
calf serum (FCS; Invitrogen, Batch: 41Q7218K). Cells were
mechanically dissociated by three forced passages through the tip
of a 10-ml pipette. Cells were then centrifuged at 515 g for 10 min
at 4.degree. C. The supernatant was discarded, and the pellet was
resuspended in a defined culture medium consisting of Neurobasal
medium (Invitrogen, Batch: 1636133) with a 2% solution of B27
supplement (Invitrogen, Batch: 1660670), 2 mmol/liter of
L-glutamine (Pan Biotech), 2% of PS solution, and, 1% of FCS and 10
ng/ml of platelet derived growth factor (PDGF-AA, Batch: H131205).
The cells were seeded at a density of 20 000 cells per well in 96
well plates precoated with PLL (BD corning, Batch: 6614022) and
laminine (Sigma, Batch: 083M4034V). The plates were maintained at
37.degree. C. into a humidified incubator, in an atmosphere of air
(95%)-CO2 (5%). Half of the medium was changed every 2 days with
fresh medium. On days 18, test compounds were pre-incubated 1 hour
before interferon-gamma (70 U/ml, 48H, R&D system, Batch:
AAL2214081) application.
Test Compounds and Interferon-Gamma Exposure
[0258] On day 18 of culture, test compounds (4 concentrations) were
solved in culture medium and then pre-incubated with
oligodendrocyte co-cultured with neurons for 1 hour before the
interferon-gamma (70 U/ml, 48H) application. One hour after test
compounds incubation, interferon-gamma was added at 70 U/ml
concentration for 48 H still in presence of test compounds. Then,
cells were fixed by a cold solution of ethanol (95%, Sigma, Batch:
SZBD3080V) and acetic acid (5%, Sigma, Batch: SZBD1760V) for 5 min
at -20.degree. C. After permeabilization with 0.1% of saponin
(Sigma, Batch: BCBJ8417V), cells were incubated for 2 h with
Monoclonal Anti-O4 antibody produced in mouse (Sigma, batch:
SLBF5997V) at dilution of 1/1000 in PBS (PAN, Batch: 8410813)
containing 1% FCS, 0.1% saponin, for 2 h at room temperature. This
antibody are revealed with Alexa Fluor 488 goat anti-mouse IgG
(Invitrogen, batch: 1664729) at the dilution 1/400 in PBS
containing 1% FCS, 0.1% saponin, for 1 h at room temperature.
Analysis of Total Number of O4 Cells
[0259] For each condition, 30 pictures per well were taken using
ImageXpress (Molecular Device) with 20.times. magnification. All
images were taken with the same conditions. Analysis of total
number of O4 cells was performed automatically by using Custom
module editor (Molecular Device). Data were expressed in percentage
of control conditions (no intoxication, no interferon-gamma=100%)
in order to express the interferon-gamma injury. All values were
expressed as mean+/-SEM (s.e.mean) (n=6 wells per condition).
1.8--In Vitro Parkinson's Disease Model: Rotenone Injured Primary
Mesencephalic Rat Neurons
Culture of Mesencephalic Dopaminergic Neurons
[0260] Rat dopaminergic neurons were cultured as described by
Schinelli et al., (1988 J. Neurochem 50 pp 1900-07) and Visanji et
al., (2008 FASEB J. 22(7) pp 2488-97). Briefly, the midbrains
obtained from 15-day old rat embryos (Janvier Labs, France) were
dissected under a microscope. The embryonic midbrains were removed
and placed in ice-cold medium of Leibovitz (L15, Pan Biotech,
Batch: 9310614) containing 2% of Penicillin-Streptomycin (PS, Pan
Biotech, Batch: 1451013) and 1% of bovine serum albumin (BSA, Pan
Biotech, Batch: h140603). The ventral portion of the mesencephalic
flexure, a region of the developing brain rich in dopaminergic
neurons, was used for the cell preparations.
[0261] The midbrains were dissociated by trypsinisation for 20 min
at 37.degree. C. (Trypsin 0.05% EDTA 0.02%, PanBiotech, Batch:
5890314). The reaction was stopped by the addition of Dulbecco's
modified Eagle's medium (DMEM, PanBiotech, Batch: 1300714)
containing DNAase I grade II (0.1 mg/ml, PanBiotech, Batch:
H140508) and 10% of foetal calf serum (FCS, Gibco, Batch:
41Q7218K). Cells were then mechanically dissociated by 3 passages
through a 10 ml pipette. Cells were then centrifuged at 180.times.g
for 10 min at +4.degree. C. on a layer of BSA (3.5%) in L15 medium.
