TWI484953B - Uses of polymer composition for the manufacture of a medicament to modulate functions of shc-1/p66 gene - Google Patents

Description

Use of a polymeric composition for the preparation of a medicament for regulating the effect of the SHC-1/p66 gene

The present invention relates to a method of inhibiting SHC-1/p66 against an aging-related disease.

The age of the world's population has increased due to the decline of infertility and the 20-year average life expectancy in the second half of the twentieth century (Centers for Disease Control and Prevention, Morbidity and Mortality Weekly Report, February 13, 2003, 52(06): 101 -106). These factors, as well as the increase in fertility in many countries during the two decades following the Second World War (ie baby boomers), will increase the number of people over the age of 65 between 2010 and 2030. The average life expectancy of the world’s population is expected to increase again to the next decade to 2050. The increase in the elderly population will also increase the demand for public health systems and medical and social welfare. How many chronic diseases that affect the elderly can lead to disability, a decline in quality of life and an increase in the cost of care and long-term care.

The relationship between the symptoms of aging and its biochemistry and neurology has been studied. In particular, a number of biological pathways have been identified to prolong life and prevent multiple age-related diseases (Trinei et al. , "P66Shc Signals to Age" Aging (2009) v. 1 (6) pages 503-510).

The method described in this case is based on the biochemical and neurological basis of aging symptoms. In particular, the method described in this case is in the SHC-1/p66 pathway, the number of reactive oxygen species (ROS) and the oxidative stress, liver function and pathology, and exercise On the motor control, it has a positive effect.

SHC-1/p66 and aging

It has been reported that mice lacking the 66 kD isoform of the SHC (Src Homology and Collagen) protein family have a 30% longer life span than the normal p66 hand and foot. Mice with p66 knockout have a longer life span and are phenotypically normal, fertile and healthy (Migliaccio et al. , "The p66 shc adaptor protein controls oxidative stress response and the life span in mammals . Nature . 1999; 402: 309-313; Alam et al. , Endocr . Relat Cancer . 2009 March; 16(1): 1.). SHC proteins are known as adapter molecules, delivered and located downstream of the growth factor receptors. The role of SHC in the transmission of insulin has also been reported by Giorgetti et al. "Involvement of Src homology/collagen (SHC) proteins in signaling through the insulin receptor and the insulin -like-growth-factor-I-receptor."Eur. J. Biochem. 1994; 223:195-202). Therefore, mice that have been knocked out of p66 have a life span formed by genetically regulating the transmission of insulin and IGF. Extended mammalian experimental examples.

However, the association between p66 and longevity currently has multiple theories and is one of the strongest arguments in support of Harman's "Aging Free Radical Theory" (Harman, D., "A biologic clock: the mitochondria?" ” Journal of the Ameriaan Geriatrics Society 1972; 20: 145-147.). In fact, p66 knockout mice and cell experiments have shown that significantly reducing ROS concentration in cells increases anti-oxidative stress.

P66shc has a completely different function from other SHC protein isoforms. In the reaction to oxidative-promoting substances and cell death substances, p66shc translocates to the granulosa and directly produces active oxides in the granules.

At the molecular level, the SHC, p66Shc, p52Shc, and p46Shc isomers generally have the same amino acid sequence at the C-terminus, and the C-terminus contains the Src homology domain (SH2), which is responsible for phosphorylation of phosphorylated tyrosine. Tyrosine-binding domain (PTB), and a region rich in glycine and proline in a region homologous to the collagen homologous (CH1) domain, homologous to collagen (Pelicci, G. et al. , "A family of Shc related proteins with conserved PTB, CH1 and SH2 regions. Oncogene. 1996; 13: 633-41).

The characteristic of p66Shc is that it has an additional CH region at the N-terminus (Pelicci, G. et al. , "A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction." Cell. 1992; 70:93 -104. Migliaccio, E. et al. , "Opposite effects on the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signaling pathway. EMBO J. 1997; 16: 706-716).

Active oxides, cell pressure and aging

Superoxides and peroxides of active oxides (ROS) perform important signaling functions in many physiological and pathological processes, including cell senescence and organism age (Afanas'ev, Igor , Oxidative Medicine and Cellular Longevity, V. 3 (2010), Issue 2, pages 77-85). ROS is an effective cell death inducer and performs a cell death process (Giorgio et al. "Electron Transfer between Cytochrome c and p66 Shc Generates Reactive Oxygen Species that Trigger Mitochondrial Apoptosis," Cell Vol. 122, 221-233, July 29, 2005).

In particular, SHC-1/p66 is stimulated by ROS message transmission. Conversely, ROS is produced by SHC-1/p66.

Therefore, it can be inferred from many results that SHC-1/p66 is involved in the molecular dynamics of diabetic-induced oxidative stress and oxidant-dependent nephropathy (Menini et al., Diabetes 55: 1642-1657 (2006)). It is also known that SHC-1/p66 is involved in diabetes-related symptoms such as endothelial dysfunction associated with high glucose concentrations, atherosclerosis, diabetic nephropathy, and myocardial lesions (Francia et al., J. Mol. Med. (2009) 87: 885-891 and Berniakovich et al. JBC Vol. 283 No. 49, pp. 34283-34293, December 5, 2008).

