KR101658357B1 - Pharmaceutical composition containing resveratrol for treating type Gaucher Diseases - Google Patents

Abstract

Currently, an enzyme treatment widely used as a treatment for Gaucher disease is not suitable for the treatment of type III Gaucher disease, which causes neurological symptoms such as epilepsy because it can not pass through the blood-brain barrier and move into the brain tissue. Resveratrol is a natural polyphenol component with various properties such as anti-inflammatory, antioxidant, and neuroprotective effects that pass through the blood-brain barrier. The present inventors evaluated the therapeutic effect of resveratrol on fibroblasts derived from type III Gaucher disease patients. The effects of AIF, Bax, truncated caspase-3, ACAT1, E3BP and CS were evaluated by western immunoblot to evaluate the effect on cell survival rate by MTT assay. In addition, intracellular glucosylceramide (glucocerebroside) concentration of resveratrol-treated cells was confirmed by thin film chromatography. As a result, survival rate of resveratrol-treated cells was significantly increased as compared with control cells. The levels of the apoptotic factors AIF, Bax and truncated caspase-3 after resveratrol treatment decreased in a dose-dependent manner, and the levels of ACAT1, E3BP and CS expression increased in a dose-dependent manner. TLC results showed that glucosyl ceramide, a causative agent of Gaucher disease, was significantly reduced in cells of resveratrol treated type III Gaucher disease patients. These results suggest that resveratrol can reduce cell death and reduce glucosyl ceramide levels and may be used as a treatment for type III Gaucher disease with neurological symptoms.

Description

[0001] The present invention relates to a pharmaceutical composition for treating type III Gaucher Disease containing resveratrol,

The present invention relates to a pharmaceutical composition for treating type III Gaucher disease containing resveratrol, wherein the treatment with resveratrol significantly reduces glucocerebroside in the cells of the type III Gaucher disease patient and significantly reduces the expression of the apoptotic factor.

BACKGROUND ART Gaucher disease (GD) is a disease caused by a loss of enzyme caused by a mutation of a gene encoding a glucocerebrosidase (GCase) in a cell, and glucocerebroside (hereinafter, referred to as "glucosylceramide" , Liver, lungs, bone marrow, and brain. The liver and spleen are enlarged, anemia accompanied by fatigue symptoms is observed. In addition to the common anemia in which hemostasis is not performed due to thrombocytopenia, excessive lipid accumulation causes brain It is known that cell damage is caused (Beutler and Grabowski, 2001). Gaucher disease is classified into three major types. The first type I is a chronic non-neurological disease with liver disease, skeletal system abnormality, anemia, platelet reduction, and other blood diseases. The second type Ⅱ is called acute neuropathic form. The progression of the disease is rapid, and the symptom starts within 1 year of birth and the abnormal symptoms occur in the nervous system. In the brain of infant and neonatal period, Neurological complications due to rosace accumulation are characteristic. It is known that spleen enlargement occurs within 6 months of birth, aphthous sclerosis occurs, growth is delayed, and most of them die within 3 years. Third, type III, called the subacute neuropathic form, occurs at a frequency of approximately one in every 50,000 people in a wide range of races worldwide. The symptoms of the nervous system are relatively slower than those of the Ⅱ type, and the timing of symptoms is very diverse, but generally appears in childhood or adolescence. Neurological complications include mental retardation, loss of motor ability, and seizures of the liver, and interstitial lung disease may also occur. Both types Ⅱ and Ⅲ exhibit features that cause epilepsy and seizures due to impairment of brain function (Beutler and Grabowski, 2001).

One of the causes of Gaucher disease is the accumulation of mutant glucocerebrosideases in the endoplasmic reticulum. Since the defective GCase is unable to migrate from the endoplasmic reticulum to the lysosome and accumulates in the endoplasmic reticulum, it activates enzymes involved in the self-apoptotic pathway such as Bcl-2 family members and caspases by inducing ER stress Leading to apoptosis (Zong et al., 2003; Wei et al., 2008). In addition, Gaucher disease also exhibits insulin resistance and impairment of metabolic responses such as abnormal lipid metabolism (Wennekes et al., 2009).

