NL2020792B1 - Cantharidinimides for treating a demyelinating disease - Google Patents
Cantharidinimides for treating a demyelinating disease Download PDFInfo
- Publication number
- NL2020792B1 NL2020792B1 NL2020792A NL2020792A NL2020792B1 NL 2020792 B1 NL2020792 B1 NL 2020792B1 NL 2020792 A NL2020792 A NL 2020792A NL 2020792 A NL2020792 A NL 2020792A NL 2020792 B1 NL2020792 B1 NL 2020792B1
- Authority
- NL
- Netherlands
- Prior art keywords
- cantharidinimides
- cantharidinimide
- formula
- hydrogen
- group
- Prior art date
Links
- UFWIBTONFRDIAS-UHFFFAOYSA-N c(cc1)cc2c1cccc2 Chemical compound c(cc1)cc2c1cccc2 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to cantharidinimides for treating a demyelinating disease. In particular, cantharidinimides for treating a demyelinating disease selected from the group consisting of demyelinating myelinoclastic diseases and demyelinating leukodystrophic diseases, such as multiple sclerosis, clinically isolated syndrome, Devic's disease, Neuromyelitis optica, anti-MOG autoimmune encephalomyelitis, Chronic relapsing inflammatory optic neuritis, Acute disseminated encephalomyelitis, Acute hemorrhagic leukoencephalitis, Balo concentric sclerosis, diffuse myelinoclastic sclerosis (Schilder diffuse sclerosis), Marburg multiple sclerosis, Tumefactive multiple sclerosis and Solitary sclerosis.
Description
DESCRIPTION
The present invention relates to cantharidinimides for treating a demyelinating disease.
Demyelinating diseases are diseases of the nervous system in which the myelin sheath of neurons is damaged. This damage impairs the conduction of signals in the affected nerves. In turn, the reduction in conduction ability causes deficiency in sensation, movement, cognition, or other functions depending on which nerves are involved. Some demyelinating diseases are caused by genetics, some by infectious agents, some by autoimmune reactions, some by unknown factors, and some by a combination of (some of) these factors.
Demyelinating diseases are traditionally classified in two kinds: demyelinating myelinoclastic diseases and demyelinating leukodystrophic diseases. In the first group a normal and healthy myelin is destroyed by a toxic, chemical or autoimmune substance. In the second group (also denominated as dysmyelinating diseases), myelin is abnormal and degenerates.
The best-known example of a demyelinating disease is multiple sclerosis (MS). MS is a chronic inflammatory and degenerative disease of the central nervous system (CNS) with a highly heterogeneous course of disease progression, and caused by a complex interplay of genetic and environmental factors. The inflammatory and neurodegenerative processes cause damage to nerve cells (neurons), which eventually leads to a loss of the electrically isolating myelin sheet around the axons, the neuronal extensions that transmit brain signals. This loss of myelin and hence neuronal functionality gives rise to a variety of neurological symptoms and impairments, depending on the brain region where the demyelination occurs.
MS symptoms are diverse with large heterogeneity among patients, making an accurate diagnosis and prognosis cumbersome. Effective MS treatments reduce the number and severity of relapses, and disease burden.
Despite the broadening range of available treatments, the response of MS patients to medication remains unpredictable and heterogeneous. Furthermore, it has been suggested in the art that genetic heterogeneity influences the pathogenesis of disease, and is involved in the disease progression, i.e. the number of relapses, the rate of disease progression and the overall disease burden.
In MS (and most other demyelinating diseases), the myelin sheath surrounding neurons is gradually degraded and/or not repaired anymore, a process that together with the autoimmune reaction characterizes MS and causes the neurological symptoms of the disease.
In order to improve the patient’s quality of life and slow down the rate of demyelination, there is a need for a therapy to treat demyelinating diseases by stopping the demyelination process and/or inducing a remyelination mechanism of the affected nerves.
The present invention provides hereto cantharidinimides for treating a demyelinating disease, wherein the cantharidinimides are selected from compounds of formula (A):
N~(CH2)n
Ar ch3q (A), wherein:
n is independently selected from 0, 1, 2, 3, 4 or 5; and
Ar is independently selected from the group consisting of compounds of formula (B-G):
wherein:
R represents one or more substituents independently selected from hydrogen, fluoro, chloro, bromo, iodo, hydroxy, nitro, cyano, amino, C1.4 alkyl, C14 alkoxy, C1-4 alkylamino, C1-4 alkylthio, aryl, benzyl, phenoxy or benzoxy;
formula C is selected from the group consisting of R-substituted 1-naphthyl and R-substituted 2-naphthyl;
formula E is selected from the group consisting of R-substituted 2-pyridyl, R-substituted 3-pyridyl and R-substituted 4-pyridyl;
formula F is selected from the group consisting of R-substituted 2-pyrimidinyl, R-substituted 4-pyrimidinyl and R-substituted 5-pyrimidinyl; and formula G is selected from the group consisting of R-substituted 2-indolyl and R-substituted 3-indolyl.
