Patent Application
20150064287
This application claims the benefits of the Taiwan Patent Application Serial Number 102130549, filed on Aug. 27, 2013, the subject matter of which is incorporated herein by reference.
1. Field of the Invention
The present invention relates to a method for treating abnormal polyglutamine-mediated diseases comprising: administering a pharmaceutical composition comprising a Gardenia jasminoides extract which can reduce reactive oxygen species to suppress polyglutamine aggregation.
2. Description of Related Art
The spinocerebellar atrophy is referred as spinocerebellar ataxias (SCA), which is a complex group of heterogeneous autosomal dominant neurodegenerative disorder. Spinocerebellar ataxias, caused by expanded CAG repeats encoding a long polyglutamine (polyQ) tract in mutant proteins, and the misfolded polyQ proteins accumulate in the nucleus and cytoplasm of neural cells. The clinical symptom of spinocerebellar atrophy comprises cerebellar degeneration, dysfunctions of nervous system and other parts.
So far, no commercial drug is available for curing or mitigating progressive cerebellar ataxia, which involves following irreversible symptoms: in the beginning, patients cannot properly control their actions; with disease progression, patients gradually become unable to walk and pick up a pen, and eventually unable to speak and swallow; and in the worst case, patients die. However, even the cerebellum, brainstem and spinal cord may atrophy, normal functions of the brain and intelligence are usually not affected, so that the patients can clearly recognize the fact that their bodies gradually become inactive.
Moreover, the modern medical treatment is based on surgery, radiation therapy, chemotherapy, hormonal therapy, biological therapy and so on. It often causes serious side effects on the patients' body, and result in weak patients. On the other hand, many people expect traditional Chinese medical treatment could be helpful since it is relatively mild, generally recognized by people and having a quite high market acceptance.
In view of the gradually increased global population suffering from cerebellar atrophy, what is needed is to find a method capable of reducing the accumulation of polyglutamine used for the manufacturing of pharmaceutical compositions for abnormal accumulated polyglutamine-mediated diseases, serving as an adjuvant therapy for neurodegenerative disease, such as cerebellar atrophy, thereby effectively slowing down the disease progression, and providing the patients with a better quality of life.
An object of the present invention is to provide a method for treating polyglutamine-mediated diseases, to thereby serve as an adjuvant therapy for neurodegenerative disease, such as cerebellar atrophy.
Another object of the present invention is to provide a method for reducing reactive oxygen species induced by the accumulation of polyglutamine.
To achieve the above objects, the present invention provides a method for treating abnormal polyglutamine-mediated diseases, comprising: administering a pharmaceutical composition comprising one or more selected from the group consisting of a Gardenia jasminoides extract, geniposide and crocin, wherein geniposide and crocin are the active ingredients extracted from Gardenia jasminoides, but may also be extracted from other Chinese medicine, and the Gardenia jasminoides extract, geniposide and crocin are all commercially available.
In addition, the pharmaceutical composition has the function of reducing reactive oxygen species (ROS). In the polyglutamine-mediated diseases, the misfolded polyglutamine causes increased reactive oxygen species and accumulation of abnormal proteins. Accordingly, the pharmaceutical composition reduces the accumulation of polyglutamine by reducing reactive oxygen species through increasing NFE2L2 (nuclear factor, erythroid 2-like 2) protein expression, and in turn, increasing the expression of the downstream proteins, such as NQO01 (NAD(P)H dehydrogenase, quinone 1), GCLC (glutamate-cysteine ligase, catalytic subunit) and GSTP1 (glutathione S-transferase pi 1).
Examples of the abnormal polyglutamine-mediated diseases are not limited, known to include Huntington's disease, spinal bulbar muscular atrophy and preferably cerebellar atrophy. Eight kinds of cerebellar atrophy are known to be caused by expanded CAG repeats of polyglutamine, including SCA1, SCA2, SCA3, SCA6, SCAT, SCA8, SCA17 and DRPLA (dentatorubral-pallidoluysian atrophy). Such SCAs associated with expanded polyglutamine show a selective and progressive degeneration of cerebellum, brainstem and spinal cord. In the degenerated nerve, expanded polyglutamine proteins significantly accumulate in the nucleus and cytoplasm, resulting in functional insufficiency and degradation of some specific neurons.