The most widely used enzyme treatment, the Gaucher disease treatment, lacks the therapeutic effect of Type II or III Gaucher disease with neurological symptoms due to the inability to pass through the blood-brain barrier, which leads to a more effective The development of therapeutic agents for Gaucher disease is urgent.

Resveratrol (trans-3,5,4'-Trihydroxystilbene) is a phytoalexin-based polyphenol substance that many plants produce to protect themselves from various external environments such as fungi and bacterial infections. Antioxidant activity such as inhibition and free radical scavenging function, anti-inflammatory action such as inhibition of cyclooxygenase, inhibition of cancer cell growth and cancer, efficacy of platelet aggregation inhibition, prevention of cardiovascular disease, (Liu et al., 2011), found in grape skin, blueberries, and peanuts. In addition, resveratrol has the effect of regulating lipid and glucose metabolism through the blood-brain barrier (Baur et al., 2006; Bradamante et al., 2004; Vallianou et al., 2013). However, the therapeutic and metabolic effects of resveratrol in type III Gaucher disease with characteristic neurological symptoms are not yet known.

Korean Patent Publication No. 10-2014-0138850 Korea Patent Publication No. 10-2009-0001069

Cox TM. Gaucher disease: clinical profile and therapeutic developments. Biologics 2010; 4: 299-113. Ron I, Horowitz M. ER retention and degradation as the molecular basis underlying Gaucher disease heterogeneity. Hum Mol Genet 2005; 14: 2387-98. Wei H, Kim SJ, Zhang Z, et al. ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neurodegenerative lysosomal storage disorders and are alleviated by chemical chaperones. Hum Mol Genet 2008; 17: 469-77. Zong WX, Li C, Hatzivassiliou G, et al. Bax and Bak can localize to endoplasmic reticulum to initiate apoptosis. J Cell Biol 2003; 162: 59-69. Wennekes T, Berg R, Boot R, et al. Glycosphingo-lipids nature, function, and pharmacological modulation. Angew Chem Int Ed Engl 2009; 48: 8848-69. Liu C, Shi Z, Fan L, et al. Resveratrol improves neuron protection and functional recovery in rat model of spinal cord injury. Brain Res 2011; 1374: 100-9. Saha A, Sarkar C, Singh SP, et al. The blood-brain barrier is disrupted in a mouse model of infantile neuronal ceroid lipofuscinosis: amelioration by resveratrol. Hum Mol Genet 2012; 21: 2233-44. Vallianou NG, Evangelopoulosa, Kazazis C. Resveratrol and diabetes. Rev Diabet Stud 2013; 10: 236-42. Bradamante S, Barenghi L, Villa A. Cardiovascular protective effects of resveratrol. Cardiovasc Drug Rev 2004; 22: 169-88. Kim SJ, Kang SY, Kim JB. Possible therapeutic effects of myxobacterial metabolites on type I Gaucher disease. Gene 2012; 504: 156-9. Bravo R, Gutierrez T, Paredes F, et al. Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics. Int J Biochem Cell Biol 2012; 44: 16-20. Szegezdi E, Logue S, Gormana, et al. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep 2006; 7: 880-5. Susin SA, Lorenzo HK, Zamzami N. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999; 397: 441-6. Sullivan A, Singh M, Srere P, et al. Reactivity and inhibitor potential of hydroxycitrate isomers with citrate synthase, citrate lyase, and ATP citrate lyase. J Biol Chem 1977; 252: 7583-90. Roche TE, Baker JC, Yan X, et al. Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms. Prog Nucleic Acid Res Mol Biol 2001; 70: 33-75. Buhaescu I, Izzedine H. Mevalonate pathway: a review of clinical and therapeutical implications. Clin Biochem 2007; 40: 575-84. Baur JA, Pearson KJ, Price NL, et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature 2006; 444: 337-42. Marambaud P, Zhao H, Davies P. Resveratrol promotes clearance of Alzheimer's disease amyloid-beta peptides. J Biol Chem 2005; 280: 37377-82. Okawara M, Katsuki H, Kurimoto E, et al. Resveratrol protects dopaminergic neurons in midbrain slice culture from multiple insults. Biochem Pharmacol 2007; 73: 550-60.