It was found that the cantharidinimides of the present invention stop the demyelination process and/or induce a remyelination mechanism. Thus, the cantharidinimides of the present invention are good drug candidates for treating demyelinating diseases. Demyelinating diseases may be selected from the group consisting of demyelinating myelinoclastic diseases and demyelinating leukodystrophic diseases.
It is noted that the cantharidinimides of the present invention are good drug candidates for treating inflammatory demyelinating diseases in particular. Examples of inflammatory demyelinating diseases include, but are not limited to, multiple sclerosis, clinically isolated syndrome, Devic's disease, Neuromyelitis optica, anti-MOG autoimmune encephalomyelitis, Chronic relapsing inflammatory optic neuritis, Acute disseminated encephalomyelitis, Acute hemorrhagic leukoencephalitis, Balo concentric sclerosis, diffuse myelinoclastic sclerosis (Schilder diffuse sclerosis), Marburg multiple sclerosis, Tumefactive multiple sclerosis and Solitary sclerosis.
In an embodiment of the present invention, the cantharidinimides of the present invention are good drug candidates for treating demyelinating diseases selected from the group consisting of standard multiple sclerosis, relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis and benign multiple sclerosis.
The present invention also relates to a formulation for treating a demyelinating disease, wherein the formulation comprises at least one cantharidinimide of the present invention. Such formulation may comprise any suitable form for administration of at least one cantharidinimide to a subject suffering from the demyelinating disease. The subject may be a mammal including human beings.
The present invention also relates to a method of treating a subject suffering from a demyelinating disease by administration of a pharmacologically effective amount of at least one cantharidinimide of the present invention.
The present invention further relates to the use of at least one cantharidinimide of the present invention in the treatment of a subject suffering from a demyelinating disease.
Given the cantharidinimides of the present invention as defined above, it is noted that the C-i-4 alkyl may be selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. Further, the C1-4 alkoxy may be selected from the group consisting of methoxy, ethoxy, propoxy, isopropoxy, butoxy and tert-butoxy. Methoxy is the preferred C1-4 alkoxy. With regard to the C1-4 alkylamino it was found that the Ci4 alkylamino may be selected from the group consisting of monomethylamino, dimethylamino, monoethylamino and diethylamino. Dimethylamino and diethylamino are preferred substituents. Further, C1-4 alkylthio may be selected from the group consisting of methylthio, ethylthio, propylthio, isopropylthio, butylthio and tertbutylthio.
With regard to the aryl substituent it is noted that the aryl may be selected from the group consisting of phenyl, tolyl, and xylyl. Preferably the aryl substituent comprises a phenyl.
With regard to the formulas C, F and G it is noted that the cantharidinimides of the present invention may have different configurations. However, for formula C an Rsubstituted 2-naphthyl is preferred, for formula F an R-substituted 2-pyrimidinyl is preferred and for formula G an R-substituted 3-indolyl is preferred.
In an embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from compounds of formula (AB):
(AB), wherein n is independently selected from 0, 1, 2, 3, 4 or 5; and
R1 represents one or more substituents independently selected from hydrogen, fluoro, chloro, bromo, iodo, hydroxy, nitro, cyano, amino, C1.4 alkyl, C1.4 alkoxy, C1-4 alkylamino, C1.4 alkylthio, aryl, benzyl, phenoxy or benzoxy in the ortho, meta and/or para position.
Preferred cantharidinimides selected from compounds of formula AB may include cantharidinimides wherein R1 is selected from hydrogen, amino, hydroxy, methoxy, benzoxy, nitro or phenyl in the ortho, meta and/or para position. More specifically, even further preferred cantharidinimides selected from compounds of formula AB may include cantharidinimides wherein R1 is selected from hydrogen, ortho-, meta- or paraamino, ortho- or para-hydroxy, para-nitro, para-phenyl, para-methoxy, meta-paradimethoxy, meta-benzoxy-para-methoxy or meta-methoxy-para-benzoxy.
In an embodiment of the present invention, the cantharidinimides selected from compounds of formula AB may include cantharidinimides wherein n is independently selected from 0, 1,2, or 4 and R1 is selected from hydrogen, ortho-amino, meta-amino, para-amino, meta-hydroxy, para-hydroxy, meta-para-dimethoxy, para-methoxy, metabenzoxy-para-methoxy, meta-methoxy-para-benzoxy, para-nitro or para-phenyl.