The concentration of the Gardenia jasminoides extract is not particularly limited, and can be adjusted depending on the use requirement (for example, disease severity or co-medication). In an embodiment of the present invention, the Gardenia jasminoides extract preferably has a concentration of 1 μg/mL to 100 μg/mL, more preferably 1 μg/mL to 50 μg/mL, and most preferably 1 μg/mL to 10 μg/mL, wherein each of the geniposide and crocin has a concentration of 50 nM to 5 μM, and more preferably 100 ng/mL to 1 μg/mL. In an embodiment of the present invention, geniposide preferably has a concentration of about 500 nM, and crocin preferably has a concentration of about 100 nM.
In the Gardenia jasminoides extract, the active ingredient, geniposide, is transformed into genipin by a reaction of intestinal bacteria (Eubacterium sp. A-44, Reference: Akao, T., Kobashi, K., Aburada, M., 1994. Enzymic studies on the animal and intestinal bacterial metabolism of geniposide. Biol. Pharm. Bull. 17, 1573-1576.), wherein the intestinal bacterial species may be known for those having ordinary skill in the art. Accordingly, the actions and transformation in vivo of the Gardenia jasminoides extract, and the active ingredients, geniposide and crocin, are not limited in the present invention. However, from the following examples, it can be learned that geniposide can reduce the accumulation of polyglutamine and the level of the reactive oxygen species.
In other words, the effective dosage of Gardenia jasminoides extract, geniposide and crocin included in the pharmaceutical composition may be varied according to the administration path, excipients and possibly, the co-used agents, and those having ordinary skill in the art may adjust the dosage thereof to generate the desired effect on the target.
Accordingly, the invention further provides a method for reducing reactive oxygen species, comprising: providing a target; administering a pharmaceutical composition to the target, wherein the pharmaceutical composition comprises one or more selected from the group consisting of a Gardenia jasminoides extract, geniposide and crocin.
The term “reduce” or “reduction” refer to the case that the pharmaceutical composition of the present invention is applied to a subject suffering from polyglutamine-mediated diseases, having symptoms of disease, or having a tendency of development of disease, in order to achieve the treatment, mitigation, slowing, therapy, improvement, or recovery of the tendency of the disease and symptoms.
To implement the method according to the present invention, the above pharmaceutical composition can be administered via oral administering, parenteral administering, inhalation spray administering, topical administering, rectal administering, nasal administering, sublingual administering, vaginal administering, or implanted reservoir, and so on. The term “parenteral” used here refers to subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intra-articular injection, intraarterial injection, joint fluid injection, intrathoracic injection, intrathecal injection, injection at morbid site, and intracranial injection or injection technique.
The Gardenia jasminoides extract of the present invention may be purchased from the market, or obtained by water extraction under heating and filtering off residue, but the present invention is not limited thereto. For example, 100 g of dried Gardenia jasminoides and 1500 mL of water are mixed at a temperature of 100° C. for 30 minutes, and sieved using a 100-mesh sieve. Then, the extract is concentrated to 100 mL and filtered through again with a 200-mesh sieve. After that, the extract is dried by a vacuum speed vacuum concentrator and reserved at a temperature of −20° C. Therefore, the pharmaceutical composition formed of the Gardenia jasminoides extract may be processed into a dried extract by using any conventional technique, such as spray drying, freeze-drying, scientific Chinese herbal medicine granulation, and further manufactured into a healthy food and a clinical therapeutic pharmaceutical for the treatment and the prevention of spinocerebellar ataxia. For example, according to the requirement for use, the pharmaceutical composition of the present invention may comprise: at least one of a pharmaceutically acceptable carrier, a diluent, or an excipient in the art. For example, it is encapsulated into liposome to facilitate delivery and absorption. Alternatively, it is diluted with aqueous suspension, dispersion or solution to facilitate injection. Or, it is prepared in a form of a capsule or tablet for storage and carrying. In detail, the composition of the present invention may be formulated in a solid or liquid form. The solid formulation form may include, but is not limited to, powders, granules, tablets, capsules and suppositories. The solid formulation may comprise, but is not limited to, excipients, flavoring agents, binders, preservatives, disintegrants, glidants and fillers. The liquid formation form may include, but is not limited to, water, solutions such as propylene glycol solution, suspensions and emulsions, which may be prepared by mixing with suitable coloring agents, flavoring agents, stabilizers and viscosity-increasing agent.
