WO2007064091A1

WO2007064091A1 - Pharmaceutical composition comprising p38 map kinase inhibitor for treating diseases caused by npc1 gene deficiency or self-renewal disorder of stem cells

Info

Publication number: WO2007064091A1
Application number: PCT/KR2006/004650
Authority: WO
Inventors: Kyung Sun Kang; Jeong Chan Ra; Se Ran Yang
Original assignee: Seoul National University Industry Foundation
Priority date: 2005-12-02
Filing date: 2006-11-07
Publication date: 2007-06-07

Abstract

The present invention relates to a pharmaceutical composition for treating diseases caused by NPCl gene deficiency or self-renewal disorder of stem cells, which contains p38 MAP kinase inhibitor as an active ingredient, more specifically, a composition for treating diseases caused by NPCl gene deficiency or self-renewal disorder of stem cells, which contains any one or a mixture of two or more selected from the group consisting of SB202190, isothiocyanate, Phellinus Linteus extract, cruciferous vegetable extract, and sulporaphane extract as p38 MAP kinase inhibitor.

Description

Pharmaceutical Composition Comprising p38 MAP Kinase Inhibitor for Treating Diseases Caused by NPCl Gene Deficiency or Self-renewal

Disorder of Stem Cells

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition for treating diseases caused by NPCl gene deficiency or self-renewal disorder of stem cells, which contains p38 MAP kinase inhibitor as an active ingredient.

BACKGROUND ART

NPC (Niemann-Pick disease type C) is a recessive hereditary disease and caused by deficiency of NPCl gene located on the long arm (18qll-12) of chromosome 18 and involved in cholesterol metabolism (Carstea, E.D. et al., Science, 277:228, 1977). Furthermore, it is known that the disease occurs in perple of all races since ecologic background and genetical background are different. Especially, in the central nervous system and liver, and the like, a large amount of cholesterol is stored inside the cell to show hypocholesterolemia in patients. An estimated incidence of one out of 100,000 newborn babies was reported, but since its epidemicity shows very different aspects depending on geological circumstances, in the case of Nova Scotia state in Canada, 1% of newborn babies developed NPC and one out of 10 was a carrier. Niemann-Pick disease type C is caused by NPCl protein deficiency, which causes confusion over cholesterol transport among cells (Genomics, 65(2): 137, 2000).

Axon spheroids, which are characteristic in the NPC disease are concentrated in i regions containing long myelinated axons such as the brainstem and cerebellar white matter, but NFTs (neurofibrillary tangles) are localized to many of the same regions (hippocampus, entorhinal cortex, thalamus and basal ganglia) vulnerable to NFTs in Alzheimer's disease. In the NPC disease, the progressive neuronal loss (especially, purkinje cell), and infiltration of astrocytoma and macrophage foam cells is characteristically shown. Among these nerve cells, astrocytes help the function of neurons and maintain homeostasis of the central nerve system, but NPC development results in astrocytoma and pathological observation of fat globule accumulation.

In the case of the Niemann-Pick disease type B, it has been reported that bone- marrow transplantation was effective in several patients, and enzyme transplantation and gene therapy also has brought an excellent results through animal experiment. In the case of the Niemann-Pick disease type A, studies on methods for directly increasing ASM level in brain of paitents are being conducted, and in the case of the Niemann-Pick disease type C, a low cholesterol diet has been recommended. However, the treatment of Niemann-Pick disease is staying at the stage of in vivo using experimental animals, furthermore, an applifϊcation to human is still in the experimental stage. Especially, treatment of the type C depends merely on diet without special therapeutics, as a matter of fact, it is difficult to treat the disease by inducing a change of intercellular cholesterol metabolism or slowing down the progression of Niemann-Pick disease by low cholesterol diet and drugs lowering cholesterol level.

Degenerative nervous system diseases show a degenerative change in a nerve cells of the central nervous system, which causes several symptoms (Ross, CA. & Poirier, M. A., Nat. Med., Suppl: S 10-7, 2004; Lindvall, O. et al, Nat. Med., Suppl:S42-50, 2004). Symptoms are as follows: the diseases start slowly and continue for several years or several decades until death, once it is developed. The symptoms are bilateral and symmetrical, and have a tendency to be affected by family history. According to diseases, the nervous system which is anatomically or physiologically related to the diseases, is selectively invaded. Representative diseases thereof are Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Pick's disease or Niemann-Pick disease etc.

