Patent Grant
9358239
This application claims the benefits of U.S. Provisional Application No. 61/931,789 filed on Jan. 27, 2014 and claims priority to U.S. Pat. No. 8,642,763 published on Feb. 4, 2014, all of which are incorporated herein by reference.
1. Field of the Invention
The present invention relates to utilize NiSOD-like compound and derivatives thereof as a pharmaceutical composition. More specifically, NiSOD-like compound and derivatives thereof are used for suppressing abnormal protein aggregation, recovering cell viability, increasing mature neuron number and neurite outgrowth length and protecting dopaminergic cells by reducing oxidative stress or reactive oxygen species in brain tissues.
2. Description of the Prior Art
As the elderly population of the world continues to increase, the prevalence of neurodegenerative disorders, such as spinocerebellar ataxia (SCA), Alzheimer's disease (AD) and Parkinson's disease (PD), has been increasing at a disconcerting rate. Despite tremendous progress that has made in neurodegenerative disorders research in the past few decades, there is still no effective treatment for such diseases.
Neurodegeneration is an umbrella term for the progressive loss of structure or function of neurons, including death of neurons. As research progresses, many similarities appear that are related these diseases to one another on a cellular level. Discovering these similarities offers hope for therapeutic advances that could ameliorate many diseases simultaneously. There are many parallels between different neurodegenerative disorders including atypical protein assemblies as well as induced cell death; moreover, with age, the risk of DNA mutation increases, as well as the risk of cell damage induced by oxidative stress.
Although SCA, AD and PD manifest with different clinical features, the disease processes at the cellular level appear to be similar.
In SCA disease, the expansions of CAG repeats in the disease genes result in long polyglutamine (polyQ) tracts in the respective proteins. The accumulation of intranuclear and cytoplasmic misfolded polyQ proteins is thought to induce oxidative stress and lead to cell death. Patients with SCA find that their ability to use the affected parts of the body becomes progressively more difficult and less exact.
AD has been categorized to be a protein misfolding disease, caused by accumulation of abnormally folded A-beta and tau proteins in the brain, which leads to progressive loss of memory. A-beta protein is made up of small peptides, 39-43 amino acids in length, called beta-amyloid (also written as Aβ oligomer). Aβ oligomer is a fragment from a larger protein called amyloid precursor protein (APP), a transmembrane protein that penetrates through the neuron's membrane. APP is critical to neuron growth, survival and post-injury repair.
PD is a neurodegenerative movement disorder. In most cases it occurs as a sporadic type of disease, but there are also rare familial forms. Pathologically Parkinson's disease is characterized by loss of dopaminergic neurons in the compact part of substantia nigra. As a part of the neurodegenerative process protein aggregates will accumulate as Lewy bodies in dopaminergic neurons. In PD, research on protein misfolding and aggregation has taken center stage following the association of alpha-synuclein gene mutations with familial forms of the disease.
The above protein aggregation leads to the increase of oxidative stress which has been implicated as a factor for the initiation and progression of neurodegenerative disorders. Thus, suppression of the protein aggregation and reduction of oxidative stress in the neurodegenerative disorders are expected to inhibit a wide range of harmful downstream events and further provide an observation for identifying the potential prevention and treatment of neurodegenerative disorders.
To achieve suppression of the protein aggregation and by reduction of oxidative stress or reactive oxygen species in brain tissues in accordance with the purpose of the invention as embodied and broadly described herein, the present invention provides NiSOD-like compound and derivatives thereof for suppressing abnormal protein aggregation, recovering cell viability, increasing mature neuron number and neurite outgrowth length and protecting dopaminergic cells by reducing oxidative stress or reactive oxygen species in brain tissues in SCA, AD or PD.