The supernatant was discarded and the cell pellets were
re-suspended in a defined culture medium consisting of Neurobasal
(Invitrogen, Batch: 1636133) supplemented with B27 (2%, Invitrogen,
Batch: 1660670), L-glutamine (2 mM, PanBiotech, Batch: 8150713) and
2% of PS solution and 10 ng/ml of Brain-derived neurotrophic factor
(BDNF, PanBiotech, Batch: H140108) and 1 ng/ml of Glial-Derived
Neurotrophic Factor (GDNF, Pan Biotech, Batch: H130917). Viable
cells were counted in a Neubauer cytometer using the trypan blue
exclusion test. The cells were seeded at a density of 40 000
cells/well in 96 well-plates pre-coated with poly-L-lysine (Corning
Biocoat, Batch: 6614022) and maintained in a humidified incubator
at 37.degree. C. in 5% CO.sub.2/95% air atmosphere. Half of the
medium was changed every 2 days with fresh medium.
[0262] On day 6 of culture, the medium was removed and fresh medium
was added, without or with rotenone (Sigma, Batch: 021M2227V) at 10
nM diluted in control medium, 3 wells per condition were assessed.
Test compounds were solved in culture medium and then pre-incubated
with mesencephalic neurons for 1 hour before the rotenone
application.
[0263] After 24 hours of intoxication, cells were fixed by a
solution of 4% paraformaldehyde (Sigma, batch SLBF7274V) in PBS
(Pan Biotech, Batch: 4831114), pH=7.3 for 20 min at room
temperature. The cells were washed again twice in PBS, and then
were permeabilized and non-specific sites were blocked with a
solution of PBS containing 0.1% of saponin (Sigma, batch:
BCBJ8417V) and 1% FCS for 15 min at room temperature. Then, cells
were incubated with Monoclonal Anti-Tyrosine Hydroxylase antibody
produced in mouse (TH, Sigma, batch: 101M4796) at dilution of 1/10
000 in PBS containing 1% FCS, 0.1% saponin, for 2 h at room
temperature. This antibody was revealed with Alexa Fluor 488 goat
anti-mouse IgG (Molecular Probes, batch: 1531668) at the dilution
1/800 in PBS containing 1% FCS, 0.1% saponin, for 1 h at room
temperature.
Analysis of Total Number of TH Positive Neurons
[0264] The immunolabeled cultures were automatically examined with
ImageXpress (Molecular device USA). For each condition, 20
automatically fields per well (representing .about.80% of the total
surface of the well) from 3 wells were analyzed. The total number
of TH neurons was automatically analyzed using Custom module editor
(Molecular Devices, USA). Data were expressed in percentage of
control conditions (no intoxication, no rotenone=100%) in order to
express the rotenone injury. All values were expressed as
mean+/-SEM (s.e. mean) of the 1 culture (n=3 wells per condition
per culture).
1.9--In Vitro Alzheimer Disease Model: Amyloid-Beta 1-42 Injured
Primary Cortical Rat Neurons.
[0265] Culture of Rat Cortical Neurons Rat cortical neurons were
cultured as described by Singer et al., (1999 J. Neuroscience 19 pp
2455-63) and Callizot et al., (2013 J. Neurosci. Res. 91 pp
706-16).