Aging and liver

The aged liver exhibits increased cellular degradation compared to younger livers. Specifically, aged liver cells exhibit metabolically attenuated granules and affect the function and morphology of mitochondria (Sastre et al. "Aging of the Liver: Age-Associated Mitrochondrial Damage in Intact Hepatocytes," Hepatology November. (1996) Pp. 1199-1205 and Koch et al., "Role of the life span determinant P66 ^shcA in ethanol-induced liver damage," Laboratory investigation (2008) 88, 750-760). For example, gluconeogenesis and ketogenesis may be reduced, but granulocyte volume may increase. Degeneration of hepatocytes can be detected by pathological cell swelling or formation of "foamy" or fatty degeneration (also known as fat change, fat degradation or fat cell degradation).

The expanded cells are typically two to three times the volume of adjacent hepatocytes and are characterized by a fine bundle of clear cytoplasm on H&E stained sections. Depending on the cytoplasm and nucleus, expanded cells can be distinguished from cells that resemble fat cells. The nucleus of the expanded cell is located in the center of the cell (the cell nucleus similar to the fat cell is located around the cell). Moreover, the expanded cells have a (small) collapsed nucleus or a nucleus that is undergoing rupture (ie, is disintegrating). Cells with swelling and degeneration are similar in cytoplasm to small bundles or spider webs, while cells resembling fat cells are cytoplasm with clear cytoplasm or vacuoles.

Steatosis means that the lipids in the cells are abnormally retained, and the normal process of synthesis and removal of the triglyceride is damaged. Steatosis is thought to be associated with oxygen free radicals causing hepatocyte enlargement (Del Monte, "Swelling of hepatocytes injured by oxidative stress suggests pathological changes related to macromolecular crowding" Medical Hypotheses (2005) 64, 818-825).

The effect of aging on motor skills and motor deficits

As it ages, sensory motor control and function will gradually decline. Control of fine movements, gait and balance decline will affect the daily activities of the elderly and their independence of life (Yankner BA, Lu T, Loerch P.The Aging brain.Annu Rev Pathol 2008;3:41-66.;Twohing,Jet et al.,"Age-dependent maintenance of motor control and corticostriatal innervation by Death receptor 3.”J.Neurosci.2010.30:3782-3792.) The cause of motor deficits is multifactorial, and changes in sensory receptors, muscles, and peripheral nerves can affect the decline of the central nervous system.

For the elderly, motor performance deficits are clearly due to central and peripheral nervous system and dysfunction of the neuromuscular system (Seidler, R. et al. , “Motor control and aging: Links to age-related brain” Structural, functional, and biochemical effects. "Neuroscience and Biobehavioral Reviews 30 (2010) 721-733). Defects in motor performance, including physical coordination difficulties in the elderly compared to young people (Seidler, RD et al., “Changes in multi-joint performance with age.” Motor Control. 2002; 6(1): 19-31.), Increased motor variability (Contreras-Vidal et al., "Elderly subjects are impaired in spatial coordination in fine motor control," Acta Psychol (Amst). 1998 Nov; 100 (1-2): 25- 35; Darling et al., "Control of simple arm movements in elderly humans." Neurobiol Aging, 1989 Mar-Apr; 10(2): 149-57), slow motion (Diggles-Buckles V. "Age-related slowing. In: Stelmach GE, Homberg V, editors. "Sensorimotor impairment in the elderly. Norwell, MA: Kluwer Academic; 1993.), gait and balance difficulties, etc. (Tang PF, Woollacott MH. "Balance control in the elderly." In : Bronstein AM, Brandt T, Woollacott MH, editors. Clinical disorders of balance, posture and gait. London: Arnold; 1996.).

These deficiencies negatively affect the ability of older people to perform their daily functional activities. The gait and balance are particularly noticed, and the elderly are injured and sick. The main reason is that 20 to 30% of the elderly who fall will suffer moderate to severe injuries, which limits their activities and lowers their quality of life. There are therefore a number of factors that significantly extend the duration of exercise with age. As you get older, your exercise slows down by about 15 to 30% (cf. Diggles-Buckles, 1993). Older people exercise as a strategy to emphasize the correctness of movement but slow down the speed of movement. Slow information processing can also affect non-specific performance, such as global neural noise or other synaptic changes.

Moreover, the link between gait patterns and aging has been described, especially in aging groups, where gait shortens and slows down (Wolfson, L., et al., (1990) "Gait assessment in the elderly :A gait abnormality rating scale and its relation to falls." J. Gerontol. 45: M12-19). This change in human identifiable gait has been found to be directly related to aging-dependent gait changes in rodents (Klapdor, K. et al., (1997) "A low-cost method to analyze Footprint patterns." J. of Neuroscience Methods. 75: 49-54 and Hilber et al., (2001) "Motor skills and motor learning in lurcher mutant mice during aging." Neuroscience. 102: 615-623).

The present invention relates to a method for treating one or more symptoms of a SHC-1/p66-related disease, a method for inhibiting ROS production, or a method for preparing a medicine for use in the above treatment. According to an embodiment of the present invention, there is provided a method of treating one or more symptoms of a SHC-1/p66-associated disease, comprising administering to a mammal in need thereof a monomer unit having the following formula (I), and/or a pharmaceutical thereof Polymeric compositions that can tolerate salts, solvates, or precursor drugs:

[Wherein, R ₁ is and R ₂ is hydrogen, alkyl, or _{acyl; R 3, R 4, R} 5, R 6 and R ₇ are each hydrogen, hydroxy, alkoxy, or acyl; R ₈ is Hydrogen or a saccharide moiety, and the polymerization degree of the above monomer is 2 to 30, and the average molecular weight of the polymer is 600 to 10,000.