The NiSOD-like compound or its derivatives implemented in the present invention has a five-coordinate structural formula (I):
wherein R1 denotes H or -A-R′; wherein A denotes O or N; wherein R′ is H, an alkoxy group, an amino acid group, or a polymeric group; wherein the polymeric group is a polyethyleneoxy group, a polydimethylsiloxane group, or polyurethane; wherein R2 is H or a para-substituent of a phenyl ring, the para-substituent of the phenyl ring is selected from a group consisting of alkyl groups, alkoxy groups, silane groups, amino groups, alkyl amino groups, and a hydroxyl group; wherein R3 is H or a para-substituent of a pyridine ring, the para-substituent of the pyridine ring is selected from a group consisting of amino groups, alkyl amino groups, siloxane amino groups, and siloxane amino groups which attach to a Fe3O4/SiO2 magnetic nanoparticle; and wherein Ni is a nickel(II) or nickel(III) ion.
Besides, the NiSOD-like compound or its derivatives can be a six-coordinate derivative having a structural formula (II):
wherein R1, R2 and R3 are defined in the above paragraph, and L is acetonitrile, water or tert-butyl isocyanate. The six-coordinate derivative comprises WCt003, WCt006 or WCt021.
The WCt003 has the following structural formula:
The WCt006 has the following structural formula:
The WCt021 has the following structural formula:
The NiSOD-like compound or its derivatives can suppress abnormal protein aggregation, recovering cell viability, increasing mature neuron number and neurite outgrowth length and protecting dopaminergic cells by reducing oxidative stress or reactive oxygen species (ROS).
A detailed description of further features in the present invention is given below so that a person skilled in the art is allowed to understand and carry out the technical contents of the present invention, and can readily comprehend the objectives and advantages of the present invention after reviewing the contents disclosed herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the invention and, together with the description, serve to explain the features and advantages of the invention. In the drawings:
The present invention provides a pharmaceutical composition which comprises a NiSOD-like compound or derivatives thereof with an effective dose for suppressing abnormal protein aggregation, recovering cell viability, increasing mature neuron number and neurite outgrowth length and protecting dopaminergic cells by reducing oxidative stress or reactive oxygen species in brain tissues. The effective dose may be a prophylactically effective amount as prophylaxis.
There are several cell and animal experiment models provided below for demonstrating the capabilities of suppressing abnormal protein aggregation, recovering cell viability, increasing mature neuron number and neurite outgrowth length and protecting dopaminergic cells by reducing oxidative stress or reactive oxygen species of the NiSOD-like compound or derivatives thereof. Furthermore, the results were confirmed with brain slice culture of spinocerebellar ataxia 17 (SCA17) transgenic mice and Alzheimer's disease (AD) mice, brain tissues immunostaining of Parkinson's disease (PD) mice, and SCA17 transgenic, AD and PD mice.
I. Preparation of Nisod-Like Compound and Derivatives Thereof
The NiSOD-like compound or derivatives thereof mentioned and the producing method thereof are disclosed in U.S. Pat. No. 8,642,763, which is incorporated herein by reference. In U.S. Pat. No. 8,642,763, a nickel complex or derivatives thereof is used for mimicking the active site of the nickel-containing superoxide dismutase (NiSOD), and is considered as a NiSOD-like compound. The present invention provides a novel use of the NiSOD-like compound for suppressing abnormal protein aggregation, recovering cell viability, increasing mature neuron number and neurite outgrowth length and protecting dopaminergic cells by reducing oxidative stress or reactive oxygen species in brain tissues in neurodegenerative disorders comprising spinocerebellar ataxia, Alzheimer's disease or Parkinson's disease.
The preparation method of the NiSOD-like compound is to sequentially react [2,6-bis(((S)-2-(diphenylhydroxymethyl)-1-pyrrolidinyl)methyl)pyri-dine] (H2BDPP) or the derivative of H2BDPP with NaH and [Ni(CH3CN)6](ClO4)2 or only with [Ni(CH3CN)6](ClO4)2. For example,
This example adopted a reaction precursor OH-BDPP wherein the pyrrolidinyl group was attached with a hydroxyl group. According to the preparation method, let 0.128 g (0.2 mmol) OH-BDPP sequentially react with 0.012 g (0.5 mmol) NaH and 0.101 g (0.2 mmol) [Ni(CH3CN)6](ClO4)2 at ambient temperature for 2 hours to obtain a five-coordinate nickel(II) complex, Ni—OH-BDPP. Two derivatives, a five-coordinate nickel(III) complex, [Ni—OH-BDPP]PF6, and a six-coordinate nickel(II) complex, Ni—OH—H2BDPP (WCt006), were obtained from further fabrication of the abovementioned five-coordinate nickel(II) complex, Ni—OH-BDPP. With the proposed preparation method, WCt003 and WCt021 have been also prepared for the following experiments.
