Patent Application
20120009131
1. Field of the Invention
The present invention generally relates to a method for preventing or treating amyloid beta peptide (amyloid β0 peptide; Aβ) associated diseases or conditions, and a method for inhibiting formation, accumulation or aggregation of amyloid beta peptides.
2. Description of the Prior Art
Alzheimer's disease is considered to be related to accumulation of peptides of 39-43 amino acids, and such peptides are called as amyloid β peptides, which are hydrolysis products of amyloid precursor protein (APP). Among the Aβ, Aβ1-40 is the most abundant form, while Aβ1-42 is more toxic to neurons and highly fibrillogenic and is considered as the most relevant Aβ form to Alzheimer's disease. Aβ monomers can form soluble Aβ oligomers through oligomerization, and further form insoluble fibrils or senile plaques extracellularly.
It is generally considered that Aβ-associated diseases or conditions comprise Down syndrome, hereditary cerebral hemorrhage with amyloid (HCHWA) Dutch, Parkinsonism-dementia complex on Guam (Clinical Neurology and Neurosurgery (1990) 92: 305-310); Cerebral amyloid angiopathy (J. Neuropath. Exp. Neuro. (2002) 61: 282-293); inclusion body myositis (Neurology (2006) 66: 65-68); frontotemporal dementia (Neuroreport (2002) 13-5: 719-723); age-related macular degeneration (Experimental Eye Research (2004) 78: 243-256); Pick's disease (Neuroscience Letters (1994) 171: 63-66), and others.
Aβ-associated diseases or conditions as described are generally related to formation, accumulation or aggregation of Aβ, leading to abnormal quantity of Aβ or Aβ aggregates present in organisms caused by congenital factors (e.g., inheritance) or acquired factors (e.g., aging or environmental effects).
It is generally believed that inhibition of Aβ formation, accumulation or aggregation can be used as an approach for effectively preventing or treating Alzheimer's disease or other Aβ-associated diseases or conditions.
Since Aβ and its aggregates are likely to cause various diseases or conditions in organisms, one object of the present invention is to provide an active ingredient for inhibiting formation, accumulation or aggregation of Aβ, and such active ingredient can be used as an additive in food, drinks, chewing gums, patches, skin care products, etc. Another object of the present invention is to provide a drug and a method for preventing or treating Aβ-associated diseases or conditions.
To achieve the above objects, the present invention discloses use of isoacteoside or a pharmaceutically acceptable salt thereof in inhibiting the formation, accumulation or aggregation of Aβ, and in preparing a drug for preventing or treating Aβ-associated diseases or conditions.
Preferably, said isoacteoside or the pharmaceutically acceptable salt thereof is provided for inhibiting neuronal damage or apoptosis caused by the amyloid β peptides, so as to retain, improve or restore learning and memory abilities.
Preferably, an effective dosage of said drug to a person is equivalent to per day 0.2 mg to 4.0 mg of isoacteoside or the pharmaceutically acceptable salt thereof per kg of body weight.
To better understand the above and other objects, features and advantages of the present invention, the present invention will be described in detail below taken from the examples with reference to the annexed drawings.
Various diseases caused by Aβ have a common feature: formation of Aβ aggregates. These Aβ aggregates present in shapes such as fibrils or plaques, and deposit in systems, organs, tissues or body fluids of organisms, causing various diseases or conditions. It is therefore supposed that inhibition of Aβ formation, accumulation or aggregation can be used as an approach for effectively preventing or treating Aβ-associated diseases or conditions.
In view of the above, the present invention discloses using isoacteoside having a structure shown below or a pharmaceutically acceptable salt thereof as an active ingredient for inhibiting (e.g., reducing or preventing) Aβ formation, accumulation or aggregation, and in particular Aβ extracellular formation, accumulation or aggregation.
U.S. Pat. No. 7,087,252 B2 discloses a medicinal preparation, which comprises 10-70 weight percent of echinacoside and 1-40 weight percent of acteoside, prepared from fleshy stems of Cistanche tubulosa (Schenk) Wight, and is provided against senile dementia. Isoacteoside and various other phenylethanoid glycosides are known to be included in the medicinal preparation.
In the present invention, hydrates or other solvates, prodrugs or metabolites of isoacteoside are deemed as functional equivalents of isoacteoside. The prodrugs described herein mean a precursor compound which can produce isoacteoside under biological conditions (in vivo or in vitro) by hydrolysis, oxidation or other reactions. The metabolites of isoacteoside described herein mean a compound which can be produced by metabolism of isoacteoside in cells or in organisms.