It is known that MAP kinase (mitogen-activated protine kinase) plays an important role in cell proliferation, differentiation and apoptosis, and also plays an essential role in embryogenesis, deformation and differentiation of the central nerve system. Such MAP kinase family comprises various proteins and has a similar pathway as that of tyrosine like a mechanism of growth factor such as EGF and FGF, of which p38 MAP kinase and ERKl/2 play a main role in the mechanism of MAP kinase. In the case of p38 MAP kinase, it is involved in stress pathway to respond to stress factor such as UV, but each one of them plays a different role depending on a specific cell and circumstances.

Expecially, in the case of p38 MAP kinase, kinases such as MKK3, MKK4, MKK6 etc. activated by various extracellular stimulus, for example, changes in osmotic pressure, inflammatory cytokine, ultraviolet, thermal stimulus, several growth fators, phosphorylates the p38 to activate. At a cell level, the activation of p38 controls transcription, protein synthesis, expression of cell membrane receptor, expression of cell cycle regulatory proteins and apoptosis. Moreover, since p38 MAP kinase is considered that it relates to various important diseases and plays an essential role in physiologic function such as cytokine signal transduction, inflammatory reaction, immune and expansion function, oxidative stress reaction or the reaction to infection, p38 MAP inhibitor has a possibility to be used for the treatment of diseases.

Stem cells are a general term for undifferentiated cells before differentiating into each cell constituting each tissue. A stem cell differentiates into a specialized cell by specific differentiation stimulus. Stem cells can self-renew and proliferate by cell division unlike differentiated cells in which cell division was stopped, and they differentiate into a specialized cell, but it can also differentiate into a different cell type by different circumstance or different differentiation stimulus to have a differential plasticity. Therefore, stem cells are classified based on embryogenesis process according to differential plasticity. An inner cell mass of blastocyte which is an early stage of embryogenesis, is a part which will later turn into an embryo, and an embryonic stem cell derived from the inner cell mass can be theoretically considered as stem cells having the potential capable of being differentiated into cells of all tissues consistuting the body. As embryogenesis proceeds to reach the stage of forming each organ of embryo, tissue specific stem cells exist in each organ to participate in differentiation process forming each organ. In this regard, generally, differential capability of the tissue specific stem cells is limited to the cells constituting a specific tissue (multipotent), and even after becoming an adult, the tissue specific stem cells remains in most organs to restore cell loss generated normally or pathologically.

About 10 years ago, stem cells are considered that they do not exist in the central nervous system unlike other most of tissues, which is a reason why damaged cerebral nervous tissue is not cured by itself. However, these days, it is proven that stem cells (cerebral nerve stem cells) exist in adult brain tissue, from which neuron is formed. The multipotent neural stem cell is differentiated into neuron, astrocyte and oligodendrocyte constituting a cerebral nervous tissue upon inducing differentiation.

Since neural stem cells exist in every site of each subtissue of embryonic central nervous system in the process of embryogenesis, it is possible that desired embryonic cerebral nervous tissue is taken to isolate and culture neural stem cells thereof. In the case of an adult, adult neural stem cells can be isolated from SVZ of lateral ventricle in the brain or hippocampus to culture, and such studies can provide a lot of information to understand the development process of the normal nervous system from neural stem cells.

Neural stem cells are capable of differentiating into neurons, oligodentrocytes and astrocytes, and has a self-renewal ability as well. These neural stem cells show characteristic property called neurosphere in vitro experiment, which is a lump of neural stem cells formed by reaction with nestin (a marker of neural stem cells) (Gage, F.H., Science, 287:1433, 2000).