In the present invention, Student's t-test is used, and all P values are two-tailed, with vales of P<0.05 considered significant. For each set of values, data are expressed as the means±standard deviation (SD), and significance is expressed as * or #.
II. Experiments of Spinocerebellar Ataxia (SCA)
In SCA, the expansions of CAG repeats in the disease genes result in long polyglutamine (polyQ) tracts in the respective proteins. The accumulation of intranuclear and cytoplasmic misfolded polyQ proteins is thought to induce oxidative stress and lead to cell death. In the cell and animal experiment models, synthetic compounds WCt003, WCt006 and WCt021 were applied to demonstrate how WCt003, WCt006 and WCt021 are likely to work in protein aggregation suppression and oxidative stress reduction.
A. Cell Culture and Cytotoxicity of NiSOD-Like Compounds Assay
Human embryonic kidney HEK-293 cells (ATCC No. CRL-1573) were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). HEK-293 cells were cultivated at 37° C. incubator containing 5% CO2 and cell proliferation was measured based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2-yL]-2,5-diphenyl-tetrazolium bromide (MTT). MTT viability assay was used for accessing the cytotoxicity of WCt003, WCt006 and WCt021 and comparing the cytotoxicity of WCt003, WCt006 and WCt021 with that of SAHA (suberoylanilide hydroxamic acid, Cayman Chemical) which is a well-known HDAC (Histone Deacetylase) inhibitor for reducing SDS (Sodium dodecyl sulfate)-insoluble polyQ aggregate.
HEK-293 cells were plated into 48-well (5×104/well) dishes, grown for 20 hours and treated with different concentrations of SAHA, WCt003, WCt006 and WCt021 (0 nM, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM) respectively. After one day, 20 μL MTT (5 mg/ml in PBS, Sigma) was added to cells and incubated for 2 hours. The absorbance of the purple formazan dye was measured at 570 nm by a Bio-Tek μQuant Universal Microplate Spectrophotometer. For each set of values, data are expressed as the means±standard deviation (SD).
As MTT viability assays were performed with human embryonic kidney 293 cells after treatment with the tested NiSOD-like compounds for one day, the IC50 was calculated using the interpolation method. Refer to
B. Construction of 293 Cells Expressing ATXN3/Q75 Aggregates
For therapies toward the polyQ diseases, we aimed to screen compounds potentially inhibiting polyQ aggregation. As removal of the N-terminal ATXN3 was required for ATXN3 aggregation, GFP-tagged ATXN3/Q14-75 C-terminal fragment was cloned to establish Flp-In 293 cells with inducible ATXN3/Q14-75-GFP expression. Refer to
C. NiSOD-Like Compounds Treatment Reduces ATXN3/Q75 Aggregation on 293 Cell Model
Refer to
D. NiSOD-Like Compounds Treatment Reduces ROS Production on 293 Cell Model
Production of reactive oxygen species (ROS) is a particularly destructive aspect of oxidative stress; such species include free radicals, peroxides and superoxide. To evaluate whether NiSOD-like compounds reduced ROS formation in 293 ATXN3/Q75 cells, the cellular production of ROS was measured by using a red fluorescent probe from Molecular Probes. After treating with WCt003, WCt006 and WCt021 in concentration of 10 nM to 100 nM for 8 hours and inducing ATXN3/Q75-GFP expression for 6 days, the red fluorescent probe exhibited bright fluorescence upon oxidation by ROS. With absorption/emission maxima at 644/665 nm, the fluorescence of the red fluorescent probe can be detected using fluorescence microscopy and analyzed by HCA system. Refer to