In one embodiment of the invention, the above method relates to treating one or more symptoms of a disease affected by SHC-1/p66 expression or activity. In one embodiment, "SHC-1/p66-related disease" means a disease that improves symptoms by reducing the performance or activity of SHC-1/p66. Such diseases include aging, diabetes, and reperfusion injury after ischemia. The method of the present invention relates to treating one or more symptoms of aging, such as cell degeneration, hepatocyte enlargement, mitochondrial dysfunction, age-related deficits in movement, and/or reduction in gait width; Or more than one symptom, such as high glucose concentration-related endothelial dysfunction, atherosclerosis, nephropathy, and/or cardiomyopathy; and one or more symptoms of treating reperfusion injury after ischemia, the above diseases Will include an increase in active oxides and cell death.

In one embodiment, the method of the invention comprises administering a composition comprising BEL-X and/or a pharmaceutically acceptable salt, solvate, or prodrug thereof. In one embodiment, the above composition is administered in an amount of 50 to 1,500 mg per kg per day. the amount.

The detailed description of the present invention and its specific embodiments are described and illustrated with reference to the drawings.

Figures 1A to 1C show the results of hepatoxylin and eosin staining of liver samples after formaldehyde fixation and paraffin embedding. Figure 1A shows young mice (two months old). Liver section samples, Figure 1B shows liver section samples of aged, untreated mice (20 months old), and Figure 1C shows aged mice (20 months old) orally administered 1000 mg/kg/day of BEL-X A sample of the liver slice after the drug.

Figure 2 is a graph showing the expression of SHC-1/p66 detected by RT-qPCR, showing that BEL-X reduces the performance of SHC-1/p66.

Figure 3 shows the detection of SHC-1/p66 protein in the liver of mice using Western blotting.

Figure 4 shows the effect of detecting BEL-X drug treatment on the amount of ROS produced in cells.

This panel shows that human hepatoma cell line Huh7 is treated with H ₂ O ₂ to induce ROS production (Figs. 4A and 4C). This cell was treated with BEL-X drug after induction with H ₂ O ₂ (Fig. 4B and 4D). Figures 4A and 4C show cells under microscope observation with similar numbers of cells on each slide. These cells were visualized by immunofluorescence in Figure 4B, and H ₂ O ₂ induced a large amount of ROS production. However, in BEL-X drug-treated cells, ROS production was significantly reduced, indicating that BEL-X reduced ROS. Production to avoid cell carcinoma (Fig. 4D).

Figure 5 is a graph showing the reduction of ROS in cells. Human hepatoma cell line Huh7, human rectal cancer cell line HRT-18 and human normal skin cell line WS1 were detected. All of the different cell types tested showed that cells treated with BEL-X significantly reduced ROS produced by H ₂ O ₂ induction. * in the figure indicates p < 0.05.

Figures 6A-6C show the results of ¹³ C nuclear magnetic resonance spectroscopy of proanthocyanidins purified from ram-X from Boehmeria nivea (L.) Gaud.

Figures 7A-7B show the bonding relationship between two monomers of proanthocyanidins.

For the purposes of further explanation, the following detailed description of several specific forms are provided to provide a complete understanding of the disclosed embodiments. However, one or more embodiments of these embodiments may be practiced without these specific details. On the other hand, conventional structures and devices are drawn to simplify the performance of the schema.

Embodiments of the present invention comprise a monomer of formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof thereof: [Wherein, R ₁ is and R ₂ is hydrogen, alkyl, or _{acyl; R 3, R 4, R} 5, R 6 and R ₇ are each hydrogen, hydroxy, alkoxy, or acyl; R ₈ is Hydrogen or a saccharide moiety].

In the above formula (I), the monomer in the polymer compound may have one or more of the following characteristics: R ₁ and R ₂ are each hydrogen; R ₃ and R ₇ are each hydrogen; and R ₄ , R ₅ and R ₆ are respectively Is a hydroxy or alkoxy group; and R ₈ is hydrogen.

In the polymerized compound, a bond between any two atoms of different monomer units, such as a C4-C8 bond (eg, a carbon atom C4 of one monomer unit is bonded to a carbon atom C8 of another monomer unit) , a C4-C6 bond (eg, a carbon atom C4 of one monomer unit is bonded to a carbon atom C6 of another monomer unit), or a C2-O7 bond (eg, a carbon atom C4 of one monomer unit and another monomer unit) The oxygen atom O7 bonds), the monomer units can be covalently linked to each other. In one embodiment, all of the monomer units are covalently bonded to each other by C4-C8 bonding. In another embodiment, all of the monomer units are covalently bonded to each other by C4-C6 bonding. It should be noted that the atomic number of the cyclic compound is well known and customary for chemical structure nomenclature. The atomic number of the core structure of the polymer compound of the formula (I) is shown below:

In one embodiment, the compositions of the present invention comprise low oligomers of monomeric units of formula (I), such as dimers, trimers, and tetramers. In other embodiments, the purified composition comprises a mixture of polymeric compounds of varying degrees of polymerization of the monomeric units of formula (I).

The degree of polymerization of the monomer unit may range from 2 to 30, preferably from 3 to 20. The average molecular weight is preferably in the range of from 600 to 10,000.

In one embodiment, the composition of the present invention (including BEL-X) may comprise a A mixture of the above monomer units of the formula (I). In one embodiment, the polymer can be homopolymers. In one embodiment, the composition can comprise a mixture of different homopolymers. In one embodiment, the polymer can be a heteropolymer comprising a plurality of different monomeric compounds as shown in formula (I).