Accordingly, the present inventors have paid attention to the fact that NPC disease develops at an early age including new born babies and made an extensive effort to identify the function of NPCl gene in neural stem cells in early embryogenesis and the influence of NPCl gene on intracellular signal transduction after differentiation of the neural stem cells, and as a result, found that the induction of abnormal differentiation of the neural stem cells is related to p38 MAP kinase activity and confrmed that p38 MAP kinase inhibitor is effective to treat diseases caused by NPCl gene deficiency, thereby completing the present invention.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a pharmaceutical composition for treating diseases caused by NPCl gene deficiency or self-renewal disorder of stem cells, which contaions p38 MAP kinase inhibitor as an active ingredient, wherein the p38 MAP kinase inhibitor inhibits p38 MAP kinase activity inducing abnormalities of differentiation such as diseases caused by NPCl gene deficiency or self-renewal disorder of stem cells when neural stem cells are differentiated.

To achieve the above objects, the present invention provides a pharmaceutical composition for treating diseases caused by NPC 1 gene deficiency or self-renewal disorder of stem cells, which contaions p38 MAP kinase inhibitor as an active ingredient.

In the present invention, the disease caused by NPCl gene deficiency is preferably a lipid metabolic disorder, and the lipid metabolic disease is preferably Niemann-Pick disease type C (NPC). Moreover, the disease caused by self-renewal disorder of stem cells is preferably degenerative disease of the nervous system or myocardial infarction, but it is not limited thereto. The degenerative diseases of the nervous system include Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Pick's disease or Niemann-Pick disease and the like.

In the present invention, the p38 MAP kinase inhibitor is preferably any one or a mixture of two or more selected from the group consisting of: SB202190, isothiocyanate, Phellinus Linteus extract, cruciferous vegetable extract, and sulporaphane (hereinafter called SFN) extract, but it is not limited thereto. Moreover, the cruciferous vegetable is preferably selected from the group consisting of: cabbage, Chinese cabbage, broccoli and kale.

Another features and embodiments of the present invention will be more clarified from the following "detailed description" and the appended "claims".

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an analysis of tail DNA genotyping of fetal mice transformed with NPCl gene using RT-PCR (+/+: wild type; -/-: homo type; +/- :hetero type).

FIG. 2 shows neurospheres formed in NPC1+/+, NPCl+/- and NPCl -/-, FIG. 3 and FIG. 4 shows the number and size of neurospheres, respectively. FIG. 5 is a comparison of expression levels of proteins involved in MAP kinase pathway in NPCl ^'+/+, NPCl+/- and NPCl-/- mice.

FIG. 6 shows the expression of GFAP/b-actin, and FIG 7 is a ratio of phosphorylated ERKl (pERKl) and phosphorylated ERK2(pERK2) to total ERKl and ERK2.

FIG. 8 shows neurospheres formed when NPCl-/- mouse derived neural stem cells were treated with PD98059 (PD₅ specific inhibitor of ERK1/2) and SB202190 (SB, specific inhibitor of p38).

FIG. 9 and FIG. 10 show the number and size of neurospheres, respectively.

FIG. 11 is a comparison of expressions between proteins involved in the mechanism of MAP kinase and GFAP(glial fibrillary acidic protein) of cells differentiated from neurospheres formed when NPCl ^'+/+, NPCl+/- and NPCl-/- mice were treated with PD and SB.

FIG. 12 is the ratio of p38 to phosphorylated ρ38 (ρp38).

FIG. 13 is a comparison of expression of GFAP (a maker for astrocytes) using an immunochemistry staining after forming neurospheres by isolating of stem cells from fetal cerebrums of NPCl +/+ and NPCl-/- mice to differentiate.

FIG. 14 is schematic figure on expression and function of NPCl gene mediated by MAP kinase in differentiation regulation and self-renewality of neural stem cells.

FIG. 15 shows phosphorylated p38 MAP kinase when H₂O₂ inducing phosphorylation of p38 MAP kinase was treated with PL. FIG. 16 shows phosphorylated p38 MAP kinase when H₂O₂ inducing phosphorylation of p38 MAP kinase was treated with SB202190.

FIG. 17 shows phosphorylated ERX 1/2 and p38 MAP kinase when H₂O₂ inducing phosphorylation of ERK1/2 and p38 MAP kinase was treated with DFl and DF2 .

FIG. 18 shows phosphorylated ERKl /2 and p38 MAP kinase when H₂O₂ inducing phosphorylation of ERK1/2 and p38 MAP kinase was treated with SFN.