E. Superoxide Dismutase (SOD)-Like and DPPH Free Radical Scavenging Activities of NiSOD-Like Compounds
For SOD activity, SOD enzyme from human erythrocytes (Sigma, catalog number 59636) and HEK-293 cell lysate (5×106/ml) were used as comparison. The SOD Assay Kit-WST from Dojindo was used to determine SOD activity. The assay utilizes WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4)-disulfophenyl-2H-tetrazolium, monosodium salt] that produces a water-soluble formazan dye upon reduction with the superoxide anion. The rate of the reduction of WST-1 with O2− was linearly related to the xanthine oxidase (XO) activity, and this reduction was inhibited by SOD. Therefore, the IC50 (50% inhibition activity of SOD or SOD-like materials) can be determined by the colorimetric method. Refer to
For DPPH (1,1-diphenyl-2-picryl hydrazyl) scavenging activity, the odd electron in the stable DPPH free radical gives a strong absorption maximum at 517 nm and is purple in color. The DPPH assay measured scavenging ability of the antioxidants towards the stable radical. The free radical DPPH was reduced when it reacted with antioxidants and results in decrease in absorbance at 517 nm. The degree of discoloration indicated the scavenging potential of the antioxidant compounds. Refer to
F. Organotypic Cerebellar Slice Culture and Immunofluorescent Staining
The cerebellar slices were prepared from postnatal day 7 SCA17 transgenic (TG) mice and cultured on 0.4 μm pore size culture plate inserts in six-well plates (Millipore). NiSOD-like compounds, WCt003, WCt006 and WCt021, were applied in concentration of 10 nM to 20 nM to the slices at day 2. After culture for 7 days, slices were immunostained with primary antibodies IP3R-1 for Purkinje cells and 1TBP18 for TBP aggregation, fluorescence-conjugated secondary antibodies and DAPI for nuclei. The staining results were observed by confocal microscope. Three independent experiments were performed and differences between groups were evaluated by Student's t-test. Refer to
G. Mouse Rotarod Performance During NiSOD-Like Compounds Treatment
Both SCA17 transgenic (TG) mice (N=60) and their wild-type (WT) littermates (N=60) were divided into 4 groups (N=15/per group). Mouse rotarod performance was evaluated at 4 weeks old prior to NiSOD-like compounds treatment. NiSOD-like compounds, WCt003, WCt006 and WCt021 (2 mg/kg, respectively), or vehicle (DMSO) were intraperitoneal injected into the mice every day for 10 weeks. Rotarod performance was conducted every month. Refer to
H. NiSOD-Like Compounds Treatment Reduces Oxidative Response of SCA17 Mice
Glutathione (GSH) plays a critical role in the cellular defense against oxidative stress in mammalian cells. Plasma was collected from the peripheral blood of 14-week old mice and the GSH level was measured by GSH assay kit (Cayman Chemical, Ann Arbor, Mich., USA). Refer to
Refer to
Refer to
I. NiSOD-Like Compounds Treatment Reduces Mutant TBP Protein Aggregation
Refer to
J. NiSOD-like Compounds Treatment Increases Chaperone Expression in SCA17 Mouse Cerebellum
Refer to
III. Experiments of Alzheimer's Disease (AD)
In the cell experiment models of AD, synthetic compounds WCt003, WCt006 and WCt021 were applied to demonstrate how WCt003, WCt006 and WCt021 are likely to work in suppressing the accumulation of abnormally folded Aβ oligomer and tau proteins and further increase the cell survival.