In a further embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from compounds of formula (I):
wherein
O),
R7 represents one or more substituents independently selected from hydrogen, hydroxy, preferably meta- and/or para-hydroxy, nitro, preferably para-nitro, and/or phenyl, preferably para-phenyl.
In another embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from compounds of formula (II):
wherein
R8 represents one or more substituents independently selected from hydrogen, hydroxy, preferably para-hydroxy, and/or amino, preferably ortho-, meta- and/or paraamino.
In an embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from compounds of formula (III):
(HI).
wherein
R9 represents one or more substituents independently selected from hydrogen, hydroxy, preferably para-hydroxy, methoxy, preferably meta- and/or para-methoxy, and/or benzoxy, preferably meta- and/or para-benzoxy.
Further, in an embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from a compound of formula (IV):
(IV).
In an embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from compounds of formula (AC):
(AC), wherein n is independently selected from 0, 1, 2, 3, 4 or 5; and
R2 represents one or more substituents independently selected from hydrogen, fluoro, chloro, bromo, iodo, hydroxy, nitro, cyano, amino, C1-4 alkyl, C1.4 alkoxy, C1.4 alkylamino, C1.4 alkylthio, aryl, benzyl, phenoxy or benzoxy in the 5, 6, 7 and/or 8 position.
Preferred cantharidinimides selected from compounds of formula AC may include cantharidinimides wherein R2 is selected from hydrogen, hydroxy, methoxy, in the 5, 6, 7 and/or 8 position.
The cantharidinimides for treating a demyelinating disease may be selected from a compound of formula (V):
(V).
In an embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from compounds of formula (AD):
(AD), wherein n is independently selected from 0, 1, 2, 3, 4 or 5; and
R3 represents one or more substituents independently selected from hydrogen, fluoro, chloro, bromo, iodo, hydroxy, nitro, cyano, amino, C1-4 alkyl, C1-4 alkoxy, C1.4 alkylamino, C1.4 alkylthio, aryl, benzyl, phenoxy or benzoxy in the 4, 5, 6 and/or 7 position.
Preferred cantharidinimides selected from compounds of formula AD may include cantharidinimides wherein R3 is selected from hydrogen, hydroxy or methoxy, in the 5, 6, 7 and/or 8 position, preferably selected from hydrogen or 5-methoxy.
The cantharidinimides for treating a demyelinating disease may be selected from a compound of formula (VI):
(VI), wherein
R10 represents one or more substituents independently selected from hydrogen, and/or methoxy, preferably 5-methoxy.
In an embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from compounds of formula (AE):
wherein n is independently selected from 0, 1, 2, 3, 4 or 5; and
R4 represents one or more substituents independently selected from hydrogen, fluoro, chloro, bromo, iodo, hydroxy, nitro, cyano, amino, C1-4 alkyl, C1-4 alkoxy, C1-4 alkylamino, C1-4 alkylthio, aryl, benzyl, phenoxy or benzoxy.
Preferred cantharidinimides selected from compounds of formula AE may include cantharidinimides wherein R4 is hydrogen.
The cantharidinimides for treating a demyelinating disease may be selected from the compounds of formulas (VII, VIII, IX):
(VIII); and (IX).
In an embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from compounds of formula (AF):
(AF), n is independently selected from 0, 1, 2, 3, 4 or 5; and
R5 represents one or more substituents independently selected from hydrogen, fluoro, chloro, bromo, iodo, hydroxy, nitro, cyano, amino, C1-4 alkyl, C1.4 alkoxy, C1-4 alkylamino, C1-4 alkylthio, aryl, benzyl, phenoxy or benzoxy.
Preferred cantharidinimides selected from compounds of formula AF may include cantharidinimides wherein R5 is hydrogen.
In an embodiment of the present invention, the cantharidinimides for treating a demyelinating disease may be selected from compounds of formula (AG):
(AG), wherein n is independently selected from 0, 1, 2, 3, 4 or 5; and
R6 represents one or more substituents independently selected from hydrogen, fluoro, chloro, bromo, iodo, hydroxy, nitro, cyano, amino, C1-4 alkyl, C1-4 alkoxy, C1-4 alkylamino, C1-4 alkylthio, aryl, benzyl, phenoxy or benzoxy.
Preferred cantharidinimides selected from compounds of formula AG may include cantharidinimides wherein R6 is hydrogen.
The cantharidinimides for treating a demyelinating disease may be selected from the compounds of formulas (X):
(X).