The present inventors intend to provide a pharmaceutical composition for the treatment of Type III Gaucher Disease, which can ameliorate or treat neurological symptoms of type III Gaucher disease without appropriate therapeutic agents.

The present inventors have evaluated the viability of the cells derived from type III Gaucher disease patients, evaluated the biomarkers related to the metabolic and apoptotic pathways, and measured the glucocerebroside concentration to investigate the therapeutic potential of resveratrol.

None of the previous studies on the useful activity of resveratrol have involved Gaucher disease. The endoplasmic reticulum is where protein synthesis, processing and maturation take place. Incorrect processing of mutant GCase accumulates this protein in the endoplasmic reticulum lumen, which can cause endoplasmic reticulum stress. Ongoing ER stress can activate the apoptotic pathway, which ultimately activates the effector caspase, such as caspase-3, leading to cell death. The results of the present invention have found that resveratrol not only increases cell viability in Type II and Type III Gaucher cells but also dose-dependently decreases the expression levels of AIF, Bax and cleaved caspase-3. AIF mediates a caspase-independent apoptotic pathway. Thus, this result suggests that resveratrol has a broad range of metabolic spectrum that affects both caspase-dependent and caspase-independent apoptotic pathways. In addition, metabolic abnormalities such as insulin resistance and lipid metabolism damage have been reported in Gaucher disease (Wennekes et al., 2009), and there was a report that resveratrol improved glucose and lipid metabolism (Vallianou et al., 2013; Bradamante et al., 2004), we evaluated the effect of resveratrol on factors associated with this metabolic pathway in Type II and Type III Gassler cells. As a result, glucocerebroside concentration was remarkably dose-dependently reduced in resveratrol-treated Gaucher cells, and E3BP and CS expression, which are involved in the carbohydrate metabolism and citric acid circuits, were dose-dependently increased , And the expression of ACAT1, an enzyme involved in lipid metabolism, also increased in a dose-dependent manner (Fig. 3). CS is known to be a marker of intact mitochondria as a regulatory enzyme involved in the first step of the citric acid cycle and catalyzes the synthesis of citric acid by reacting acetyl-CoA with oxaloacetate (Sullivan et al., 1977) E3BP catalyzes the irreversible oxidative dicarboxylation of pyruvic acid to acetyl-CoA, linking sugars and citric acid circuits (Roche et al., 2001). Glucocerebroside is normally metabolized into lysosomal glucose and ceramide, and the glucose produced in this process can be used as a major source of energy through the citric acid circuit. The results of the present example show that resveratrol reduces intracellular stress by decreasing side-chain accumulation of glucocereb from the cell, and the cell can increase metabolic pathway activity by increasing acetyl-CoA demand, which can be restored in pathology 5). The ACAT1 enzyme catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA, which is the basis for molecular synthesis required for a variety of cellular processes, including growth, differentiation and maintenance through the mevalonate pathway (Buhaescu and Izzedine, 2007). Therefore, the upregulation of ACAT1 by resveratrol treatment in GD cells results in the conversion of acetyl-CoA, which is a byproduct of glucocerebroside degradation, to acetoacetyl-CoA, which is the molecule required for cell growth, proliferation and differentiation by the mevalonate pathway Can be used to produce In the present invention, resveratrol did not increase GCase activity (data not shown). Thus, glucocerebroside reduction in type II fibroblasts and type III GD fibroblasts appears to be due to substrate degradation, which is involved in the upregulation of glucose and lipid metabolism pathways by producing glucose.