A. Cell Viability Assay and NiSOD-Like Compounds Treatment Increases the Cell Survival, Mature Neuron Number and Neurite Outgrowth Length of AD Hippocampal Primary Culture Added with Aβ Oligomer
For the Aβ oligomer experiment models, primary neurons were isolated from mouse hippocampus at embryonic day 16 to 18, and cultured on 48-well culture dishes. Thirty minutes before Aβ oligomer addition, NiSOD-like compounds were applied to the primary culture with the concentration of 0.25 μM, 0.5 μM and 1 μM. Primary cells were harvested for MTT cell viability assay and immunofluorescent staining after Aβ oligomer addition with concentration of 1 μM for 3 hours. Refer to
Refer to
B. Cell Viability Assay and NiSOD-Like Compounds Treatment Increases the Cell Survival, Mature Neuron Number and Neurite Outgrowth Length of AD Hippocampal Primary Culture Added with WT and GFX
For tau proteins experiment models, primary neurons were isolated from mouse hippocampus at embryonic day 16 to 18, and cultured on 48-well culture dishes. Thirty minutes before WT (wortmannin, 10 μM) and GFX (GF-109203X, 10 μM) addition, NiSOD-like compounds were applied to the primary culture with the concentration of 0.25 μM, 0.5 μM and 1 μM. Primary cells were harvested for MTT cell viability assay and immunofluorescent staining after WT and GFX addition with concentration of 10 μM for 3 hours. Refer to
Refer to
IV. Experiments of Parkinson's Disease (PD)
In the cell and animal experiment models of PD, synthetic compounds WCt003, WCt006 and WCt021 were applied to demonstrate how WCt003, WCt006 and WCt021 are likely to work in protecting dopaminergic cells and reducing ROS production.
A. NiSOD-like Compound Reduces the 6-OHDA-Induced Apoptosis of PC12 Cells
PC 12 cells were treated with NiSOD-like compound, WCt006, with concentration of 1 μM, 10 μM and 20 μM for 5 minutes and then co-incubated with 50 μM of 6-OHDA (6-hydroxydopamine) for 24 hours. The 6-OHDA is not an ROS, but it can induce ROS causing damage to PC12 cells. Refer to
Refer to
B. NiSOD-Like Compound Protects Dopaminergic Cells from 6-OHDA-Induced Injury
10-week-old rats were pre-injected with 10 μg/kg and 50 μg/kg of NiSOD-like compound, WCt006, for 2 weeks and then treated with 6-OHDA (2 mg/ml) in the left brain. Refer to
Refer to 23B. The data of left and right brain were shown as means±S.E. Statistical significance between left and right brain was analyzed by using Fisher's LSD. In
C. NiSOD-Like Compound Attenuates the Production of Nitric Oxide Under the Stress of 6-OHDA
10-week-old rats were pre-injected with 50 μg/kg of NiSOD-like compound, WCt006, for 2 weeks and then treated with 6-OHDA (2 mg/ml) in the left brain. Refer to
Refer to
D. NiSOD-Like Compound Reduces Ipsiversive Rotation Caused by 6-OHDA
After damaged by 6-OHDA, the apomophine (0.75 mg apomophine and 0.2% Vitamine C dissolved in 1 ml saline) which stimulates ipsiversive rotation was intraperitoneal injected to the rats either with or without the treatment of WCt006. Refer to
The experiments and results presented above in the present invention demonstrate that the NiSOD-like compounds suppress abnormal protein aggregation, recover cell viability, increase mature neuron number and neurite outgrowth length and protect dopaminergic cells by reducing oxidative stress or reactive oxygen species in brain tissues. The present invention may be applied to other neurodegenerative disorders with similar pathogenic mechanism as well. The present invention utilizes the NiSOD-like compound or its derivatives suppressing abnormal protein aggregation, recovering cell viability, increasing mature neuron number and neurite outgrowth length and protecting dopaminergic cells by reducing oxidative stress or reactive oxygen species in brain tissues in neurodegenerative disorders including spinocerebellar ataxia, Alzheimer's disease or Parkinson's disease, and provides the effective dose according to the experiments and results presented above in a concentration between 1 nM to 4 μM (0.5 μg/kg to 2 mg/kg).
The foregoing embodiments are illustrative of the characteristics of the present invention so as to enable a person skilled in the art to understand the disclosed subject matter and implement the present invention accordingly. The embodiments, however, are not intended to restrict the scope of the present invention. Hence, all equivalent modifications and variations made in the foregoing embodiments without departing from the spirit and principle of the present invention should fall within the scope of the appended claims.
| Number | Name | Date | Kind |
|---|---|---|---|
| 8642763 | Lee | Feb 2014 | B2 |
| Number | Date | Country | |
|---|---|---|---|
| 20150209370 A1 | Jul 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61931789 | Jan 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13492657 | Jun 2012 | US |
| Child | 14605968 | US |