For example, a powder formulation may be prepared by simply mixing the compound of the present invention with suitable pharmaceutically acceptable excipients such as sucrose, starch and microcrystalline cellulose. A granule formulation may be prepared by mixing the compound of the present invention, suitable pharmaceutically acceptable excipients, suitable pharmaceutically acceptable binders such as polyvinyl pyrrolidone and hydroxypropyl cellulose, followed by wet granulation method using a solvent such as water, ethanol and isopropanol, or dry granulation method using compression force. Also, a tablet formulation may be prepared by mixing the granule formulation with suitable pharmaceutically acceptable glidants such as magnesium stearate, followed by tableting using a tablet machine.
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
In the following description, numerous specific details are set forth to provide a thorough understanding of embodiments of the present disclosure. However, one having an ordinary skill in the art will recognize that embodiments of the disclosure can be practiced without these specific details. In some instances, well-known structures and processes are not described in detail to avoid unnecessarily obscuring embodiments of the present disclosure.
Gardenia jasminoides Extract Preparation and HPLC Analysis
In the following experiment, the Gardenia jasminoides extract was provided by Sun-Ten Pharmaceutical Company (Taipei, Taiwan) as described (Chang et al., 2013; Aqueous extract of Paeonia lactiflora and paeoniflorin as aggregation reducers targeting chaperone in cell models of spinocerebellar ataxia 3. Evidence-based Complementary and Alternative Medicine 2013:471659). High pressure liquid chromatography (HPLC) was performed using a LaChrom Elite HPLC system (Hitachi), consisting of a photo diode array detector. The chromatographic separation of Gardenia jasminoides extract (50 μL, 1 mg/mL) was carried out on a Hypersil ODS (C18) column (250×4.6 mm, 5 μm), eluted with the mixture of water (A), acetonitrile (B) or methanol (C). The linear gradient elution program for A:B:C (v/v/v) was set as follows: 80:5:15 (0-8 min), 70:0:30 (8-30 min), 15:0:85 (30-50 min), 80:5:15 (50-60 min) with a flow rate of 1 mL/min. Then, absorbance was monitored at 240, 440 nm and the scan range for photo diode array was 190-600 nm, wherein geniposide and crocin (50 μL, 0.01-1 mM) were used as reference compounds for Gardenia jasminoides.
Cell Culture and Cell Proliferation 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). Human neuroblastoma SH-SYSY cells (ATCC No. CRL-2266) were maintained in DMEM F12 supplemented with 10% FBS. Cells (5×104/well in 48-well dishes) were cultivated at 37° C. incubator containing 5% CO2 for 20 hr and treated with different concentrations of the Gardenia jasminoides extract (5-30 mg/mL) or pure compound (100 nM-1 mM). After one day, 20 μL MTT (3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, 5 mg/mL in PBS; Sigma-Aldrich) was added to cells and incubated for 2 hr. The absorbance of the purple formazan dye was measured at 570 nm by a Bio-Tek μQuant Universal Microplate Spectrophotometer.
ATXN3 cDNA Constructs and Isogenic Cell Lines
200 ng of polyadenylation (polyadenylated) RNA isolated from the neuroblastoma SK-N-SH cells was subjected to the reverse transcription using SuperScript™ III reverse transcriptase (purchased from Invitrogen). The forward and reverse primers for amplification of ATXN3/Q14 cDNA (+826 to +1152, NM—004993) were SEQ ID NO: 1, 5′-ATTCAGCTAAGTATGCAAGGTAGTTCCA (the lined letters are Met257 codon), and SEQ ID NO: 2, 5′-CATGCCATGGCATGTTTTTTTCCTTCTGTT (the lined letters are NcoI enzyme site). Then, the amplified 3′ polyQ-containing cDNA fragment (which encoded amino acids 257-361) was cloned into pGEM-T Easy (purchased from Promega) and sequenced. ATXN3/Q14 cDNA cDNA fragment was cut using EcoRI and NcoI, and subcloned into pEGFP-N1 (purchased from Clontech). Then, ATXN3/Q14-EGFP-containing DNA fragment was cut using HindIII-NotI and subcloned into pcDNA5/FRT/TO. In addition, ATXN3/Q75 cDNA was prepared by replacing the ATXN3/Q14 BsmBI-BsmFI fragment of 88 by with the ATXN3/Q75 fragment of 271 by from the cDNA clone of a SCA3 patient.