The term "isolation preparation" means a composition containing one or more of the above-mentioned polymerized compounds which are obtained by partitioning from a natural resource or a synthetic mixture.

The term "alkyl" means a straight or branched hydrocarbon having from 1 to 10 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, or a t-butyl group, but are not limited thereto. The term "mercapto" means -C(O)-alkyl or -C(O)-aryl. Examples of the fluorenyl group include -C(O)-CH ₃ or -C(O)-ph, but are not limited thereto. The term "alkoxy" means -O-alkyl. Examples of alkoxy groups include -OCH ₃ or -OC ₂ CH ₃ .

The alkyl group described herein may be substituted or unsubstituted. Examples of the substituent include halogen, hydroxy, amine, cyano, nitro, thiol, alkoxycarbonyl, decylamino, carboxy, alkanesulfonyl, alkylcarbonyl, carbamido, Carbamyl, carboxyl, thioureido, thiocyanato, sulfonamido, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl a group, a cycloalkyl group, a heterocyclic group, wherein the alkyl group, the alkenyl group, the alkynyl group, the alkoxy group, the aryl group, the heteroaryl group, the cyclic group, and the heterocyclic group are optionally an alkyl group, an aryl group or a heterocyclic group. Substituted by a halogen, a hydroxyl group, an amine group, a thiol group, a cyano group or a nitro group.

The term "glycosyl ligand" means a carbohydrate group. Can be monosaccharides (such as allose, altrose, glucose, mannose, gulo Gumose, idose, galactose, talose, ribuose, psicose, fructose, sorbose, or tagatose Tagatose)), disaccharides (such as sucrose, lactulose, lactose, maltose, trehalose or cellobiose), oligosaccharides (containing 3-10 monosaccharides), or polysaccharides ( Contains more than 10 monosaccharides).

Further, the monomer represented by the formula (I) may contain a flavonoid. Flavonoids may include catechin, epicatechin, epiafzetechin, gallocatechin, gallococcal catechin ( Galloepicatechin), epigallocatechin, gallate gallates, flavonols, flavandiols, leucocyanidins, or procyynins. In one embodiment, the monomer of formula (I) may comprise flavan-3-ol or flavanones derivatives. Specific examples include 3-flavanol, 3,4-flavanol, catechin ((2R, 3S) and (2S, 3R)) and epicatechin Prime ((2S, 3S) and (2R, 3R)).

The above polymeric compound comprises the above monomers or polymerized compounds, as well as implementable salts, precursors and solvates. Salts, for example, are formed between an anion of a polymeric compound and a positively charged group such as an ammonium ion. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, succinate, malate Ionic, tosylate ion, tartrate ion, fumarate ion, glutamate ion, alduronate ion, lactate ion, glutarate ion, and maleate ion. Similarly, a salt may also be formed between a cation on a polymeric compound and a negatively charged group such as a phenolic or carboxyl group. Suitable cations include sodium ions, potassium ions, magnesium ions, calcium ions, and ammonium ions. The compounds may also be in the form of prodrugs and solvates. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives which provide the active compound when administered to an individual. Solvates represent complexes of active compounds with pharmaceutically acceptable solvents. Examples of the pharmaceutically acceptable solvent include water, ethanol, isopropanol, ethyl acetate, acetic acid or ethanolamine.

The monomer of the polymeric compound contains an asymmetric center. Thus racemates and racemic mixtures, single pairs of enorganizers, individual diastereoisomers, and mixtures of diastereoisomers can occur. The isomeric forms described herein are all considered. In one embodiment, the monomer comprises an (R) or (S) optical isomer at the C4 position.

The invention may also be used as a method for inhibiting SHC-1 expression, inhibiting hepatocyte enlargement, inhibiting ROS production, or reducing the occurrence or severity of age-related motor deficits by using the above The pharmaceutical composition obtained by isolating and mixing with a pharmaceutically acceptable carrier is administered to a subject in need thereof.

The invention of the present invention encompasses the use of the above-described separation preparation, a method for treating an SHC-1/p66-related disease, or a preparation for the above-mentioned improvement, treatment, and promotion of medicine.

In another aspect, the compositions of the present invention can be administered to an individual in a food product, such as a nutritional product, a nutritional preparation, a health food, or a supplement.

Various embodiments of the present invention are described in detail below. From the instructions and rights Other features, objects, and advantages of the invention will be apparent from the claims.

The polymeric compound of formula (I), as described above, can be administered to a subject in need thereof to treat SHC-1/p66 related diseases and to inhibit ROS production.

The composition of the present invention can be extracted from plants or artificially modified or synthesized. In one embodiment, the plant may comprise belonging to the family Leguminosae, Crassulaceae, Combretaceae, Asclepiadaceae, Rosaceae, Lamiaceae. , Polygonaceae, Ericaceae, Pinaceae, Vitaceae, or Urticaceae, preferably Urticaceae, Boehmeria nivea (L) .) Gaud). The extractable plant parts may comprise roots, stems, leaves and/or fruit parts.

The extraction method is described in U.S. Patent Publication No. US 2010/0168221 and U.S. Patent Application Serial No. The extract may be further partially or completely purified as needed.