In conclusion, the present inventors evaluated the effect of resveratrol on type II and type III Ghosian cells in Invitro. The results showed that resveratrol reduced the accumulation of glucocerebroside, decreased the expression of apoptotic factors, and increased the cell survival rate and the expression of enzymes related to glucose and lipid metabolism in Type II and Type III Gaucher cells. Resveratrol can cross the blood-brain barrier and exhibit protective activity in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Therefore, resveratrol is considered to be useful for treating intractable neurological symptoms of Ⅱ type Gaucher disease and Ⅲ type Gaucher disease such as epilepsy.

The present invention provides a pharmaceutical composition for treating Type III Gaucher Disease comprising resveratrol as an active ingredient. The pharmaceutical composition for treating type III Gaucher Disease containing resveratrol as an active ingredient can be formulated together with a carrier that is conventionally accepted in the pharmaceutical field and can be formulated by oral methods or injection forms by a conventional method. Oral compositions include, for example, tablets and gelatin capsules, which may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, , Magnesium stearate, stearic acid and its magnesium or calcium salt and / or polyethylene glycol) and the tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone ), And may optionally contain a disintegrant (e.g., starch, agar, alginic acid or a sodium salt thereof) or a boiling mixture and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The injectable composition is preferably an isotonic aqueous solution or suspension, and the composition mentioned is sterilized and / or contains adjuvants such as preservatives, stabilizers, wetting or emulsifying solution accelerators, salts for controlling osmotic pressure and / or buffering agents. They may also contain other therapeutically valuable substances.

Further, the present invention provides a pharmaceutical composition for treating type III Gaucher disease, which comprises the resveratrol as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutical preparations thus prepared may be administered orally or parenterally, that is, intravenously, subcutaneously, intraperitoneally, or topically, as desired. The dose may be administered in a single dose of 0.01 to 100 mg / kg per day. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, time of administration, method of administration, excretion rate, severity of disease, and the like.

The present invention also provides a health functional food composition for preventing or ameliorating a Type III Gaucher disease using the resveratrol as an active ingredient.

According to the present invention, when resveratrol is administered to a cell of a Type III Gaucher disease patient who does not have a proper therapeutic agent at present, the intracellular glucocerebroside concentration is markedly decreased, thereby increasing the cell survival rate.

In addition, according to the present invention, when resveratrol is administered to cells of the type III Gaucher disease patient, the expression of the apoptotic factor is markedly lowered compared with the control, thereby increasing the cell survival rate.

Therefore, when a pharmaceutical composition containing resveratrol is administered to a patient with type III Gaucher disease, resveratrol having a low molecular weight can pass across the blood-brain barrier to improve specific neurological symptoms in patients with type III Gaucher disease, Since resveratrol is a natural polyphenol component, it is expected that a significant improvement in the symptom of type III Gaucher disease, which does not have a proper therapeutic agent at present, can be expected because cell survival rate is increased and glucocerebroside accumulation is decreased without any adverse side effects.