[0266] Pregnant females (Wistar; Janvier Labs) at 15 days of
gestation were killed by cervical dislocation. Fetuses were
collected and immediately placed in ice-cold L15 Leibovitz medium
(Pan Biotech, Batch: 9310614) with a 2% penicillin (10,000 U/ml)
and streptomycin (10 mg/ml) solution (PS; Pan Biotech, Batch:
1451013) and 1% bovine serum albumin (BSA; Pan Biotech, Batch:
h140603). Cortex was treated for 20 min at 37.degree. C. with a
trypsin-EDTA (Pan Biotech, Batch: 5890314) solution at a final
concentration of 0.05% trypsin and 0.02% EDTA. The dissociation was
stopped by addition of Dulbecco's modified Eagle's medium (DMEM)
with 4.5 g/liter of glucose (Pan Biotech, batch: 1300714),
containing DNAse I grade II (final concentration 0.5 mg/ml; Pan
Biotech, Batch: h140508) and 10% fetal calf serum (FCS; Invitrogen,
Batch: 41Q7218K). Cells were mechanically dissociated by three
forced passages through the tip of a 10-ml pipette. Cells were then
centrifuged at 515 g for 10 min at 4.degree. C. The supernatant was
discarded, and the pellet was resuspended in a defined culture
medium consisting of Neurobasal medium (Invitrogen, Batch: 1636133)
with a 2% solution of B27 supplement (Invitrogen, Batch: 1660670),
2 mmol/liter of L-glutamine (Pan Biotech, Batch: 8150713), 2% of PS
solution, and 10 ng/ml of brain-derived neurotrophic factor (BDNF;
Pan Biotech, Batch: H140108). Viable cells were counted in a
Neubauer cytometer, using the trypan blue exclusion test. The cells
were seeded at a density of 30,000 per well in 96-well plates
precoated with poly-L-lysine (Corning Biocoat, Batch: 6614022) and
were cultured at 37.degree. C. in an air (95%)-CO.sub.2 (5%)
incubator. The medium was changed every 2 days. The cortical
neurons were intoxicated with A-beta solutions (see below) after 11
days of culture.
Test Compounds and Amyloid-Beta 1-42 Exposure
[0267] The Amyloid-beta1-42 preparation was done following the
procedure described by Callizot et al., 2013. Briefly, Amyloid-beta
1-42 peptide (Bachem, Batch: 1014012) was dissolved in the defined
culture medium mentioned above, devoid of serum, at an initial
concentration of 40 .mu.mol/liter. This solution was agitated for 3
days at 37.degree. C. in the dark and immediately used after being
properly diluted in culture medium to the concentrations used.
[0268] Test compounds were solved in culture medium and then
pre-incubated with primary cortical neurons for 1 hour before the
Amyloid-beta 1-42 application. Amyloid-beta 1-42 preparation was
added to a final concentration of 20 .mu.M (including to .about.2
.mu.M of toxic oligomers measured by WB) diluted in control medium
in presence of drugs. After 24 hours of intoxication, cells were
fixed by a cold solution of ethanol (95%, Sigma, Batch: SZBD3080V)
and acetic acid (5%, Sigma, Batch: SZBD1760V) for 5 min at
-20.degree. C. After permeabilization with 0.1% of saponin (Sigma,
Batch: BCBJ8417V), cells were incubated for 2 h with mouse
monoclonal antibody anti microtubule-associated-protein 2 (MAP-2;
Sigma, Batch: 063M4802) at dilution of 1/400 in PBS (Pan biotech,
Batch: 4831114) containing 1% foetal calf serum (Invitrogen, Batch:
41Q7218K) and 0.1% of saponin. This antibody was revealed with
Alexa Fluor 488 goat anti-mouse IgG (Molecular probe, Batch:
1572559) at the dilution of 1/400 in PBS containing 1% foetal calf
serum and 0.1% of saponin for 1 H at room temperature.
Analysis of Total Number of Neurons
[0269] The immunolabeled cultures were automatically examined with
ImageXpress (Molecular device USA) at .times.20 magnification. For
each condition, 30 automatically fields per well (representing
.about.80% of the total surface of the well) from 3 wells were
analyzed. The total number of neurons was automatically analyzed
using Custom module editor (Molecular Devices, USA). Data were
expressed in percentage of control conditions (no intoxication, no
Amyloid-beta 1-42=100%) in order to express the A-beta 1-42 injury.
All values were expressed as mean+/-SEM (s.e.mean) (n=3 wells per
condition per culture).