DETAILED DESCRIPTION OF THE INVENTION, AND PPRFERRED EMBODIMENTS

In the present invention, the fuction of NPCl gene was identified using mouse models with NPCl gene deficiency. As a result, the number and size of nurospheres, a clump of neural stem cells, were significantly decreased in neural stem cells derived from NPC-defϊcient mice, from which it was confirmed that NPCl gene is involved in the self-renewal of neural stem cells. Furthermore, NPC r^/- mouse derived neural stem cells showed a completely defferent immunoactivity pattern from NPC1^+/+ mouse derived neural stem cells for GFAP (glial fibrillary acidic protein). That is, while astrocytes differentiated from NPCl^{+ +} mouse derived neural stem cells showed a typical polygonal morphology, astrocytes differentiated from NPCl^" mouse derived neural stem cells showed clumpy and rounded apperarance. Therefore, it was confirmed that NPC disease is caused by NPCl gene deficiency.

The NPClgene located on the a long arm (18qll-12) of human chromosome 18 is involved in cholesterol metabolism, and causes NPC disease (Carstea, E.D. et al., Science, 277:228, 1977; Loftus, S.K. et al., Science 277:232, 1997). In the present invention, the influence of MAP kinase on self- renewal capability of neural stem cell was examined by treating cells isolated from fetal NPCT mouse brain with MAP kinase inhibitor such as PD98059 and SB202190. As a result, it was confirmed that when the NPCl gene is expressed normally, the activity of MAP kinase family proteins is suppressed and when the NPCl gene is not expressed, the MAP kinase is activated to induce neural stem cells to differentiate into astrocytes upon the differentiation of a neural stem cell, but the differentiation results in abnomal phenotype thereof. It was also confirmed that, in the case when NPCl gene is deficient, the abnormal differentiation of neural stem cell is related to the activity of p38 MAP kinase. Consquently, when the p38 MAP kinase is inhibited, the self renewal ability of neural stem cells is improved, and it is possible to inhibit the induction of phathological morphology upon differentiation from neural stem cells into astrocytes. Therefore, p38 MAP kinase inhibitor is useful for treating diseases caused by NPCl gene deficiency or self renewal disorder of the stem cells.

In the present invention, the p38 MAP kinase belongs to a MAP kinase family, and proteins belonging to MAP kinase family include MKK1/2, ERK1/2, MKK3/6, MKK4, p38, Mkk4/7, JNK and the like. These proteins induce transcription factors such as ATF2, EIK-I, STAT1/3, ATF2, EIK-I, MEF-2C, c-Jun, respectively, and influence cell proliferation, differentitation, growth, inflammation, apoptosis, and stress reaction, etc (Ono, K.& Han, J., Cell Singalling, 12:1, 2000).

In the present invention, a matrial effectively inhibiting p38 MAP kinase was identified and the effect thereof was confirmed using an extract from cruciferous vegetables (broccoli, cabbage, Chinese cabbage, radish, etc); sulforaphane (SFN) existing in cruciferous vegetables as a major ingredient; and an extract from Phellinus Linteus.

Among yellow, green leafy vegetables belonging to the cruciferous family of vegetables, kale, broccoli, cabbage, and Chinese cabbage, and the like are known to have excellent anticancer effect, and sulforaphane (SFN), a phytochemical found in cruciferous vegetables, has the function of making carcinogenic materials to be decomposed and removed in liver by activating 'phase II enzymes' involved in removing carcinogen in liver. Additionally, like sulforaphane, other phytochemicals such as indol-3-carbinol, isothiocyanante, ellagic acid, etc found in cruciferous vegetables also have anticancer effect.