The cantharidinimides are preferably selected from the group consisting of NBenzylcantharidinimide, N-(2-Aminobenzyl)cantharidinimide, N-(3Aminobenzyl)cantharidinimide, N-(4-Aminobenzyl)cantharidinimide, N-[2-(4’Hydroxyphenyl)ethyl] cantharidinimide, N-(4-Phenylbutyl)cantharidinimide, N-[2-(3’,4’Dimethoxyphenyl)ethyl] cantharidinimide, N-[2-(p-Methoxyphenyl)ethyl] cantharidinimide, N-[2-(3’-Methoxy-4’-benzyloxyphenyl)ethyl]cantharidinimide, N-[2(3’-Benzyloxy-4’-methoxyphenyl)ethyl]cantharidinimide, N-(4Hydroxyphenyl)cantharidinimide, N-(3-Hydroxyphenyl)cantharidinimide, N-4-(1Nitro)phenylcantharidinimde, N-(4-Biphenyl)cantharidinimide, N-[2-(2Naphthyl)ethyl]cantharidinimide, N-[2-(3’-lndolyl)ethyl]cantharidinimide, N-[2-(5’Methoxy-3’-lndolyl)ethyl] cantharidinimide, N-[2-(2’-Pyridyl)ethyl]cantharidinimide and N-[2-(3’,4’-Methylenedioxyphenyl)ethyl] cantharidinimide.
EXAMPLES
Synthesis of cantharidinimides
Cantharidinimides were prepared by heating cantharidin and primary amines, aniline derivatives or aminopyridines according to the following general reaction scheme:
H2NR, Et3N toluene, reflux
The reactants were heated to a temperature of about 200°C with 3 mL of dry toluene and about 1.5 mL of trimethylamine in a high-pressure sealed tube (Buchi glasuster 0032). After 2 hours and recrystallization from methanol the cantharidinimides listed in table 1 were obtained.
Table 1. Overview of synthesized compounds
Ex | Structure | Chemical name | |
1 | CHsy o I N—\ θΗ=ο ΛΛ | N-Benzylcantharidinimide | |
2 | ο | Ί-Ι θ lX N---v NH2 ζΧ XX -1-I3 0 c y | N-(2-Aminobenzyl)cantharidinimide |
3 | o | •>|_l O -3 // N--\ DH3 0 y--NH2 | N-(3-Aminobenzyl)cantharidinimide |
4 | o | Ί-Ι ° N—\ ζΧ -¾ 0 <7 y nh2 | N-(4-Aminobenzyl)cantharidinimide |
5 | o | ~>i_i ° _ 3 // n—\ Γ/—λ ---\ /--OH DH3 O \=/ | N-[2-(4’-Hydroxyphenyl)ethyl] cantharidinimide |
6 | o | Ί-Ι θ ; 3// N--\ iX \_v=\ 3 ° λ \\ /7 | N-(4-Phenylbutyl)cantharidinimide |
7 | ch3/? | N-[2-(3’,4’-Dimethoxyphenyl)ethyl] cantharidinimide | ||
o | JJ OMe | |||
N--\ ^h 3'q | --ς —OMe | |||
?H3// | ||||
o | N—\ | .---------------------. | ||
8 | // \\ | N-[2-(2-Naphthyl)ethyl]cantharidinimide | ||
\ / \\ | ||||
θΗ3/° | ||||
q | o | N—\ | N-[2-(3’,4’-Methylenedioxyphenyl)ethyl] | |
\ /^° | cantharidinimide | |||
θΗ3 Q | '---------\ I | |||
O·^ | ||||
ch3/° | ||||
10 | o | N---v | N-[2-(p-Methoxyphenyl)ethyl] | |
u / \ | cantharidinimide | |||
-—OMe | ||||
θΗ3*Ο | ||||
?H3/? | ||||
11 | O | N—\ | N-[2-(3’-Methoxy-4’- | |
— | benzyloxyphenyl)ethyl]cantharidinimide | |||
CH3^ | \---/ | |||
OMe |
12 | CH^ I O Γ N---\ r--λ '--</ —oMe ch3 o O | N-[2-(3’-Benzyloxy-4’- methoxyphenyl)ethyl]cantharidinimide | |
13 | O | ->|_l O HH3// N—\ f/—X lX \ / -m3 o N—/ | N-[2-(2’-Pyridyl)ethyl]cantharidinimide |
14 | CH^ CDd^^h ch3\\ | N-[2-(3’-lndolyl)ethyl]cantharidinimide | |
15 | O | ~LJ O jj iH\\ VI -ns 0 / MeO | N-[2-(5’-Methoxy-3’-lndolyl)ethyl] cantharidinimide |
16 | CHs^ —C —oh CH3^ | N-(4-Hydroxyphenyl)cantharidinimide |
17 | ch3/° | N-(3-Hydroxyphenyl)cantharidinimide | |
o | N---(( | ||
CH3^O oh | |||
18 | ChJ? --N O 2 | N-4-(1-Nitro)phenylcantharidinimde | |
19 | o | ~|_| O ;3// N---(( J)---(( J) Xï -H3 o | N-(4-Biphenyl)cantharidinimde |
Cultures of cell lines
The neuroblastoma cell line SH-SY5Y was purchased from ATCC (Manassas, VA, USA). Human oligodendroglial HOG cells were kindly provided by Prof. Dr. José Antonio López Guerrero (Univeristy of Madrid, Spain). The MO3.13 cell line was kindly provided by Prof. Dr. Neil Cashman (University of Vancouver, Canada).