1.10--In Vivo Mouse Model of Amyotrophic Lateral Sclerosis (ALS):
SOD1-G93A Transgenic Mice
[0270] Transgenic mice expressing mutated human SOD1-G93A
transgenic (TG) mice (heterozygous TgN-SOD1-G93A-1Gur; Gurney et
al. (1994) Science 264, 1772-1775) and 5 wild-type littermates were
used for the experiments. G93A SOD1 mice were bred by Charles River
Germany by mating hemizygous TG males (strain 002726M; B6SJL TG
SOD1.times.G93A 1GUR/J, JAX) with WT females (strain 10012, JAX)
obtained from JAX Laboratories USA. Animals were grouped as follows
(the females and males were distributed equally to treatment groups
i.e. each treatment group was strive to have equal numbers of males
and females): Transgenic G93A SOD1 mice [0271] 12 transgenic G93A
SOD1 mice treated with Vehicle QD (i.e. once a day) via oral gavage
starting at 60 days of age and continuing until end-point [0272] 12
transgenic G93A SOD1 mice treated with Compound 2 (1.5 mg/kg) BID
(i.e. twice a day) via oral gavage starting at 60 days of age and
continuing until end-point. [0273] 12 transgenic G93A SOD1 mice
treated with Compound 2 (3 mg/kg) QD via oral gavage starting at 60
days of age and continuing until end-point. [0274] 12 transgenic
G93A SOD1 mice treated with Compound 2 (3 mg/kg) QD in combination
with riluzole (20 mg/kg) via oral gavage starting at 60 days of age
and continuing until end-point. [0275] 12 transgenic G93A SOD1 mice
treated with Compound 2 (10 mg/kg) QD via oral gavage starting at
60 days of age and continuing until end-point.
Behavioral Testing
[0276] Rotarod test were performed before the dosing was started
(baseline, day 60) and around day 75, 90, 105, and 120. Mice born
within 2-4 days are pooled for rotarod testing. One day session
includes a training trial of 5 min at 4 RPM on the rotarod
apparatus (AccuScan Instruments, Columbus, USA). 30 min later, the
animals are tested for 3 consecutive accelerating trials of 6 min
with the speed changing from 0 to 40 RPM over 360 seconds and an
inter-trial interval at least 30 min. The latency to fall from the
rod is recorded. Mice remaining on the rod for more than 360
seconds are removed and their time scored as 360 seconds.
1.11--In Vivo Model of Charcot-Marie-Tooth 1A Disease: PMP-22
Overexpressing Transgenic Rat
[0277] CMT1A transgenic rats were obtained from mating of male
PMP-22 rats (Laboratory of Pr Nave, Max-Planck Institut fur
experimentelle Medizin, Gottingen, Germany) and female
Sprague-Dawley rats (Elevage Janvier, France). Animals were housed
and maintained at Key-Obs (Orleans, France). Animal procedures were
conducted in strict adherence to the EU Directive of Sep. 22, 2010
(2010/63/UE).
[0278] Animals were grouped as follows (Only male animals were
included in the experiments): [0279] 8 transgenic rat treated with
Vehicle QD via oral gavage starting at week 5 of age and continuing
until end-point. [0280] 8 transgenic rat treated with Compound 2 (1
mg/kg) QD via oral gavage starting at week 5 of age and continuing
until end-point. [0281] 8 transgenic rat treated with Compound 2 (3
mg/kg) QD via oral gavage starting at week 5 of age and continuing
until end-point.
Behavioral Testing
[0282] Animals were tested in a random and blind manner for
treatment and outcome measurements. Behavioral experiments and
readouts of bar was performed and validated at Key-Obs facilities
by the examiners who were blinded for the treatment. Bar test was
performed on CMT1A rats after 3 weeks and 5 weeks of treatment. Bar
test evaluated the muscular strength of the four paws and the
equilibrium performances on a fixed rod. The rat was placed on its
four paws on the middle of the wooden rod (diameter: 2.5 cm;
length: 50 cm). The time spent on the bar (fall latency) in each
trial and the number of falls were recorded. Five successive trials
were performed (60 s max).
1.12--In Vitro Model of Leukodystrophy (PMD): Overexpression of
Mutated PLP1 and DM20 in Human Cell Line
[0283] One day before transfection, 293T cells were plated at
300,000 cells/mL. 293T cells were transfected with PLP1 and DM20
mutant constructs using Lipofectamine 2000 according to
manufacturer's procedure. After transfection, cells were treated
with molecules or left untreated. As a control, cells were
transfected with native forms of the proteins. 48 h later, cellular
lysates were harvested. Protein accumulation was assessed by
western-blot.
1.13--In Vitro Model of Type 2 Diabetes: Min6 and INS1 Cell
Lines
[0284] Cytoprotection from ER Stress
[0285] Cells were plated in 96 well plates at a density of
0.5.10.sup.6 cells/mL for Min6 cell line, 0,4.10.sup.6 cells/mL for
INS1 cell line the day before the treatment.