Figure 1 shows the effect of resveratrol on the type III Gaucher cell survival. The survival rate of resveratrol treated Gaussian cells was increased by MTT assay. The survival rate of type I Gaucher cells treated with DMSO (carrier) was defined as 100% relative cell viability.
Normal-vehicle: Normal cells treated with DMSO; GD Ⅲ, Ⅲ type Gaucher disease fibroblast. * Represents a significant difference between the carrier group and the other groups. *, P < 0.05 by Student's t-test.
FIGS. 2A and 2B show the results of Western blot and density analysis of apoptotic factors. (Fig. 2a) Resveratrol in type III GD cells reduced the expression of AIF, Bax and truncated caspase-3. (2b) Density scale analysis showed that the protein concentration decreased dose-dependently. β-actin was used as a control. The vehicle was treated with DMSO for type III GD cells and defined as a relative density of 100%. Normal-vehicle, normal cells treated with DMSO; GD I, Type I Gaucher disease fibroblast. * Represents a significant difference between the carrier group and the other groups. *, P < 0.05 by Student's t-test.
Figures 3a and 3b show the results of Western blot and density analysis for metabolic markers. (3a) In type III GD cells, resveratrol increased expression of ACAT1 (acetyl-coenzyme A acetyltransferase 1), E3BP (E3-binding protein) and CS (citrate synthase) (3b) Density analysis revealed that the protein concentration was dose dependent. β-actin was used as a control. The vehicle was treated with DMSO for type III GD cells and defined as a relative density of 100%. Normal-vehicle, normal cells treated with DMSO; GD Ⅲ, Ⅲ type Gaucher disease fibroblast. * Represents a significant difference between the carrier group and the other groups. *, P < 0.05 by Student's t-test.
4A and 4B are the results of measurement of intracellular glucocerebroside (glucosylceramide; GluCer) concentration by thin-layer chromatography (4a) and density analysis (4b). In type III GD cells, resveratrol reduced glucocerebroside concentration. The vehicle was treated with DMSO for type III GD cells and defined as a relative density of 100%. Normal-vehicle, normal cells treated with DMSO; GD Ⅲ, Ⅲ type Gaucher disease fibroblast. * Represents a significant difference between the carrier group and the other groups. *, P < 0.05 by Student's t-test.
5 is a schematic diagram showing the activity of resveratrol in type III GD cells. AIF, apoptosis-inducing factor; ACAT1, acetyl-coenzyme A acetyltransferase 1; E3BP, E3-binding protein; CS, citrate synthase.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the scope of the embodiments.

Cell line and cell culture

Type III Gaucher Disease Fibroblasts were obtained from Coriell Institute for Medical Research (GM # 20272; homozygous for the L444P mutation, Coriell, Camden, NJ, USA) , NJ, USA) were also obtained from the same place. Normal fibroblasts were obtained from Korean Cell Line Bank (KCLB # 21947, CCD-986sk; Seoul, South Korea). (Invitrogen, Carlsbad, Calif., USA), 100 units / mL penicillin, 100 mu g / mL streptomycin and 0.25 mu g / mL ampicillin, In a 100-mm dish containing Dulbecco's modified Eagle's medium (Mediatech Inc., Herndon, Va., USA). The culture was maintained at 37 ° C in an incubator with 5% CO ₂ .

Cell survival analysis

Type II and III GD fibroblasts and normal fibroblasts were cultured in 96-well plates (10 ³ cells / 200 μl) (Thermo Scientific) containing 100 μl of medium for 24 hours in a CO ₂ incubator at 37 ° C, The cells were then incubated in DMEM containing 0.1 μM, 1 μM or 10 μM resveratrol (prepared by dissolving in dimethylsulfoxide (DMSO); Sigma, Saint Louis, Mo., USA) for 24 hours at 37 ° C in a 5% CO ₂ incubator . Type II GD fibroblasts, type III GD fibroblasts and normal fibroblasts were treated with 0.1% DMSO as a carrier control. 10 μl of MTT {EZ-cytox 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide} assay solution (Daeillab service, Seoul, South Korea) was added to each well, Lt; / RTI &gt; After incubation, the amount of MTT formazan produced was determined by measuring the absorbance at 405 nm with a microplate spectrophotometer (Bio-Rad, Hercules, Calif., USA). Cell viability was expressed as a percentage of control cell viability.