All cell lines were cultured in growth medium (GM) containing Earl’s Modified Eagle Medium (EMEM, Lonza) supplemented with 15% heat-inactivated fetal bovine serum (hiFBS, Gibco), 1% antibiotic-antimycotic (Gibco) and 1% Glutamax (Gibco) in a humidified 5% CO2 incubator at 37°C. Regular mycoplasm tests (MycoAlert Detection Kit, Lonza) were performed in order to assure the bacteria-free status of the cells.
Differentiation of cell lines
SH-SY5Y
At 0 days in vitro (DIV), SH-SY5Y cells were plated at a confluency of 5.0*104 cells/cm2 in a plate or on a glass coverslip coated with Matrigel (1 : 100; Corning) diluted in Dulbecco’s Modified Eagle Medium (DMEM) for 1 hour at 37°C.
To induce differentiation, the cells were subjected for 6 DIV to differentiation medium 1 (DM1), consisting of EMEM, 2.5% hiFBS and 1% antibiotic-antimycotic, freshly supplemented at each medium change with 10 pm all-trans retinoic acid (ATRA, Sigma). Medium of the cells was changed every 48 hours.
At 7 DIV, the cells were split 1 : 1 onto a new plate coated with Matrigel (1 : 100) diluted in DMEM for 1 hour at 37°C.
At 8 DIV, the cells were subjected to differentiation medium 2 (DM2), consisting of EMEM, 1% hiFBS and 1% antibiotic-antimycotic, freshly supplemented at each medium change with 10 pm ATRA.
At 10 DIV, the cells were split 1 : 1 onto a new plate coated with Matrigel (1 : 100) diluted in DMEM for 1 hour at 37°C.
To induce final differentiation, differentiation medium 3 (DM3) was added at 11, 14 and 17 DIV. DM3 consists of Neurobasal Medium (Gibco) supplemented with 20 mM KCI (Merck), 20 mM B-27 supplement (Gibco), 1% antibiotic-antimycotic, culture-one supplement (1 : 100, Gibco), 50 ng/ml brain-derived neurotrophic factor (BDNF, Sigma), 2mM adenosine-3',5'-cyclic monophosphate (cAMP, Sigma) and 10 μΜ ATRA added freshly at each medium change.
HOG and MO3.13
At 0 DIV, the HOG cells as well as the MO3.13 cells were plated at a confluency of 1.5*104 cells/cm2 in a plate or on a glass coverslip coated with Matrigel (1:100) diluted in DMEM for 1 hour at 37°C.
To induce differentiation, the HOG and MO3.13 cells were subjected for 5 DIV to differentiation medium 4 (DM4), consisting of EMEM supplemented with 50 pg/ml apotransferrin (Sigma), 16 pg/ml putrescine (Sigma), 0.5 pg/ml human-derived insulin (Sigma), 30 nM sodium selenite (Sigma), 30 nM triiodo-L-thyronine (T3, Sigma), 500 μΜ 3-isobutyl-1-methylxanthine (IBMX, Sigma), 500 μΜ cAMP and 1% antibioticantimycotic.
Oxidative stress-exposed cell lines
Undifferentiated SH-SY5Y cells were exposed to oxidative insult of hydrogen peroxide (H2O2, Sigma) at a concentration of 100 μΜ for 18 h.
Undifferentiated HOG cells were exposed to oxidative insult of H2O2 at a concentration of 100 μΜ for 18 h.
Undifferentiated MO3.13 cells were exposed to oxidative insult of H2O2 at a concentration of 500 μΜ for 18 h.
The optimal concentrations of H2O2 were determined by dose-response experiments. Cell viability following oxidative insult experiments was determined by quantification of fluorescent cell signals with the NucBlue/NucGreen Cell Viability kit (Thermo Fisher) and fluorescence-activated cell sorting (FACS) of NucGreen-positive cells and Propidium Iodide (Sigma)-positive cells.