[0286] ER stress was elicited by addition of 2.5 .mu.g/mL
tunicamycin (Sigma Aldrich) together with phosphatases
inhibitors.
[0287] Media were changed 6 h later with fresh media and the
cytoprotection was maintained by the addition of phosphatases
inhibitors.
[0288] Cell viability was assessed by measuring the reduction of
WST-8 into formazan using Cell Counting Kit-8 (Sigma) according to
the supplier's recommendation, 72 h after tunicamycin
treatment.
Protection Against Accumulation of Misfold Prone
Insulin.sup.Akita
[0289] Min6 cells were nucleofected with Insulin.sup.Akita mutant
constructs and seeded in 96 well-plates at 300,000 cells/mL and 24
h later, cells were treated with molecules or left untreated. As a
control, cells were nucleofected with non-relevant plasmid. 6 days
later, a selective agent was added (G418).
[0290] Cell viability was assessed by measuring the reduction of
WST-8 into formazan using Cell Counting Kit-8 (Sigma) according to
the supplier's recommendation, 9 days after treatment.
1.14--In Vitro Inflammation/Infection Disease Model: Poly I:C
Induced Mouse Embryonic Fibroblasts
Experimental Protocols
[0291] Mouse Embryonic Fibroblasts (MEFs) were lipofected with poly
I:C and treated with two concentrations of compounds of the
invention (25 .mu.M) for 6 h. After 6 h of culture,
eIF2alpha-phosphorylation (eIF2a-P) and PPP1R15A (GADD34)
expression was monitored by western blotting, while type-1
Interferon(IFN)-beta production was quantified in culture
supernatants by ELISA. Control (nt) and poly I:C/DMSO are
respectively negative and positive controls.
[0292] Poly I:C (polyinosinic:polycytidylic acid or
polyinosinic-polycytidylic acid sodium salt) is an immunostimulant
used to simulate viral infections. Poly I:C which is structurally
similar to double-stranded RNA, is known to interact with toll-like
receptor 3 which is expressed in the intracellular compartments of
B-cells and dendritic cells.
[0293] Guanabenz (25 .mu.M) was used as reference inhibitory
compound.
Cell Culture
[0294] MEFs were cultured in DMEM, 10% FCS (HyClone, Perbio), 100
units/ml penicillin, 100 .mu.g/ml streptomycin, 2 mM glutamine,
1.times.MEM non-essential amino acids and 50 .mu.M
2-mercaptoethanol. MEFS were treated for the indicated time with 10
.mu.g/ml poly I:C (InvivoGen) in combination with lipofectamine
2000 (Invitrogen).
Immunoblotting
[0295] Cells were lysed in 1% Triton X-100, 50 mM Hepes, 10 mM
NaCl, 2.5 mM MgCl2, 2 mM EDTA, 10% glycerol, supplemented with
Complete Mini Protease Inhibitor Cocktail Tablets (Roche). Protein
quantification was performed using the BCA Protein Assay (Pierce).
25-50 .mu.g of Triton X-100-soluble material was loaded on 2%-12%
gradient or 8% SDS-PAGE before immunoblotting and
chemi-luminescence detection (SuperSignal West Pico
Chemi-luminescent Substrate, Pierce). Rabbit polyclonal antibodies
recognizing GADD34 (C-19) were from Santa Cruz Biotechnology and
anti-el F2alpha[pS.sup.52] were from Invitrogen.
Elisa
[0296] IFN-beta quantification in culture supernatant was performed
using the Mouse Interferon Beta ELISA kit (PBL Interferon Source)
according to manufacturer instructions.
1.15--In Vivo Retinal Ciliopathies/Retinitis Pigmentosa Symptoms:
BBS12 Knock-Out Mice Generation of Knockout Mice and Animal
Husbandry
[0297] Bbs12.sup.-/-/J mice were kept on a C57BL/6 genetic
background (Mockel et al., 2012 J. Biol. Chem. 287 pp 37483-494).
Mice were kept and bred in humidity- and temperature-controlled
rooms on a 12 hour light/dark cycle with free access to normal chow
and water. Bbs12-/- total knock-out mice were identified by
genotyping through Polymerase Chain Reaction (PCR) using KAPA Mouse
Genotyping Kit (Catalog # KK7302, Kapa Biosystems, Woburn, Mass.,
USA).