When a pharmaceutically acceptable salt of isoacteoside is administered to an individual, the pharmaceutically acceptable salt generally provides equivalent or similar therapeutic effects as isoacteoside, and is physiologically tolerable without causing adverse side effects such as allergy or the like. The pharmaceutically acceptable salt of isoacteoside may comprise but not limit to iron, calcium, and magnesium salts, etc.
The term “prevent” used herein means avoiding or delaying occurrence of a disease or condition in organisms. The terra “treat” used herein means slowing or stopping progress of a disease or condition, or making an individual return back to his improved or normal status.
The term “amyloid β peptide (Aβ)-associated diseases or conditions” generally refers to those diseases or conditions that occur relating to formation, accumulation or aggregation of Aβ, and particularly refers to the diseases or conditions that are caused by Aβ. When abnormal formation, accumulation or aggregation is found in a certain proportion of individuals with certain diseases or conditions, the diseases or conditions can be considered as being associated with Aβ. In addition, when Aβ aggregates somewhere that is close to occurrence of pathological features affected in certain diseases or conditions, the diseases or conditions can be also considered as being associated with Aβ.
The active ingredient for Aβ inhibition provided by the present invention can be used as an additive in food, drinks, etc., to facilitate the use of inhibiting formation, accumulation or aggregation of Aβ.
The drugs or pharmaceutical compositions for treating Aβ-associated diseases or conditions provided by the present invention, which comprises isoacteoside or the pharmaceutically acceptable salt thereof as an active ingredient, can comprise suitable carriers, diluents or excipients etc., and can be present in powders, granules, tablets, troches, pills, capsules, solutions, suspensions, creams, ointments, gels, aerosols, suppositories, patches or any other desired forms. The drugs or pharmaceutical combinations described above can be administered via oral, topical, parenteral, dermal, intranasal, ocular, intraocular or other routes.
In the context, the term “or” is generally defined as “and/or”, unless it is otherwise specified.
Along with the aging of the population, dementia, which is related to aging, has become one of the major concerns in the present medical researches related to senile diseases. Alzheimer's disease is the most common type of dementia. Alzheimer's disease is a chronic progressive neurodegenerative disease, characterized in that patients gradually lose their cognition ability and show abnormal behavior, followed by a possible loss of verbal and motion abilities, and thus Alzheimer's disease easily makes a great impact on the patient's and his/her family's life qualities. In view of the trend of the number of patients suffering Alzheimer's disease gradually increasing, causing severe burdens on the patient's family and the society, the present invention selects the amyloid beta peptides (Aβ) which are more related to Alzheimer's disease for carrying out experiments.
The chemical structures of echinacoside, acteoside and isoacteoside are shown below:
Wild-type human neuroblastoma cells (SH-SY5Y) were cultured in Eagle's Minimum essential Medium (EMEM)/Ham's F12 medium (1:1 mixture) (containing 10% FBS, 10 units/ml penicillin, 10 μg/ml Streptomycin). Wild-type mouse neuroblastoma Neuro-2a cells were cultured in minimum essential medium (MEM) (containing 10% FBS, 10 units/ml penicillin, 10 μg/ml Streptomycin).
The medium of the wild-type human neuroblastoma SH-SY5Y cells in Example 1 were switched into chemical defined medium (EMEM/F12 medium (Cat. No. 12500-062), Hepes 5 mM, Glucose 0.6%, NaHCO3 3 mM, Glutamine 2.5 mM, Insulin 25 μg/ml, Transferin 100 μg/ml, Progestrone 20 nM, Putrescine 60 μM, Sodium selenite 30 nM, Heparin 2 μg/ml). Each well contained 1×105 SH-SY5Y cells in 300 μl of culture medium. Thirty minutes later, each well was treated with the test samples A-D given in Table 1 respectively at a concentration of 50 μg/ml for 24 hours. After that, the level of Aβ1-40 in the medium of each well was analyzed by Human Aβ1-40 immunoassay kits (Catalog #KHB3482 Invitrogen).
Human neuroblastoma SH-SY5Y cells cause extracellular accumulation of Aβ.
Referring to
Each well contained 1×105 SH-SY5Y cells in 300 μl of culture medium. After the SH-SY5Y cells of each well were individually treated with the test samples A-D at a concentration of 50 μg/ml, cell homogenate was individually prepared from cells of each well, and the amount of Aβ1-40 in the medium of each well was determined with Human Aβ1-40 immunoassay kits (Catalog #KHB3482 Invitrogen).