Isogenic 293 and SH-SY5Y Cell Lines
Flp-In™-293 cells derived from human 293 (Invitrogen) were cultured as described above. The isogenic ATXN3/Q14-75 cell line was employed, which was formed from subjecting the Flp-In™-293 cells to targeting insertion of the pcDNA5/FRT/TO-ATXN3/Q14 and Q75 recombinant plasmids. These cell lines were grown in medium containing 5 μg/mL blasticidin and 100 μg/mL hygromycin (InvivoGen). In addition, the method for culture in establishment of the human SH-SY5Y derived Flp-In host cell line and the formation of the isogenic ATXN3/Q14-75 cell line was the same.
ATXN3/Q75 Aggregation Assay
293 ATXN3/Q75-GFP cells were plated into 96-well (2×104/well) dishes, grown for 24 hr and treated with different concentrations of the Gardenia jasminoides extract (1-100 μg/mL) or suberoylanilide hydroxamic acid (SAHA, Cayman Chemical), genipin, crocin (Sigma), geniposide (ChromaDex) (50 nM-5 μM) for 8 hr. Then doxycycline (10 μg/mL, BD) and oxaliplatin (5 μM, Sigma) were added for 6 days. After that, cells were stained with Hoechst 33342 (0.1 μg/mL, Sigma-Aldrich) and aggregation percentage was assessed by high-content analysis (HCA) system (ImageXpressMICRO, Molecular Devices).
SH-SYSY ATXN3/Q75-GFP cells were seeded in 6-well (2×105/well) plate, with all trans retinoic acid (10 μM, Sigma) added at seeding time. At day 2, cells were treated with genipin, geniposide (500 nM), crocin (100 nM) or the Gardenia jasminoides extract (10 μg/mL) for 8 hr, and then doxycycline (5 μg/mL) was added to induce ATXN3/Q75-GFP expression. The cells were kept in the medium containing 10 μM trans retinoic acid, doxycycline and genipin/geniposide/crocin/Gardenia jasminoides extract for one week. After that, cells were stained with Hoechst 33342 (0.1 μg/mL) and aggregation percentage was assessed as described.
Western Blot Analysis
Total proteins were prepared using lysis buffer containing 50 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM EDTA pH8.0, 1 mM EGTA pH8.0, 0.1% SDS, 0.5% sodium deoxychalate, 1% Triton X-100 and protease inhibitor cocktail (Sigma). Proteins (25 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes by reverse electrophoresis. After blocking, the membrane was probed with NFE2L2 (1:500 dilution, Santa Cruz), NQO1 (NAD(P)H dehydrogenase, quinone 1; 1:1000 dilution, Sigma), GCLC (glutamate-cysteine ligase, catalytic subunit; 1:100 dilution, Abcam), GSTP1 (glutathione S-transferase pi 1; 1:1000 dilution, Abcam) or β-actin (ACTB) (1:5000 dilution, Millipore) at 4° C. overnight. Then the immune complexes were detected by horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG antibody (1:5000 dilution, GeneTex) and chemiluminescent substrate (Millipore).
ROS Analysis
293 ATXN3/Q75-GFP cells were plated into 6-well (105/well) dishes, grown for 24 hr and treated with different concentrations of the Gardenia jasminoides extract (10 μg/mL) or genipin, geniposide (500 nM), crocin (100 nM) for 8 hr. Then doxycycline (10 μg/mL) and oxaliplatin (2.5 μM) were added to the medium for ATXN3/Q75-GFP expression and aggregate accumulation. Six days later, fluorogenic CellROX™ Deep Red Reagent (5 μM, Molecular Probes) designed to reliably measure ROS in live cells was added to the cells and incubated at 37° C. for 30 min. The cells were then washed with PBS and analyzed for green (GFP) and red (ROS) fluorescence on a flow cytometry (Becton-Dickinson), with excitation/emission wavelengths at 488/507 (green) and 640/665 nm (red). 5×104 cells in each sample were analyzed.