Reagents for Intra-Vitreal Injection
[0298] The solution used for intravitreal injection was prepared
under sterile condition. 1.25 mM of compound 2 and 100 mM Valproic
acid (VPA) stock solutions were then diluted into PBS, pH6 to
obtain compound 2 (2.5 .mu.M)+VPA (0.2 mM) and compound 2 (2.5
.mu.M) solutions. Valproic acid was sourced (Catalog #4543,
Sigma-Aldrich).
Intra-vitreal injection
[0299] Bbs12.sup.-/- mice retinal phenotypes and mechanism were
published (Mockel et al., 2012). At postnatal day 14-16 mice were
injected intravitreally. The operation was performed under surgical
microscope. Mice were anesthetized with isoflurane. Pupils of mice
were dilated with 0.3% Atropine eye drops (Alcon). A 33-gauge
needle connected to a repeating dispenser (Hamilton Bonaduz AG,
Bonaduz, Switzerland) was inserted into the vitreous cavity from
the limbus. The location of needle was monitored through the
microscope. 1 .mu.l treatment solution was injected into the left
eye of mice, and 1 .mu.l PBS, pH6 was administered into the right
eye as control. Mice with vitreous hemorrhage or retinal damage
were excluded from analysis.
Electroretinograms
[0300] Electroretinograms (ERGs) were performed two weeks after
intravitreal injection using the HMsERG system (Ocuscience.RTM.,
Kansas City, Mo., USA). Mice were dark-adapted overnight and then
anesthetized by intraperitoneal injection of domitor (7.6 .mu.g/g
body weight) and ketamine (760 .mu.g/g body weight). Pupils were
dilated as described above. The experiments were carried out in dim
red light (catalog # R1251RR, Philips, Suresnes, France). ERGs
standard procedure was used according to manufacturer's protocol
(Ocuscience.RTM., Kansas City, Mo., USA). Briefly, the protocol
consisted in recording a dark-adapted ERG (scotopic ERG) after
photonic stimuli with intensities ranging from 0.1 to
25cds/m.sup.2. ERG results were amplified and captured digitally by
ERG View system 4.3 (Xenotec, Ocuscience.RTM., Kansas City, Mo.,
USA). The a-wave and b-wave of scotopic responses were then
measured.
Transmission Electron Microscopy
[0301] The samples were fixed by immersion in 2.5% Glutaraldehyde
and 2.5% Para formaldehyde in Cacodylate buffer (0.1M, pH 7.4), and
post fixed in 1% osmium tetroxide in 0.1M Cacodylate buffer for 1
hour at 4.degree. C. and dehydrated through graded alcohol (50, 70,
90, 100%) and propylene oxide for 30 minutes each. Samples were
embedded in Epon.TM. 812 (Sigma-Aldrich, Saint-Louis, Mo., USA).
Semi-thin sections were cut at 2 .mu.m with an ultra-microtome
(Leica Ultracut UCT Leica Biosystems, Wetzlar, Germany) and stained
with toluidine blue, and histologically analyzed by light
microscopy. Ultrathin sections were cut at 70 nm and contrasted
with uranyl acetate and lead citrate and examined at 70 kv with a
Morgagni 268D electron microscope. Images were captured digitally
by Mega View III camera (Soft Imaging System).
1.16--Hypoxia-Induced Apoptosis in Cultured Neonatal Rat
Cardiomyocytes
Cell Culture
[0302] Primary cultures of neonatal rat cardiomyocytes were
obtained from the ventricles of 1-day-old Sprague Dawley rats
(Janvier, France). The rats were euthanized and their hearts
excised. Hearts cut into small pieces (1-2 mm.sup.3) and
enzymatically digested using the Neonatal Heart Dissociation Kit
rat and the gentleMACS.TM. Dissociator (MiltenyiBiotec, Germany).
After dissociation, the homogenates were filtered (70 .mu.m) to
obtain a single-cell suspension. Isolated cells were collected by
centrifugation and resuspended in Dulbecco's Modified Eagle's
Medium (DMEM) containing 10% horse serum (HS), 5% fetal bovine
serum (FBS) and 1% penicillin/streptomycin. Cultures were enriched
with myocytes by pre-plating for 90 min to deplete the population
of non-myocytes. Non-attached cells were plated onto 6- or 96-well
plates at an appropriate cell density. The cells were cultured at
37.degree. C. in 95% air/5% CO.sub.2 for 24 h. Then the culture
medium was exchanged with fresh DMEM containing 1% FBS and
different concentrations of test compound thirty minutes before
incubation in a normal or a hypoxic (N.sub.2/CO.sub.2, 95%/5%; 0.3%
O.sub.2) culture chamber.