Following visual confirmation of the differentiated phenotype, differentiated cell lines SH-SY5Y, HOG and MO3.13 were exposed to oxidative insult of H2O2. The optimal concentrations to induce oxidative insult in differentiated SH-SY5Y cells, HOG cells and MO3.13 cells were 500 μΜ, 50 μΜ and 100 μΜ, respectively. Quantification of cell viability was performed by quantification of fluorescent cell signals with the NucBlue/NucGreen Cell Viability kit and FACS of NucGreen-positive cells and Propidium Iodide-positive cells.
Co-cultures of SH-SY5Y and HOG
Co-culture of differentiated SH-SY5Y cells with undifferentiated HOG cells
In order to generate a co-culture of differentiated SH-SY5Y cells with undifferentiated HOG cells, the protocol to generate differentiated SH-SY5Y cells was used until 10 DIV.
At 10 DIV, the differentiating SH-SY5Y cells were split 1 : 1 onto a new plate coated with Matrigel (1 : 100) diluted in DMEM for 1 hour at 37°C. SH-SY5Y cell density was determined by cell counting by hand and undifferentiated HOG cells were added at various cell densities relative to the number of SH-SY5Y cells, at a ratio of HOG : SHSY5Y cells of 1 : 2, 1 : 4, 1 : 6 and 1 : 8.
Co-culture of differentiated SH-SY5Y cells with differentiated HOG cells
First, a co-culture of differentiated SH-SY5Y cells with undifferentiated HOG cells was generated using the above protocol.
Next, in order to generate a co-culture of differentiated SH-SY5Y cells with differentiated HOG cells, differentiation medium 5 (DM5) was added at 11, 14 and 17 DIV. DM5 consists of DM3 supplemented with 50 pg/ml apo-transferrin, 16 pg/ml putrescine, 0.5 pg/ml human-derived insulin, 30 nM sodium selenite, 30 nM T3 and 500 pM IBMX.
Oxidative stress-exposed co-cultures of SH-SY5Y and HOG
To elicit oxidative stress, the above obtained co-cultures of differentiated SH-SY5Y cells and undifferentiated HOG cells and differentiated SH-SY5Y cells and differentiated HOG cells were exposed to various concentrations of H2O2 in order to determine the optimal concentrations.
Co-cultures of SH-SY5Y and MO3.13
Co-culture of differentiated SH-SY5Y cells with undifferentiated MO3.13 cells
In order to generate a co-culture of differentiated SH-SY5Y cells with undifferentiated MO3.13 cells, the protocol to generate differentiated SH-SY5Y cells was used until 10 DIV.
At 10 DIV, the differentiating SH-SY5Y cells were split 1 : 1 onto a new plate coated with Matrigel (1 : 100) diluted in DMEM for 1 hour at 37°C. SH-SY5Y cell density was determined by cell counting by hand and undifferentiated MO3.13 cells were added at various cell densities relative to the number of SH-SY5Y cells, at a ratio of MO3.13 : SH-SY5Y cells of 1 : 2, 1 : 4, 1 : 6 and 1 : 8.
Co-culture of differentiated SH-SY5Y cells with differentiated MO3.13 cells
First, a co-culture of differentiated SH-SY5Y cells with undifferentiated MO3.13 cells was generated using the above protocol.
Next, in order to generate a co-culture of differentiated SH-SY5Y cells with differentiated MO3.13 cells, DM5 was added at 11, 14 and 17 DIV.
Oxidative stress-exposed co-cultures of SH-SY5Y and MO3.13
To elicit oxidative stress, the above obtained co-cultures of differentiated SH-SY5Y cells and undifferentiated MO3.13 cells and differentiated SH-SY5Y cells and differentiated MO3.13 cells were exposed to various concentrations of H2O2 in order to determine the optimal concentrations.
Myelin-inducing activity of cantharidinimides
The cantharidinimides of table 1 were tested in the above-obtained cell cultures.
Hereto various concentrations of cantharidinimides (1 to 20 μΜ) were added to: cultures of undifferentiated and differentiated SH-SY5Y cells; cultures of undifferentiated and differentiated HOG cells; cultures of undifferentiated and differentiated MO3.13 cells;
cultures | of | oxidative | stress-exposed | undifferentiated | and |
differentiated SH-SY5Y cells; | |||||
cultures | of | oxidative | stress-exposed | undifferentiated | and |
differentiated HOG cells; | |||||
cultures | of | oxidative | stress-exposed | undifferentiated | and |
differentiated MO3.13 cells;
co-culture of differentiated SH-SY5Y cells with undifferentiated
HOG cells;
co-culture of differentiated SH-SY5Y cells with differentiated
HOG cells;
co-culture of differentiated SH-SY5Y cells with undifferentiated
MO3.13 cells;
co-culture of differentiated SH-SY5Y cells with differentiated
MO3.13 cells;
oxidative stress-exposed co-culture of differentiated
SH-SY5Y cells with undifferentiated HOG cells;
oxidative stress-exposed co-culture of differentiated
SH-SY5Y cells with differentiated HOG cells;
oxidative stress-exposed co-culture of differentiated
SH-SY5Y cells with undifferentiated MO3.13 cells; and oxidative stress-exposed co-culture of differentiated
SH-SY5Y cells with differentiated MO3.13 cells.