The term "alleviation" or "inhibition" includes alleviating the severity or occurrence of symptoms. In one embodiment, the mitigation or inhibition system measures the percentage of the untreated control group and/or the untreated young control group that are comparable to the age. In one embodiment, the occurrence of symptoms can be reduced by at least 25%, 50%, or 75% compared to an age-matched control group. In one embodiment, the severity of a particular symptom can be reduced by at least 25%, 50%, or 75-99%. A 100% reduction in the severity of the symptoms indicates that the symptoms no longer exist or can be detected. In one embodiment, "alleviation or inhibition" can measure a "fold" increase in the occurrence or severity of the condition relative to young mice. Preferably, the fold increase in the onset or severity of the condition is at least 25% relative to younger mice.

The term "significant" is a significant value for general statistics. Statistical significance can be confirmed by generally known methods.

The term "significant" means using statistical methods to compare treatment groups with untreated groups or different age groups.

The term "treating SHC-1/p66 related diseases" may mean treating one or more symptoms of a disease affected by SHC-1/p66 expression or activity. In particular, "SHC-1/p66-related diseases" are diseases in which symptoms are alleviated by reducing the expression or activity of SHC-1/p66. The disease includes aging, diabetes, and post-ischemic reperfusion injury.

In particular, the invention relates to the treatment of one or more symptoms of aging, such as cellular degeneration, hepatocyte swelling, mitochondrial dysfunction, aging motor deficit (age) -related motor deficits) and/or reduced stride length; etc.; one or more symptoms of treating diabetes, such as high glucose associated endothelial dysfunction, arteries Aceogenesis, nephropathy, and/or cardiomyopathy; and one or more symptoms associated with treating reperfusion injury after ischemia, including increased active oxides and cell death.

The term "aging motor deficit" may include a decrease in the speed of the activity, a decrease in the width of the gait, or a decrease in the range of the range of motion. A reduction in aging motor deficits may be indicative of delaying the onset of aging motor deficits or reducing the severity of aging motor deficits. In one embodiment, the occurrence of human aging motor deficits can be delayed for at least 5 years, 10 years, 20 years, 30 years, 40 years, or 50 years. In an embodiment, The reduction in the severity of aging motor deficits may be at least 50% to 100% improvement in the range of dynamometer speed and/or activity compared to the severity of motion defects in an untreated, age-matched control group (including The value).

The individual can be an animal, more specifically a mammal, more specifically a mouse, a rat, a rabbit, a sheep or a human.

Formulation

For carrying out the method of the present invention, a composition comprising one or more of the above polymeric compounds or a constituent monomer thereof can be administered parenterally, orally (e.g., p.o.), nasally, rectally, topically, or buccally. The term "parenteral tract" means subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intramedullary, intralesional or intracranial injection and any suitable injection technique.

The sterile injectable compositions can be a non-toxic parenterally acceptable diluent or a solution or suspension in a solvent such as a solution in 1,3-butanediol. Acceptable carrier and solvent are mannitol and water. Moreover, it is known to use a fixed oil as a solvent or a suspension medium (for example, a synthetic mono- or di-glyceride). Fatty acids such as oleic acid and its glycerol derivatives are useful in the preparation of injectables, natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in the form of their polyethylene oxides. These oil solutions or suspensions may also contain long chain alcohol diluents or suspensions also containing long chain alcohol diluents or dispersants, carboxymethylcellulose, or similar dispersing agents. Other surfactants commonly used in pharmaceutically acceptable solid, liquid or other dosage forms such as Tween or Spans or other similar emulsifiers or bioactive accelerators can also be used in the formulation.

Oral compositions can be in any orally acceptable dosage form including capsules, troches, emulsions or aqueous suspensions, dispersions and solutions. When it is a tablet, The commonly used carriers are lactose and corn starch. Lubricants such as magnesium stearate are also typically added. Effective diluents for oral capsules include lactose and dried corn starch. Oral water-soluble suspensions or emulsions, the active ingredient being suspended or dissolved in the oil phase in association with an emulsifier or suspension. Sweeteners, flavoring agents, or coloring agents can also be added if desired.

Nasal vaporized spray or inhalation compositions can be prepared according to conventional pharmaceutical formulation techniques. For example, a solution such as a solution in physiological saline, benzyl alcohol or other suitable preservative, absorption enhancer may be added to enhance bioavailability, such as fluorocarbons, and/or other co-solvents or dispersants conventional in the art. Compositions having more than one active compound can also be administered rectally in the form of a suppository.

The composition of the present invention can be administered at a dose of 50 to 1,500 mg/kg per day. In one embodiment, the composition of the present invention is administered at a dose of 250 to 1,000 mg/kg per day.

A preferred dosage range for increasing the width of the gait is 50 to 1000 mg/kg per day.

The preferred dosage range for reducing hepatocyte enlargement is 100 to 1000 mg/kg per day.

The carrier in the pharmaceutical composition must be &quot;acceptable&quot;, which means that it must be compatible with the active ingredient of the composition (preferably to stabilize the active ingredient) and not deleterious to the subject being treated. More than one co-solvent can be a pharmaceutical excipient for delivery of the active compound. Examples of other carriers include colloidal cerium oxide, magnesium stearate, cellulose, sodium laurate, D&C Yellow #10.

In another aspect, the composition can be consumed in a food product, such as a nutritional product, a nutritional preparation, a health food or a supplement.