The Phellinus Linteus is also known as an effective food for treating cancer, and enhances immune function when it is used during chemotherapy after tumor excision of digestive system cancer such as stomach cancer, esophageal cancer, duodenal cancer, colon cancer, rectal cancer and the like, as well as liver cancer. Futhermore, the Phellinus Linteus is effective in controlling uterine bleeding, leucorrhea, irregular menstrual cycles, intestinal hemorrhage, and in improving the function of five viscera (heart, liver, spleen, lung, and kidney) and stomach, and also effective in detoxification.

p38 MAP kinase is considered to play an important role with respect to such anticancer effect and thus whether said matrials (cruciferous vegetable extracts, sulforaphane and Phellinus Linteus extract) act as p38 MAP kinase inhibitor was examined by an experiment to identify the functional mechanism thereof. The functional mechanism was identified through the fact that said matrials prevent the inhibition of GJIC and simultaneously inactivate MAP kinase family proteins activated by H₂O₂, which resulted from an experiment to prevent the GJIC (gap junctional intercellular communication) inhibition induced by hydrogen peroxide (H₂O₂) which is a tumor promoter.

Examples

Hereinafter, the present invention will be described in more detail by examples. However, it is obvious to a person skilled in the art that these examples are for illustrative purpose only and are not construed to limit the scope of the present invention.

Example 1: Neurosphere formation ability of NPCl-deficient mice

1-1. Preparation of NPCl-deficient mice and analysis of genotype thereof

After deleting NPCl genes on chromosomes 18 of Balb/c mice to form a heterozygous type, two mice were mated to obtaion NPCl knockout mice. Cerebrums obtained from fetal mice on the 14^th day, preferably 16^th day of pregnancy of NPCl transgenic mice, were collected and tail DNA was taken to anaylize genotype thereof with RT-PCR using the primary DNA kit (GOMA, Japan).

As a result, as shown in FIG. 1, wild geno type (+/+), hetero geno type (+/-) and homo geno type ( -/-) were confirmed.

1-2. self renewal ability of the neural stem cells derived from NPCl-deficient mice

Each fetal cerebrum, whose genotype had been analized, was collected and separated to move to a new dish. The separated cerebrum was treated with 0.5 % of trypsin and cells were cultured for 20 min, then DNase and culture broth were added and pipetted several times using a pipette. The obtained cells were seeded into a dish for cell culture to add a neural medium, DMEM/F12 containing 20 ng/ml of FGF (fibroblast groth factor), 10 ng/ml EGF (epidermal growth factor) and 2% of B27 supplement, and cultured in a 5% CO2 incubator at 37 ^°C for 7 days to observe formed neurospheres using optical microscope. Herein, neurospheres were subcultured two to three times.

As a result, as shown in FIG. 2 and 4, it was confirmed that the number and size of neurospheres formed in homo genotype mice were significantly decreased compared to those in wild-genotype mice. Since the homo genotype is derived from NPCl -deficient (NPCl-/-) mice, a decrease in the number and size of neurospheres, a clump of neural stem cells in homo genotype mice indicates that NPCl gene is involved in self renewal of neural stem cells.

Example 2: Relationshop between NPCl gene and MAP kinase

Since a number of different MAP kinase-dependent pathways mediate the cell differentiation of astroglia, the expression of proteins involved in MAP kinase pathways was examined in neural stem cells derived from NPCl ^'+/+, NPCl+/- and NPCl-/- mice.

The neural stem cells isolated from fetal cerebrums of 16-day-old, NPCl-/- mice were cultured according to the same method as the example 1-2 to observe neurospheres. The neurospheres were moved to 8 well chamber slide coated with poly-D-lysine using 1% FBS, and cultured for a week in an incubator to induce differentiation. Then, in order to carry out Western Blot analysis, treatment was performed in the presence of an appropriate test material depending on the group and treated with ultrasound for lOsec everytime, 6 times using 20% SDS containing ImM of PMSF (phenylmethylsulfonyl fluoride), lμM of leupetin, lμM of antipain, 0.1 μM of sodium orthovanadate, and 5mM of sodium fluoride. Proteins were quantitated using DC protein Kit (Bio-Rad). The proteins were separated on a 12.5% SDS polyacrylamide gel for 1 hr at 200V and transferd to PVDF membrane for 1 hr at 100V and 350mA. The membrane was washed with T-PBS containing 5% dried skim milk, and then primary antibody and secondary antibody were cultured for 1 hr, respectively, and washed with T-PBS four times at each step, followed by observing with ECL chemiluminiscenct detection reagent and the membrane was exposed to X-ray film for 15 sec ~ lmin depending on the condition thereof.