Following treatment with cantharidinimides, the exposed cells were analysed with:
microscopy on cells stained with antibodies directed towards the myelin-related proteins myelin-basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), myelin proteolipid protein (PLP1) and myelin-associated glycoprotein (MAG), and quantification of the signals with Cellomics software (Thermo Fisher), wherein an increase in the amount of staining reflects myelin-inducing activity of the cantharidinimides;
live-cell imaging of living cells using the fluorescent myelin stain FluoroMyelin Red (Thermo Fisher) and quantification of the signals with Cellomics, wherein an increase in the amount of fluorescent staining reflects myelin-inducing activity of the cantharidinimides;
mutli-electrode arrays (MEAs) to quantify electrical activity of the living cells as a measure of the degree and quality of myelination induced by the cantharidinimides;
imaging cellular Ca2+ signals with organic fluorescent Ca2+ indicators for functional analysis of the neuronal circuits induced by the cantharidinimides;
western blot protein analyses of protein lysates of the cells using antibodies against MBP, MOG, PLP1, MAG and the “housekeeping gene” glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and quantification of protein bands by an Odyssey imaging system to collect data in digital form, wherein an increase in the amount of myelin-related proteins reflects myelin-inducing activity of the cantharidinimides; and quantitative polymerase chain reaction (qPCR) analysis of RNA extracted from the cells and using primers specific for MBP, MOG, PLP1, MAG and the “housekeeping genes” GAPDH, peptidylprolyl isomerase A (PPIA), tyrosine 3monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ), and eukaryotic translation initiation factor 4a2 (EIF4A2), wherein an increase in the amount of myelin-related RNAs reflects myelin-inducing activity of the cantharidinimides.
The results of the above tests show myelin-inducing activity of the cantharidinimides tested.
Claims (22)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NL2020792A NL2020792B1 (en) | 2018-04-19 | 2018-04-19 | Cantharidinimides for treating a demyelinating disease |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NL2020792A NL2020792B1 (en) | 2018-04-19 | 2018-04-19 | Cantharidinimides for treating a demyelinating disease |
Publications (1)
Publication Number | Publication Date |
---|---|
NL2020792B1 true NL2020792B1 (en) | 2019-10-28 |
Family
ID=62873531
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NL2020792A NL2020792B1 (en) | 2018-04-19 | 2018-04-19 | Cantharidinimides for treating a demyelinating disease |
Country Status (1)
Country | Link |
---|---|
NL (1) | NL2020792B1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017173052A1 (en) * | 2016-03-31 | 2017-10-05 | Merck Patent Gmbh | Compounds for the inhibition of cyclophilins and uses thereof |
-
2018
- 2018-04-19 NL NL2020792A patent/NL2020792B1/en active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017173052A1 (en) * | 2016-03-31 | 2017-10-05 | Merck Patent Gmbh | Compounds for the inhibition of cyclophilins and uses thereof |
Non-Patent Citations (7)
Title |
---|
DENG: "Exploiting protein phosphatase inhibitors based on cantharidin analogues for cancer drug discovery", MINI REV MED CHEM. 2013 JUN 1;13(8):1166-76., 1 January 2013 (2013-01-01), XP055520730, Retrieved from the Internet <URL:http://www.eurekaselect.com/109851/article> [retrieved on 20181102] * |
GIJBELS K ET AL: "REVERSAL OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS WITH A HYDROXAMATE INHIBITOR OF MATRIX METALLOPROTEASES", JOURNAL OF CLINICAL INVESTIGATION,, vol. 94, no. 6, 1 December 1994 (1994-12-01), pages 2177 - 2182, XP000574655, ISSN: 0021-9738, DOI: 10.1172/JCI117578 * |
ING-JY TSENG ET AL: "Characterization of Novel Aminobenzylcantharidinimides and Related Imides by Proton NMR Spectra and Their Effects on NO Induction", JOURNAL OF THE CHINESE CHEMICAL SOCIETY., vol. 62, no. 1, 9 October 2014 (2014-10-09), CHINA, pages 59 - 63, XP055519201, ISSN: 0009-4536, DOI: 10.1002/jccs.201400228 * |