Each well contained 1×105 SH-SY5Y cells in 300 μl of culture medium. After the SH-SY5Y cells of each well were individually treated with the test samples A-D at a concentration of 50 μg/ml for 24 hours, the cells were homogenized in homogenize buffer (50 mM Hepes pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 5 μg/ml aprotinin, 10 μg/ml leupeptin). Cell debris was removed by centrifugation. Proteins were separated by SDS-polyacrylamide gel electrophoresis and were then transferred to PVDF membrane, followed by evaluating holoAPP amounts by immunoblot (antiAβ1-17, 6E10 or anti-APP C-terminal antibody)
Mouse neuroblastoma Neuro-2a cells will release Aβ-degrading enzymes in the chemical defined medium, but will not produce detectable amount of Aβ in the medium. The Neuro 2a cells were incubated in the medium for 24 hours, and then the medium were drawn out without cells. The medium were individually treated with the test samples A-D at a concentration of 50 μg/ml and 10 ng synthetic Aβ1-40 for 24 hours, and then the effects of each test sample on promoting the enzymes in the medium were examined. The remaining Aβ1-40 amount of each well was analyzed by Human Aβ1-40 Immunoassay kits (Catalog #KHB3482 Invitrogen), expressing as percentage, as compared with the Vehicle control group. Referring to
Dried Human Aβ1-42 was taken out from the refrigerator and equilibrated to room temperature. Aβ1-42 was dissolved in 1,1,1,3,3,3-Hexa-fluro-2-propanol (HFIP) to a concentration of 1 mM, and was then placed at room temperature for one hour. The Aβ1-42/HFIP solution was aliquoted by Hamilton syringe, and was then dried under a stream of nitrogen gas, followed by storing at a temperature of −20□. Aβ1-42 treated with HFIP was dissolved in PBS, and was vibration-incubated with treatment of each test sample at a concentration of 50 μg/ml and at 4□ for 24 hours to prepare Aβ1-42 oligomers. The level of Aβ1-42 oligomerization was analyzed by thioflavin T fluorescence (Ex=450 nm, Em=482 nm).
In summary, isoacteoside (D) or its pharmaceutically acceptable salts can be used as an active ingredient for inhibiting formation, accumulation or aggregation of Aβ. Therefore, it is expected that isoacteoside (D) can be provided for effectively inhibiting neuronal damage or apoptosis caused by Aβ, and further for retaining, improving or restoring learning or memory abilities. In addition, according to those results above, isoacteoside (D) or the pharmaceutically acceptable salts thereof can be provided for preventing or treating Aβ-associated diseases or conditions, as well as for inhibiting formation, accumulation or aggregation of Aβ.
The described Aβ-associated diseases or conditions comprise but not limit to Alzheimer's disease, mild cognitive impairment, Lewy body dementia, Down syndrome, hereditary cerebral hemorrhage with amyloid (HCHWA) Dutch, Parkinsonism-dementia complex on Guam, Cerebral amyloid angiopathy, inclusion body myositis, frontotemporal dementia, age-related macular degeneration, Pick's disease, and others. In addition, even though the described Aβ is exemplified by Aβ1-40 at most or highly fibrillogenic Aβ1-42, the Aβ can also comprise other peptide fragments.
Among the test samples above, isoacteoside (D) possesses excellent effects on reducing the formation, accumulation or aggregation of Aβ. In the following Examples 7 to 11 illustrated below, male Sprague-Dawley (SD) rats, weighed 250-300 gm, were obtained from BioLASCO Taiwan Co. Ltd. The SD rats were infused intracerebroventricularly with Aβ1-42 to cause neuronal damage, affecting their memory and learning ability, and leading to form senile plaque-like aggregates in the rat's brain, being used as an animal model of Alzheimer's disease. Alzheimer's disease animal model induced by Aβ can be referred to, for example, Nabeshima et al. (Neuroscience Letters (1994) 170: 63-66)(British Journal of Pharmacology (1999) 126: 235-244).
The rats were anesthetized for implantation of an infusion cannula into the left cerebral ventricle thereof. After sewing up the incisions, the rats were returned to their cages.
In Examples 7 to 11, the results were shown in mean±standard deviation (SD) form. Significant difference between the Aβ1-42 control group and the other experimental groups were indicated by *., P<0.05, *.*, P<0.01, and. ***., P<0.001.