As a result, as shown in FIG. 5 to 7, levels of MKK3, ρ38, pMKK3/6, and ρρ38 in the neural stem cells derived from NPCl+/- and NPC-I- mice were significantly increased compared to those in the neural stem cells derived from NPCl ^'+/+ mice. Moreover, as shown in FIG. 7, levels of ERK1/2 and phosphorylated ERK1/2 were also increased in neural stem cells derived from NPCl-/- mice, compare to those in neural stem cells derived from NPCl +/+ mice. From these results, it was confirmed that the activity of the MAP kinase family proteins is inhibited when NPCl gene is expressed normally, and when NPCl gene is not expressed, MAP kinase is activated to induce neural stem cells to differentiate into astrocytes, but the differentiated astrocytes are abnomal phenotype.

Example 3: self renewal ability of neural stem cell by p38MAP kinase inhibitor in neurospheres derived from NPCl-/- mice

The following experiment was performed in order to examine the possibility of a corelationship between MAP kinase and NPCl gene involving in self renewal ability of neural stem cells. The neural stem cells isolated from cerebrums of NPCl-I- mice were treated with 2μM of PD98059 (MEK inhibitor) and 2μM of SB202190 (p38 MAP kinase inhibitor), respectively.

As a result, as shown in FIG. 8 to 10, the number and size of neurospheres in the group treated with SB202190 were increased, and the number of neurospheres in the group treated with PD98059 was slightly larger than that of the non-treated group, but the size thereof was significantly increased. From these results, it was confirmed that p38 MAP kinase inhibits the self- renewal of neural stem cells.

Moreover, in order to examine whether an increase in proteins associated with MAP kinase is specific expression, preotein expression in cells induced from neurospheres, which is obtained by culturing after NPCl homo genotype (-/-) neurospheres were treated with 2μM PD98059 and 2μM SB202190, was analized by performing the same method as the above western blot. As a result, as shown in FIG. 11, the expressions of GFAP, ERK1/2 and pERKl/2 in groups threated with SB202190 and PD98059 was similar to the control group (non-treated with kinase inhibitor). However, the expression of proteins associated with phophorylated MAP kinase (pMKK3/6, pp38) was prominently decreased in the group treated with SB202190. Although the expression of pERKl/2 was increased in the neural stem cells from NPCl+/- and NPCl-/-, compare to that in the neural stem cells from NPC1+/+ (Fig. 5), the pERKl/2 expression in the group treated the neural stem cells from NPCl-/- with PD98059 was not increased compare to the control. Futhermore, as shown in FIG. 12, the expression of pp38 was also significantly decreased compared with that of p38.

From these results, it was confirmed that abnormal differentiation of neural stem cells in the case of NPCl gene deficiency was related to p38MAP kinase activity.

Example 4: Differentiation of NPCl-/- mouse derived neural stem cells

The neural stem cells isolated from fetal cerebrums of 16-day old NPCl-/- mice were cultured according to the same method as the example 1-2 to observe neurospheres. The neurospheres were moved to 8 well chamber slide coated with poly-D-lysine using 1% FBS, and cultured for a week in an incubator to induce differentiation.

4% paraformaldehyde was added to each chamber and fixed neurospheres for 10 min, followed by washing with PBS three times and permiabilizing with 0.3% of triton X-100 for 5 min. And then, the neurospheres were precultured for lhr with 10% if NGS (normal goat serum), allowed to react with primary antibody (anti- GFAP, 1:200, Chemicon, Temecula, CA, USA) for 2 hrs, and washed with PBS three times, followed by allowing it to react with secondary antibody (anti-mouse TRITC, 1:200, Zymed Lab. Inc., CA, USA) for 1 hr to wash three times with PBS. Next, tissue tek was removed and dried completely, followed by covering with mountant to photograph with WG and WB filters. As a control, the neural stem cells isolated from fetal cerebrums of NPCl wild-genotype mice were subjected to the same method as described above to perform immunochemistry staining.