1,1-diphenyl-2-picryl hydrazyl (DPPH) Assay
100 μM DPPH radical solution (purchased from Sigma) and 10-200 μM of genipin, geniposide, crocin or 0.2-4 mg/mL of Gardenia jasminoides extract were vortexed for 15 seconds and then left to stand at room temperature for 30 min. Then, the scavenging capacity was measured by monitoring the decrease in absorbance at 517 nm by a Thermo Scientific Multiskan GO Microplate Spectrophotometer. The radical scavenging activity was calculated using the formula: 1-(absorbance of sample/absorbance of control)×100%. The antioxidative activity was expressed as EC50, which was defined as the concentration of the compounds required for inhibition of the formation of DPPH radicals by 50%.
The NFE2L2 cDNA (BC011558) in pOTB7 was obtained from Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute, Taiwan. The cDNA was excised with BamHI and XhoI and subcloned into pcDNA3 (Invitrogen). For transient overexpression, cells were plated into 12-well (5×105/well) dishes, grown for 20 hr, and transfected with using T-Pro reagent with pcDNAS/FRT/TO-ATXN3/Q75 and overnight later with pcDNA3-NFE2L2 or pcDNA3 vector plasmids (1.5 μg each).
Statistical Analysis
For each set of values, data were expressed as the means±standard deviation (SD). Three independent experiments were performed and non-categorical variables were compared using the Student's t-test. All P-values were two-tailed, with values of P<0.05 considered significant.
Results
The chemical properties of the Gardenia jasminoides extract were analyzed and quantified by full-spectrum analytic HPLC. The chromatographic patterns in
In MTT assays, the cytotoxicity of the Gardenia jasminoides extract, geniposide and crocin against human embryonic kidney 293 and human neuroblastoma SH-SY5Y cells was shown in
293 ATXN3/Q75 Cells and Reduced NFE2L2 Expression
In this experiment, ATXN3/Q14-75 fragment with C terminal marked by GFP was established, to form the Flp-In 293 cells with ATXN3/Q14-75-GFP expression in an inducible fashion, wherein ATXN3/Q14 was the control group of normal protein expression. As shown in
PolyQ mutation may induce reactive oxygen species and NFE2L2 plays a central role in the regulation of the cellular redox status. Please refer to
Effects of Gardenia jasminoides Extract and its Derivatives on 293 ATXN3/Q75 Aggregation
In this experiment, influence of genipin, geniposide, crocin, Gardenia jasminoides extract, and SAHA on ATXN3/Q75 was tested using ATXN3/Q75-GFP cells. As shown in
Effects of Gardenia jasminoides Extract and its Derivatives on NFE2L2, NQO1, GCLC and GSTP1 Expression
The protein expression levels of NFE2L2, NQO1, GCLC and GSTP1 were compared under addition of the Gardenia jasminoides extract and its active ingredients and/or doxycycline, to examine if the Gardenia jasminoides extract and its active ingredients up-regulated the NFE2L2 and the downstream protein expression levels of NQO1, GCLC and GSTP1. ATXN3/Q75 cells were treated with genipin, geniposide, crocin and G. jasminoides extract for 8 hr and induced by doxycycline for 6 days.
Effects of Gardenia jasminoides Extract and its Derivatives on ROS Production
The fluorescence and ROS levels of ATXN3/Q75-GFP cells were compared under the addition of the Gardenia jasminoides extract and its active ingredients and/or doxycycline. As shown in
Radical Scavenging Activity of Gardenia jasminoides Extract and its Derivatives
In this experiment, kaempferol with strong antioxidant property was chosen as the reference antioxidant. As shown in
NFE2L2 Overexpression to Reduce ATXN3/Q75 Aggregation
As shown in
Effect of Gardenia jasminoides Extract and its Derivatives on SH-SY5Y ATXN3/Q75 Aggregation
After differentiation of ATXN3/Q14-75-GFP SH-SYSY cells using retinoic acid for 7 days, it can be found that the induced ATXN3/Q75-GFP formed aggregates in -1% differentiated neurons, whereas no aggregate was seen in ATXN3/Q14-GFP cells, and the aggregates were increased with time (not shown). As shown in
It should be understood that these examples are merely illustrative of the present invention and the scope of the invention should not be construed to be defined thereby, and the scope of the present invention will be limited only by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102130549 | Aug 2013 | TW | national |