LEE JI-YEON ET AL: "A novel cantharidin analogN-Benzylcantharidinamide reduces the expression of MMP-9 and invasive potentials of Hep3B via inhibiting cytosolic translocation of HuR", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 447, no. 2, 2014, pages 371 - 377, XP028659909, ISSN: 0006-291X, DOI: 10.1016/J.BBRC.2014.04.035 * |
LIN: "Effects of cantharidinimides on human carcinoma cells", CHEMICAL AND PHARMACEUTICAL BULLETIN 2004 VOL. 52 # 7 P. 855 - 857, 1 January 2004 (2004-01-01), pages 855 - 857, XP055519211, Retrieved from the Internet <URL:https://www.jstage.jst.go.jp/article/cpb/52/7/52_7_855/_pdf/-char/en> [retrieved on 20181025] * |
ONWUHA-EKPETE: "Selective inhibition of matrix metalloproteinase-9 in CD4+ T-cells reduces clinical severity in a murine model of Multiple Sclerosis. | The Journal of Immunology", J IMMUNOL MAY 1, 2017, 198 (1 SUPPLEMENT) 219.8;, 1 May 2017 (2017-05-01), XP055519282, Retrieved from the Internet <URL:http://www.jimmunol.org/content/198/1_Supplement/219.8> [retrieved on 20181026] * |
T BRENNER ET AL: "Inhibition of nitric oxide synthase for treatment of experimental autoimmune encephalomyelitis", THE JOURNAL OF IMMUNOLOGY, 15 March 1997 (1997-03-15), United States, pages 2940 - 2946, XP055519293, Retrieved from the Internet <URL:http://www.jimmunol.org/content/jimmunol/158/6/2940.full.pdf> * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Soria-Valles et al. | NF-κB activation impairs somatic cell reprogramming in ageing | |
Pentinmikko et al. | Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium | |
Lee et al. | Modeling treatment response for lamin A/C related dilated cardiomyopathy in human induced pluripotent stem cells | |
Egashira et al. | Disease characterization using LQTS-specific induced pluripotent stem cells | |
Sancho-Martinez et al. | Establishment of human iPSC-based models for the study and targeting of glioma initiating cells | |
Bach‐Ngohou et al. | Enteric glia modulate epithelial cell proliferation and differentiation through 15‐deoxy‐Δ12, 14‐prostaglandin J2 | |
Garcia et al. | A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Down's syndrome | |
Ballas et al. | Non–cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology | |
Dugas et al. | The T3-induced gene KLF9 regulates oligodendrocyte differentiation and myelin regeneration | |
Merson et al. | The transcriptional coactivator Querkopf controls adult neurogenesis | |
Sher et al. | Differentiation of neural stem cells into oligodendrocytes: involvement of the polycomb group protein Ezh2 | |
Bellucci et al. | Alpha‐synuclein aggregation and cell death triggered by energy deprivation and dopamine overload are counteracted by D2/D3 receptor activation | |
Kim et al. | Loss of ATM impairs proliferation of neural stem cells through oxidative stress-mediated p38 MAPK signaling | |
Loghman-Adham et al. | Immortalized epithelial cells from human autosomal dominant polycystic kidney cysts | |
Lombes et al. | Myoclonic epilepsy and ragged‐red fibers with cytochrome oxidase deficiency: neuropathology, biochemistry, and molecular genetics | |
Yadirgi et al. | Conditional activation of Bmi1 expression regulates self-renewal, apoptosis, and differentiation of neural stem/progenitor cells in vitro and in vivo | |
Gómez et al. | Breast cancer–associated macrophages promote tumorigenesis by suppressing succinate dehydrogenase in tumor cells | |
US9410945B2 (en) | Brown adipocyte progenitors in human skeletal muscle | |
Miao et al. | Peritoneal Milky Spots Serve as a Hypoxic Niche and Favor Gastric Cancer Stem/Progenitor Cell Peritoneal Dissemination Through Hypoxia-Inducible Factor 1 α | |
Gao et al. | Suppression of glioblastoma by a drug cocktail reprogramming tumor cells into neuronal like cells | |
Zhang et al. | Urine-derived induced pluripotent stem cells as a modeling tool for paroxysmal kinesigenic dyskinesia | |
Han et al. | The long noncoding RNA MALAT1 modulates adipose loss in cancer-associated cachexia by suppressing adipogenesis through PPAR-γ | |
Shah et al. | A chromatin modulator sustains self-renewal and enables differentiation of postnatal neural stem and progenitor cells | |
CA2325603A1 (en) | Pns cell lines and methods of use therefor | |
Okabe et al. | Neural development of methyl-CpG-binding protein 2 null embryonic stem cells: a system for studying Rett syndrome |