As a result, as shown in FIG. 13, the neural stem cells from NPC1+/+ and NPCl-/- showed completely different immunoactivity patterns for GFAP(glial fibrillary acidic protein). Astrocytes differentiated from NP Cl+/+ mouse derived neural stem cells showed a typical polygonal morphology, whereas astrocytes differentiated from NPCl-/- mouse derived neural stem cells showed a clumpy and rounded apperarance, which is consistent with the prior document describing abnormalities of differentiation (Griffin, L.F. et al, Nat. Med., 10:704, 2004; Burns, M. et al, Neurorx, 1 :394, 2004).

Example 5: p38 MAP kinase inhibitor

1-1. p38 MAP kinase inhibitory action of Phellinus Linteus

Phellinus Linteus was extracted using 30% ethanol for 4 hrs at 90 ^"C , to filter with 270 mesh filter. The filtered extract was extracted once more using the same method as descrived above, then concentrated using Eyela rotating vacuum evaporator at

60 °C , and 70cmHg. Liver epithelial cells of WB RAT provided by Dr. J.E.Trosko in

Michigan state university (USA) were cultured in D-media (Formula No. 78-

5470EF, Gibco BRL, Grand Island, NY) containing 50μgM of gentamicin and 5% of FBS(fetal bovine serum). The epithelial cells were cultured in 5% CO₂ incubator at 37°C and each medium was replaced with a new one every second day.

The epithelial cells were pretreated with PL for 24 hrs and treated with H₂O₂ for 1 hr to activate p38 MAP kinase. The cells were treated according to the same method as SL/DT(scrape loading/dye transfer) assay in order to measure the ability of cells performing GJIC (gap junction intercellular communication), and Cx43 (GJIC protein) was analyized by Wstern blot. The protein was quantified with DC protein Kit (Bio-Rad) using 20% SDS containing ImM of PMSF (phenylmethylsulfonyl fluoride), 1OmL of iodoacetoamide, lμM of leupetin, lμM of antipain, 0.1 μM of sodium orthovanadate, and 5mM of sodium fluoride. The protein was separated by Laemmli method on 12.5% SDS polyacrylamide gel, and transferd to nitrocellulose membrane for 1 hr at 100V and 350mA.

As a result, as shown in FIG. 15, when H₂O₂ inducing p38 MAP kinase phospholylation was treated with 25//g/m£ of PL, phosphorylated p38 MAP kinase are significantly decreased.

In order to examine whether PL is specific to the inhibition of p38 MAP kinase activity or not, H₂O₂ was treated with SB202190 which is known in the art as p38 MAP kinase inhibitor according to the same method as described above to perform

Western Blot. As shown in FIG. 16, when H₂O₂ was treated with 4μM of

SB202190, phosphorylated p38 MAP kinases are significantly decreased.

Consequently, it was confirmed that PL has an effect to inhibit p38 MAP kinase activity like SB202190 (p38 MAP kinase inhibitor).

1-2. p38 MAP kinase inhibitory action of extract from Chinese cabbage and SFN

Chinese cabbage (Brassica pekinensis) used in the experiment was provided by the East HanNong Corporation (Ansung, Korea). DFl was extracted from B. pekinensis and DF2 was extracted from Agarobacterium B. pekinensis. 3g of Chinese cabbage leaves were extracted using methanol for 3 days at room temperature to filter and the fϊlterate was concentrated using a rotary evaporator (Eyela, Tokyo, Japan). The concentrated extract of each Chinese cabbage was diluted with 10m# of dimethylsulfoxide (DMSO) to use. Sulforaphane (SFN) was provided by Alexis Biochemicals. Liver epithelial cells of WB RAT provided by Dr. J.E.Trosko at Michigan state university (USA) was cultured in D-media (Formula No. 78-5470EF, Gibco BRL, Grand Island, NY) containing a mixture of penicillin- streptomycin- neomycin and and 5% of FBS(fetal bovine serum). The epithelial cells were cultured in 5% CO₂ incubator at 37 ^°C and each medium was replaced with a new one every second day.