Western Blat

Approximately 1x10 ⁶ type II and type III GD fibroblasts and normal fibroblasts were cultured at 37 ° C in a 100-mm dish containing culture medium containing resveratrol (0.1 μM, 1 μM or 10 μM). Both type II and type III GD fibroblasts and normal fibroblasts were also cultured in 0.1% DMSO (carrier control). After 24 hours of incubation, the cells were washed with cold PBS and incubated with total cell lysis buffer (20 mM HEPES pH 7.5, 0.5 mM EDTA, 0.1 mM EGTA, 350 mM NaCl, 10 mM NaF, 1 mM MgCl ₂ And 1% Triton X-100 [Sigma]). Protein concentrations were measured by Coomassie + protein assay (Pierce, Rockford, IL, USA). 30 μg of protein was collected from each sample, run on an electrophoresis gel, and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were incubated with AIF antibody (Abcam, Cambridge, MA, USA), Bax (Bcl-2-associated X protein) antibody (Cell Signaling, Beverly, USA), truncated caspase- 3 antibody, E3BP (Abcam), citrate synthase (CS) antibody (Abcam), ACAT1 (acetyl-coenzyme A acetyltransferase 1) antibody (Abcam) and β-actin antibody (Sigma). The membranes were then incubated with anti-mouse secondary antibody (Abcam) and anti-rabbit secondary antibody (Abcam). The band was visualized with an enhanced chemiluminescence system (SuperSignalWest Pico Luminol / Enhancer Solution, Pierce). Density analysis was performed to quantify the protein concentration with TINA 2.0 software (Raytest Isotopenmessgerate GmbH, Straubenhardt, Germany).

Lipid extraction and Glucocerebroside  Concentration measurement

Quantitative changes in glucocerebroside concentration of cells treated with resveratrol or DMSO (carrier control) were measured by thin layer chromatography (TLC). Lipid extraction and glucocerebroside separation were performed according to the prior art (Kim et al., 2012). Density analysis was performed to determine glucocerebroside concentration using TINA 2.0 software (Raytest Isotopenmessgerate GmbH).

Statistical analysis

Each experiment was performed independently from at least three to at most six times. Student's t-test was performed to confirm that there were significant differences. P <0.05 was considered significant.

Result 1: Resveratrol  Cell viability after treatment

Type III GD fibroblasts were treated with 0.1 μM, 1 μM and 10 μM resveratrol and the effect on cell viability was analyzed by MTT assay. Treatment of 0.1 μM, 1 μM and 10 μM resveratrol increased the survival rate of type III GD fibroblasts by 17-46% (P <0.05, FIG. 1) in comparison with GD fibroblasts treated with DMSO. To identify the protective activity of resveratrol, expression patterns of AIF, Bax and truncated caspase-3 associated with apoptosis were evaluated. Treatment of type III GD fibroblasts with 0.1 μM, 1 μM and 10 μM resveratrol resulted in AIF (34-63%; P <0.05), Bax (23-46%; P <0.05) and truncated caspase- %; P < 0.05) expression was dose-dependent as compared to DMSO-treated GD fibroblasts (Fig.

Result 2: Metabolism On the marker  About Western Blat  analysis

Because metabolic abnormalities such as insulin resistance and lipid metabolism damage have been reported in Gaucher's disease (Wennekes et al., 2009), regulatory effects of resveratrol on glucose and lipid metabolism have been reported (Vallianou et al., 2013; Bradamante et al., 2004) investigated whether resveratrol in the type III GD fibroblasts regulates the concentration of factors involved in the metabolic pathway. After 24 hours of treatment with resveratrol by concentration, the levels of ACAT1 and E3BP expression increased in a dose-dependent manner (23-58% and 19-67%, respectively, for type III cells) compared to the carrier control. In addition, CS expression, a mitochondrial marker enzyme, increased 10-54% in a dose dependent manner on type III cells (P &lt; 0.05, Figs. 3a and 3b).

Result 3: Intracellular Glucocerebroside  Concentration change measurement

Quantitative changes in glucocerebroside in type III GD fibroblasts were assessed by thin-layer chromatography. Glucocerebroside concentration increased in GD cells treated with DMSO compared with normal cells. Glucocerebroside accumulation was dose-dependently reduced in GD cells treated with increasing resveratrol concentration (21-38% reduction in type III cells by relative density scan, P < 0.05, Figures 4a, 4b).

Claims (2)

A pharmaceutical composition for treating type III Gaucher disease containing resveratrol.
A health functional food composition for preventing or ameliorating Type III Gaucher disease containing resveratrol.

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