Treatment with Test Compound
[0303] Purified neonatal rat cardiomyocytes were seeded in a
96-well plate at 10.sup.6 cells/2 mL for flow cytometry
experiments.
[0304] After 24 hours, the cardiomyocytes were treated with
different concentrations of test compound in culture medium with
0.1% DMSO. The positive controls cells were treated with culture
medium (0.1% DMSO). Thirty minutes after starting the treatments,
the cells were incubated in the hypoxic culture chamber
(N.sub.2/00.sub.2, 95%/5%; final measured O.sub.2: 0.3%) for 36
hours.
[0305] The negative controls cells were left in normoxic conditions
at 37.degree. C. with culture medium (1% FBS, 0.1% DMSO) for the
same time periods.
Apoptotic Cell Measurement
[0306] At the end of the treatment period, flow cytometry were
performed to measure the amount of apoptotic cells. The Annexin
V-fluorescein isothiocyanate (FITC) apoptosis detection kit from
Miltenyi was used. Cells were washed twice with PBS and
re-suspended in binding buffer. FITC-Annexin V and propidium iodide
were added according to the manufacturer's protocol. The mixture
was incubated for 15 min in the dark at room temperature, and
cellular fluorescence was then measured by FACS scan flow
cytometry.
2--Results
2.1--Cytoprotection & Compound Selectivity
[0307] The results of the different assays ran with selected
compounds of the invention are shown below in Table 1.
[0308] As example, FIG. 1 represents the cytoprotective effect of
compound 1 after the stress induced by an exposure of
tunicamycin.
TABLE-US-00002 TABLE 1 Cytoprotection Translation from ER stress
inhibition in Translation recovery compared to non-stressed after
Tunicamycin Functional adrenergic Compound No guanabenz cells
treatment alpha2 receptor assay 1 ++ no effect prolongs EC50 >
33.3 .mu.M 2 ++ no effect prolongs EC50 > 33.3 .mu.M 3 ++ no
effect 4 + 5 + 6 + no effect 7 ++ no effect EC50 > 33.3 .mu.M 8
++ no effect 9 - 10 + 11 +
2.2--Multiple Sclerosis
[0309] FIG. 2 shows dose dependent protection of interferon-gamma
injured rat oligodendrocytes by compound 1 of the invention.
[0310] These data show that the compounds of this invention are
promising effective treatment of Multiple Sclerosis.
2.3--Parkinson's Disease (PD)
[0311] FIG. 3 shows dose dependent protection of rotenone injured
primary mesencephalic rat neurons by compound 2 of the
invention.
[0312] These data show that the compounds of this invention are
promising effective treatment of synucleopathies, and more
specifically Parkinson's disease.
2.4--Alzheimer Disease (AD) & Amyloidosis
[0313] FIG. 4 shows dose dependent protection of amyloid-beta 1-42
injured primary cortical rat neurons by compound 2 of the
invention.
[0314] These data show that the compounds of this invention are
promising effective treatment of Amyloidosis and more specifically
Alzheimer disease.
2.5--Amyotrophic Lateral Sclerosis (ALS)
[0315] FIG. 5 represents the results of rotarod test at day 90 of
transgenic SOD1 G93A mice with compound 2 of the invention.
[0316] These data show that the compounds of this invention,
specifically compounds 1 and 2, rescue motor deficit of transgenic
mice and are promising effective treatment of ALS.
2.6--Charcot-Marie-Tooth 1A (CMT-1A)
[0317] FIG. 6 represents the results of bar test at weeks 3 and 5
of transgenic rat overexpressing PMP22 treated with compound 2 of
the invention.
[0318] These data show that the compounds of this invention,
specifically compounds 1 and 2, rescue motor deficit of transgenic
rat overexpressing PMP22 and are promising effective treatment of
demyelinating disorders like CMT, more specifically CMT1A and
CMT1B.