The potency of the compounds can be determined by testing in vitro or in vivo assays. For example, the compounds of the present invention can be initially screened by in vitro assay to test the biological activity of the compound in relation to oxidative stress. Compounds which have been shown to be highly potent by preliminary screening can be further evaluated by in vivo assays known in the art to assess the activity of the compound to reduce or inhibit the expression or transcription of aging genes (e.g., SHC-1/p66). .

The following specific examples are for illustrative purposes only and are not intended to limit the disclosure. Without further elaboration, it is believed that one skilled in the art can use the invention to the fullest extent. The disclosures of this reference are hereby incorporated by reference in entirety in entirety.

Example

Example 1: Manufacture and characteristics of BEL-X

Preparation of extract of Boehmeria nivea (L.) Gaud containing Proanthocyanidins

method 1

Wash the roots and stems of the mountain nettle ( Boehmeria nivea (L.) Gaud) and dry in the natural environment. The dried ramie was cut into 5 mm thick sheets and stored at 4 °C. The stored mountain ramie powder is then passed through a 20 mesh sieve. The sieved powder was collected, added with 95% ethanol (1:10 w/v), heated to reflux for 2 hours (two times), and then cooled to room temperature. This extraction solution obtained by heating and cooling to room temperature was centrifuged again. The filtered solution was vacuum dried at a temperature below 40 ° C and then freeze dried. The freeze-dried extract is a pharmaceutical composition containing proanthocyanidins.

Method 2

As in Method 1, the mountain ramie powder stored at 4 ° C was passed through a 20 mesh sieve. The sieved powder was collected, RO water (1:10 w/v) was added, and the mixture was heated under reflux for 2 hours (two times), and then cooled to room temperature. This extracted solution obtained by heating and cooling to room temperature was added to an aqueous ethanol solution (95-50%) and mixed. After the extraction solution is cooled and precipitated, the upper layer solution is subjected to centrifugal filtration. The filtered solution was vacuum dried at a temperature below 40 ° C and then freeze dried. The freeze-dried extract is a pharmaceutical composition containing proanthocyanidins.

Purification of mountain nettle extract

method 1

Solvent extraction 1

The cumin containing procyanidins was added to hexane (1:10 w/v) and heated under reflux for 6 hours to remove the lipids in the extract. The solid extract is dissolved in a 70% aqueous methanol solution and/or a 0.3% vitamin C solution and vacuum dried at a temperature below 40 ° C to remove the solvent. The extract was then added to chloroform (extract: chloroform = 1:1 v/v) and shaken for 30 minutes (several extracts were carried out). The obtained aqueous layer was added to ethyl acetate (extract: ethyl acetate = 1:1 v/v) and shaken for 30 minutes (several extracts). The resulting aqueous layer was vacuum dried at a temperature below 40 ° C and then freeze dried.

Method 2

Solvent extraction 2

The mountain ramie containing proanthocyanidin is dissolved in water/ethanol solution, dried under vacuum at a temperature lower than 40 ° C to remove ethanol, and hexane (1:10 v/v) is added for 30 minutes. Several extracts) to remove lipids from the extract. The obtained aqueous layer was added to ethyl acetate (aqueous layer: ethyl acetate = 1:1 v/v) and shaken for 30 minutes (this step was subjected to multiple extractions). The resulting aqueous layer was added to 1-butanol (1:1 v/v) and shaken for 30 minutes (this step was subjected to multiple extractions). The resulting aqueous layer was vacuum dried at a temperature below 40 ° C and then freeze dried.

Method 3

Gel permeation chromatography

The partially purified proanthocyanidin-containing ramie from method 1 was subjected to gel permeation chromatography (4 cm diameter x 45 cm long Sephadex LH-20), and was extracted using a solvent having different polarities to remove impurities. Separation. 2.5 g of partially purified ramie was dissolved in 0.5 ml of 95% ethanol, added to a gel filtration column, and then continuously flushed with a series of solvents. Collect the extracts eluted with different solvents. The solvent was 300 ml of 95% ethanol, 300 ml of 95% ethanol/methanol (1/1, v/v), 300 ml of methanol, 300 ml of 50% aqueous methanol solution, 300 ml of 50% aqueous acetone solution, and 300 ml of acetone. Except that the extract was washed with 300 ml of 95% ethanol, all other extracts were vacuum dried at a temperature below 40 ° C and then freeze dried. The lyophilized material was stored at -20 ° C until use. The physicochemical properties of the freeze-dried ramie with partially purified and/or purified proanthocyanidins were analyzed. The freeze-dried extract has a partially purified and/or purified proanthocyanidin component.

¹³ C and ¹ H nuclear magnetic resonance spectroscopy

The purified proanthocyanidin samples were detected by ¹³ C NMR spectroscopy and ¹ H NMR spectroscopy. ¹³ C NMR spectroscopy is shown in Figures 6A to 6C, in which there is only a double peak at the position of 145.2-145.7 ppm. Therefore, the monomer has anthocyanins, but no delphindin, that is, the B ring has three OH groups, and the results are consistent with the EGA/MS analysis results. In Fig. 6, R ₁ = H or OH, and R ₂ = H, OH or OCH.

According to the results of ¹³ C NMR spectroscopy and ¹ H NMR spectroscopy, the bond between two adjacent monomers of proanthocyanidin mainly occurs in C4 and C8 carbon-carbon bonds. The C4, C6 carbon-carbon bonds and the C2, C7 oxygen bond units are shown in Figures 7A and 7B.

Example 2 Effect of BEL-X

Experimental fauna : Male and female C57BL/6 species were used. Divided into 4 groups:

(1) Unfed male rats (control).