The epithelial cells were pretreated with extract of Chinese cabbage (DFl and DF2) or SFN for 24 hrs, and treated with H₂O₂ for 1 hr to activate p38 MAP kinase. The cells were treated according to the same method as SL/DT(scrape loading/dye transfer) assay in order to measure the ability of cells performing GJIC (gap junction intercellular communication), and Cx43 (GJIC protein) was analyized by Wstern blot. The protein was quantified with DC protein Kit (Bio-Rad) using 20% SDS containing ImM of PMSF (phenylmethylsulfonyl fluoride), 1OmM of iodoacetoamide, lμM of leupetin, lμM of antipain, 0.1 μM of sodium orthovanadate, and 5mM of sodium fluoride. The protein was separated by Laemmli method on 12.5% SDS polyacrylamide gel, and transferd to nitrocellulose membrane for 1 hr at 100V and 350mA.

As a result, it was found that when DFl and DF2 were 500μg/mt, phospholation of Cx43(GJIC protein) was significantly decreased, thus assuming that when MAP kinases playing an important role in GJIC are activated, they might have an influence on GJIC to perform Western blot using the same method as described above. As shown in FIG. 17, when H₂O₂ inducing phosphorylation of ERK1/2 and p38 MAP kinase was treated with 500μgM of DFl and DF2, phosphorylated ERK1/2 was significantly decreased. Moreover, while phosphorylated p38 MAP kinases in the sample group treated with 250/^g/m£ of DFl and DF2 did not show any change, phosphorylated p38 MAP kinases in the sample group treated with 500μg/m£ of DFl and DF2 showed a significant decrease. Furthermore, as shown in FIG. 18, when pretreated with SFN, both of phosphorylated ERKl/2 and p38 MAP kinases were significantly decreased. Accordingly, it was conformed that all of SFN, DFl and DF2 have an effect to inhibit p38 MAP kinase activity, especially, SFN was more efficient to inhibit p38 MAP kinase activity than DFl and DF2.

As previously stated in detail, differentiation and self renewal ability of neural stem cells depend on the existence of NPCl gene. When NPCl gene exists, the gene inhibits the expression of p38 MAP kinases to have excellent self renewal ability, but when the NPCl gene is deficient, p38 MAP kinases are activated to cause a decrease in self renewal ability and induce morphological change of astrocytes (Fig 14). Therefore, materials which inhibit p38 MAP kinases can be extracted from natural foods (Phellinus Linteus, Chinese cabbage and broccoli etc.) to use as an inhibitor. The inhibitor will be effective to increase self renewal ability of neural stem cells when it is clinically applied to diseases caused by NPCl gene deficiency.

INDUSTRIAL APPLICABILITY

As previously described in detail, the present invention has an effect to provide a pharmaceutical composition for treating diseases caused by NPCl gene deficiency or self-renewal disorder of stem cells, which contains p38 MAP kinase inhibitor as an active ingredient. The composition according to the present invention is useful for treating diseases caused by NPCl gene deficiency such as lipid metabolic disease, as well as diseases caused by self-renewal disorder of stem cells such as degenerative diseases of the nervous system, myocardial infarction and the like. Furthermore, since the p38 MAP kinase inhibitor is obtained from natural foods, it is possible to mass-produce p38 MAP kinase inhibitor efficiently.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMSWhat is Claimed is:

1. A pharmaceutical composition for treating diseases caused by NPCl gene deficiency or self-renewal disorder of stem cells, which contains p38 MAP kinase inhibitor as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the disease caused by NPCl gene deficiency is a lipid metabolic disorder.

3. The pharmaceutical composition according to claim 2, wherein the lipid metabolic disorder is Niemann-Pick disease type C (NPC).

4. The pharmaceutical composition according to claim 1, wherein the disease caused by self-renewal disorder of stem cells is degenerative disease of the nervous system or myocardial infarction.

5. The pharmaceutical composition according to claim 4, wherein the degenerative diseases of the nervous system is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALS), Pick's disease and Niemann-Pick disease.

6. The pharmaceutical composition according to claim 1, wherein the p38 MAP kinase inhibitor is any one or a mixture of two or more selected from the group consisting of: SB202190, isothiocyanate, Phellinus Linteus extract, cruciferous vegetable extract, and sulporaphane extract.

7. The pharmaceutical composition according to claim 6, wherein the cruciferous vegetable is any one selected from the group consisting of: cabbage, Chinese cabbage, broccoli and kale.