2.7--Leukodystrophy: Pelizaeus-Merzbacher Disease (PMD),
[0319] T181P and L223P mutations in PLP1 and DM20 proteins have
been described to cause a severe phenotype of Pelizaeus-Merzbacher
disease (Strautnieks et al. 1992, Am. J. Hum. Genet. 51 (4):
871-878; Gow and Lazzarini, 1996 Nat Genet. 13(4):422-8).
[0320] The Compound 1 of the invention (5 microM) is able to
prevent the accumulation of T181P mutated DM20 protein expressed in
Human 293T cell (FIG. 7).
[0321] These data show that the compounds of this invention,
specifically compounds 1 and 2, are promising effective treatment
of demyelinating disorders like leukodystrophies, more specifically
PMD.
2.8--Type 2 Diabetes
[0322] FIG. 8 represents the results of over expression of
pre-pro-insulin bearing Akita mutation in Min6 cells with compound
6 of the invention.
[0323] The Compound 1 and compound 6 at different concentrations
prevent Min6 insulinoma cell death associated with accumulation of
misfolded protein induced by 6 hour exposure to tunicamycin (FIG.
9)
[0324] The compound 1 and compound 6 at different concentrations
prevent INS1 insulinoma cell death associated with accumulation of
misfolded protein induced by 6 hour exposure to tunicamycin. (FIG.
10).
[0325] These data show that the compounds of the invention are
promising effective treatment of pre-diabetes and diabetes,
preferably type 2 pre-diabetes and type 2 diabetes.
2.9--Retinal Ciliopathies/Bardet Biedl Syndrome
[0326] The compound 2 is able to protect photoreceptors against
apoptosis and preserves light detection (FIG. 11) and to decrease
protein load in the Endoplasmic Reticulum (FIG. 12) in BBS12-/-
mice. These results show an increased ERG response when treated
with the Compound 2 and Valproic acid (VPA) combination or with
compound 2 alone in the Bbs12-/- mice (FIG. 11). FIG. 12 shows a
representative transmitted electron microscopy picture of ER of the
photoreceptors of BBS12-/- mouse in response to the indicated
administrated treatment or genotype. Dilatation is observed when
PBS only is injected (left) whereas Compound 2 (2.5 microM) in
combination with VPA (valproic acid) (0.2 mM) are able to decrease
this dilatation after one single intra-vitreal injection.
[0327] These data show that the compounds of this invention in
association with a compound increasing the expression and/or the
activity of BIP protein, such as Valproic acid, are promising
effective treatment of retinal ciliopathies such as Bardet-Biedl
syndrome and retinitis pigmentosa.
[0328] Although not tested, we hypothesized that this treatment
might also be helping in reducing other forms of cellular stress
like the photonic stress.
2.10--Infection-Related or Non-Infectious Inflammatory
Conditions
[0329] Normal response of MEFs to poly I:C is characterized by
PPP1R15A expression, increase in eIF2alpha-P (variable in time and
related to the levels PPP1R15A expression) mediated by PKR
activation and type-I IFN production (range 500 to 700 pg/ml).
Knock out PPP1R15-/- MEFs are unable to produce this cytokine in
response to poly I:C.
[0330] The potency of compounds of the invention to inhibit
PPP1R15A was evaluated by measuring the increase of eIF2alpha
phosphorylation, the decrease of PPP1R15A expression due to its own
pharmacological inhibition resulting in general protein synthesis
inhibition and type-I IFN production.
[0331] The evaluated compounds of the invention were found
efficient at 25 .mu.M to increase eIF2alpha phosphorylation, to
decrease of PPP1R15A expression and to prevent type-I IFN
production. As example, FIG. 13 shows the ability of compounds 1, 6
and 8 (at 25 microM) to prevent type-I IFN production by mouse
embryonic fibroblasts lipofected with poly I:C.
[0332] These data show that the compounds of this invention are
promising effective treatment of infection-related or
non-infectious inflammatory conditions.
2.11--Cardiac Ischemia
[0333] The compound 2 of the invention protects cultured neonatal
rat cardiomyocytes from hypoxia-induced apoptosis (FIG. 14). These
data show that the compounds of this invention are promising
effective treatment of ischemia, specifically cardiac ischemia.
[0334] Various modifications and variations of the invention will
be apparent to those skilled in the art without departing from the
scope and spirit of the invention. Although the invention has been
described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
which are obvious to those skilled in the relevant fields are
intended to be covered by the present invention.
* * * * *
References