All rats were sacrificed and their brains were removed, cut into slices and stained. Neurotransmitters were also tested.
An exploration behavior test apparatus consisted of a box (40 cm×40 cm×40 cm) and a stainless steel bottom board with 16 holes of a diameter of 3 cm, wherein these holes were spaced 4 cm apart and were at a distance of 7 cm from the side edge. The entry counts of each rat were recorded for 10 minutes.
A passive avoidance apparatus is consisted of a light compartment, a dark compartment and a guillotine door between the two compartments.
Each rat was placed in the light compartment while the guillotine door was open. The rats, which entered into the dark compartment within 90 seconds, were selected to be tested in this experiment.
On the training trial, each of the selected rats was individually put in the light compartment while the guillotine door was open. Upon entering the dark compartment, the guillotine door was closed, and an electrical shock was given to the rat in the dark compartment through the floor for five seconds. After that, the rat was removed from the dark compartment and was put back into its home cage.
Twenty four hours after the training trial, the rats were individually put in the light compartment while the guillotine door was open, and the step-through latency (STL) of each rat was recorded.
In this example, the water maze pool was divided into four quadrants, and a hidden platform was located in the fourth quadrant and was submerged 1.0 cm below the surface of water. Each rat was given two trials with an interval of four hours per day, two minutes for each trial to find the hidden platform. The first trial was performed for the spatial performance test. Each rat was allowed to swim a maximum of 120 seconds to find the hidden platform. When successful, the rat was allowed a 30-second rest period on the platform. If unsuccessful within the aborted time period, the rat was given a score of 120 seconds and then put on the platform, allowing a 30-second rest period. The spatial performance tests were performed for four consecutive days. On the fifth day, the hidden platform was taken away from the water maze pool, and each rat was placed in the first quadrant and was given a swimming period of 60 seconds. The time for each rat spent in the quadrant where the hidden platform located was recorded during the probe test.
After the water maze performance tests, all rats were decapitated and their brains were rapidly removed from the skull, and then immunohistochemical staining for Aβ1-42 in the hippocampus section of their brains.
After the water maze performance tests, all rats were decapitated and their brains were rapidly removed from the skull. According to Glowinski and Iversen, the brain cortex and hippocampus were separated, and then each cortex and hippocampus were weighed and were homogenized by addition of 50 mM Na-phosphate buffer (pH 7.8) for analyzing the concentrations of neurotransmitters and the levels of enzymes.
Table 3 and Table 4 respectively show concentrations of neurotransmitter dopamine (DA) and its metabolites (DOPAC and HVA) in brain cortex and hippocampus of the rats according to Table 2. Referring to Table 3 and Table 4, Aβ1-42 decreases DA levels in the cortex and hippocampus of the rats, and the test sample D (isoacteoside) and Aricept reverse the decrease in DA levels in the cortex and hippocampus of rats caused by A□ 1-42.
Since Aβ aggregates cause cell damage, and further lead to various diseases or conditions. According to the results from cell studies in Examples 2 to 6, isoacteoside (D) is effective in inhibiting formation, accumulation or aggregation of Aβ, further preventing Aβ from forming aggregates, and thus isoacteoside can be used in preventing or treating Alzheimer's disease or other Aβ-associated diseases or conditions.
The results from animal studies in Examples 7 to 9 show that isoacteoside possesses significant effects on improving memory or learning deficits induced by Aβ. According to Example 10, isoacteoside is effective in inhibiting Aβ forming plaques by aggregation or in cleaning plaques. Besides, according to Example 11, isoacteoside can reverse the decrease in neurotransmitter levels caused by Aβ1-42, and therefore it is effective in improving deficits of memory or learning abilities. The above cell and animal studies results indicate that isoacteoside or its equivalent pharmaceutically acceptable salt can be administered to a person in a daily therapeutically effective amount of about 0.2 mg/kg to 4 mg/kg (i.e., a recommended dosage of an adult weighted 60 kg of about 12 mg-240 mg), and used as an approach for preventing or treating Alzheimer's disease or other Aβ-associated diseases or conditions.
Although specific embodiments have been illustrated and described, it is still appreciated by those skilled in the art that various modifications may be made without departing from the scope of the present invention, which is intended to be limited solely by the appended claims.
This application claims priority benefit from U.S. Provisional Application No. 61/355,169, filed Jun. 16, 2010.
| Number | Date | Country | |
|---|---|---|---|
| 61355169 | Jun 2010 | US |