(2) Male rats fed BEL-X at 9-20 months of age.

(3) Female mice that were not fed (control).

(4) Female rats fed BEL-X at 9-20 months of age.

Preparation and dosage of the composition: The BEL-X obtained in the above method 3 was dissolved in distilled water, and the mice were fed with 1000 mg/kg daily.

Sacrifice mice to 20 months of age. Liver tissues and serum were collected for pathological and biochemical activity analysis.

Effect of A.Bel-X on aging liver

The mice were measured for body weight and liver weight at the time of sacrifice. The liver was collected and fixed with formalin and embedded in paraffin. Liver sections were stained with hemotoxylin and eosin. The histological evaluation of hepatocytes was evaluated by microscopic observation and histopathological evaluation of fixed and stained liver sections.

The results are shown in Figures 1A, 1B, and 1C. Compared with the liver of young mice (2 months old) and aged mice (20 months old), the liver of aged mice was confirmed to have hepatocyte enlargement (compare Fig. 1A, Fig. 1B). The hepatocyte enlargement of young mice and BEL-X-treated old mice was compared. The BEL-X-treated group showed little or no hepatocyte enlargement, similar to the liver of young mice (Fig. 1C). . The detailed results are shown in Table 1 below:

Specifically, for the control group (not administered) of male mice, 12 elderly mice were evaluated, and 9 of them showed hepatocyte enlargement and degeneration. Therefore, the "hepatic abnormality ratio" is the percentage of the number of mice showing hepatocyte enlargement in the total number of mice evaluated. According to this data, the higher the proportion of liver abnormalities, the more people with hepatomegaly.

Table 1 demonstrates that BEL-X administration significantly reduces the incidence of hepatocyte enlargement in male and female mice by approximately 50-75%.

B. Detection of SHC-1/p66 by RT-qPCR

RT-PCR : Total RNA was extracted from frozen mouse livers according to the Trizol RNA isolation protocol. DNA synthesis was performed using random primers and SuperScript II kits.

The salt-free forward primer for the target gene SHC1 isoform p66 was 5'-CGGAATGAGTCTCTGTCATCGCTGGA (SEQ ID NO: 1); the reverse primer was 5'-CGCCGCCTCCACTCAGCTTGTT (SEQ ID NO: 2).

The forward primer for the endogenously controlled housekeeping gene GAPDH was 5'-GAAGGTGAAGGTCGGAGT (SEQ ID NO: 3); the reverse primer was 5'-GAAGATGGTGATGGGATTTC (SEQ ID NO: 4).

To detect the degree of expression of the target gene in different liver tissues, 1 μl of total cDNA was added to 9 μl of the real-time PCR mixing reagent (Roche Molecular Biochemica 1s) in a glass capillary. The procedure was carried out using a four-step experiment: (i) denaturation step (95 ° C, 20 seconds); (ii) amplification and quantification steps, repeated cycles of SHC-1/p66 60 times, repeated cycles of GAPDH 40 times (95 ° C, 20 seconds; 62 ° C for 20 minutes for SHC-1/p66, 60 ° C for 20 seconds for GAPDH; 20 seconds for 72 ° C for 20 seconds; and 82 ° C for 20 seconds for SHC-1/p66 and GAPDH; Measurement); (iii) melting curve step (measuring at a heating rate of 0.1 ° C/sec, heating at 60-95 ° C, and continuous fluorescence measurement); (iv) cooling step, to 40 ° C.

The C _T value of the RT-PCR product was calculated using LightCycler Software 3.5 (Roche Molecular Biochemicals). The degree of expression of the relevant SHC-1/p66 was analyzed by a comparison of the C _T method (Schmittgen, TD & KJ Livak. (2008) "Analyzing real-time PCR data by the comparative CT method." Nature Protocol. 3: 1101-1108.).

Results: As shown in Fig. 2, the BEL-X administration group significantly reduced the expression of SHC-1/p66 in comparison with the expression of SHC-1/p66 in mice not administered with BEL-X. It is known that mice and cells lacking p66 exhibit a decrease in ROS concentration and an increased resistance to oxidative stress, and thus a decrease in SHC-1/p66 expression can reduce ROS production and thereby reduce oxidative stress.

C. Effect of aging and BEL-X on SHC-1/p66 protein

Western spotting method : 10 times the volume of RIPA buffer was added to the liver tissue, homogenized, and the tissue fragments were removed by centrifugation, and the protein in the solution was separated by SDS-PAGE. The protein in SDS-PAGE was transferred to a PVDF membrane (Millipore) and cultured with an anti-SHC-1/p66 antibody and an anti-GAPDH antibody, respectively. Specific protein performance was detected and analyzed using a UVP biospectrometer.

Results: As shown in Figure 3, Figure 3 demonstrates that the protein concentration of SHC-1/p66 in aged mice is approximately twice that of young mice. However, in the aged mice administered with BEL-X, the amount of SHC1/p66 protein expression in the liver was significantly reduced (the protein concentration was only increased by about 64%).

Example 3 Evaluation of motor skills

Experimental fauna : Male and female C57BL/6 species were used. Divided into 4 groups:

(1) Unadministered male rats (control).

(2) Male rats fed BEL-X at 12-20 months of age.

(3) Female rats not administered (control).

(4) Female rats fed BEL-X at 12-20 months of age.

BEL-X was dissolved in distilled water, and mice were fed 250 mg/kg daily.

Motor Skills : Studies have compared rodent gait patterns with changes in age and changes in walking patterns in older people (Wolfson L, Whipple R, Amerman P, Tobin JN. (1990) Gait assessment in the elderly: A gait abnormality rating Scale and its relation to falls.J Gerontol.45: M12-19.). Therefore, a modified gait test method is used (Klapdor, K. et al., (1997) A low-cost method to analyze footprint patterns. Journal of Neuroscience methods. 75: 49-54; Hilber, P. and J. Caston (2001) Motor skills and motor learning in lurcher mutant mice during aging. Neuroscience. 102: 615-623.) Measurement of gait width of mice of different ages. The paws of the mice were stained with foot dye and the footprints were evaluated on 40 x 10 cm paper. Each mouse was analyzed for gait width at least 3 times.

Results: As shown in Table 2, the gait of aged mice was significantly shorter than that of young mice, regardless of male or female rats. However, older mice fed BEL-X have almost the same gait width as young mice. Therefore, a daily BEL-X treatment of 250 mg/kg can help elderly people maintain their gait width.

Comparing mice to human lifespan, mice have a significantly shorter lifespan than humans. According to the Jackson Laboratory report, mice aged 3 to 6 months can be equivalent to humans 20 to 30 years old, while mice aged 16 to 24 months can be equivalent to humans 56 to 69 years old. therefore, According to the above experimental data, the treatment of BEL-X can provide an elderly person aged 56-69 with a gait width comparable to that of a young man aged 20-30.

Example 4 Effect of BEL-X Treatment on ROS Generation

Cell culture : Human liver cancer cells (Huh7) were cultured in MEM, and human rectal adenocarcinoma cells (HRT-18) and human skin cells (WS1) were cultured in DMEM, respectively. The medium was supplemented with 1% Penicillin/Streptomycin mixture and 1% non-essential amino acid, 1% GlutaMAX-I, 1 Mm sodium pyruvate and 10% fetal bovine serum. The cells were cultured in a 37 ° C, 5% CO ₂ incubator.

ROS induction : The cells were seeded in 24-well plates and cultured for 24 hours, after which 800 μM of H ₂ O _{2 was added} and cultured for 1 hour to induce production of ROS in the cells.

BEL-X treatment : After the above H ₂ O ₂ induction, BEL-X was added to the above cells for 24 hours to test the effect of BEL-X on ROS.

Detection of ROS production : 10 μM of CM-H ₂ DCFDA was added to the above cells, and cultured at 37 ° C for 45 minutes. After culturing with the fluorescent carbon needle, the cells were washed twice with PBS, and the production of ROS was observed directly under a fluorescence microscope, or the fluorescence intensity was measured by a cytolysis cell and a spectrophotometer to obtain a ROS production.

Results : As shown in Figures 4B to 4D, Huh7, a human hepatoma cell induced by H ₂ O ₂ to induce ROS production, showed a large amount of ROS production (e.g., green fluorescent spot in Fig. 4B). However, cells that induced ROS production showed almost no ROS production by BEL-X treatment (as in the green fluorescence of Figure 4D).

Moreover, as shown in Fig. 5, BEL-X significantly reduced H ₂ O ₂ -induced ROS in three cell types (human hepatoma cell Huh7, human rectal adenocarcinoma cell line HRT-18, human skin cell WS1). Production (*p<0.05). Therefore, comparing these test results with previous evaluations, it was shown that BEL-X affects SHC-1, while SHC-1 is subsequently affected by the oxidative pressure pathway. Therefore, BEL-X can reduce oxidative stress in various cell types such as hepatocytes, colon cells, and skin cells.

It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments disclosed herein. This disclosure is intended to be illustrative of the embodiments of the invention

<110> Industrial Technology Research Institute

<120> Method for inhibiting SHC-1/P66 against aging-related diseases

<130> 2668-0127PUS1

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<170> PatentIn version 3.5

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<213> Artificial sequence

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<223> Forward introduction of SHC1 isomer p66

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<212> DNA

<213> Artificial sequence

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<223> Reverse Initiation of SHC1 Isomer p66

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<212> DNA

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<223> GAPDH Forward Initiator

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<223> GAPDH reverse primer

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Claims (7)

A use of a polymeric composition for the manufacture of a medicament for modulating the action of the SHC-1/p66 gene, wherein the polymeric composition comprises a monomer having the formula (I): [wherein R ₁ and R ₂ are each hydrogen; R ₃ , R ₄ , and R ₇ and R ₈ are each hydrogen; R ₅ and R ₆ are each a hydroxyl group], and the polymerization degree of the above monomer is 2 to 30. The polymer has an average molecular weight of 600 to 10,000. The use according to claim 1, wherein the SHC-1/p66 gene acts to cause aging, diabetes or post-ischemic reperfusion injury. The use according to the first aspect of the patent application, wherein the SHC-1/p66 gene causes cell degeneration, hepatocyte enlargement, granulocyte dysfunction, age-related motor deficits, steps Reduced stride length, high glucose associated endothelial dysfunction, atherosclerosis, nephropathy or myocardial lesions. The use according to any one of claims 1 to 3, wherein the medicament is administered by injection, orally, nasally, rectally, topically or orally. The use according to any one of claims 1 to 3, wherein the polymeric composition is in the form of a nutraceutical, a nutritional preparation, a health food or a supplement. The use of any one of claims 1 to 3, wherein the drug system Invest in mammals. The use according to claim 6, wherein the polymeric composition is administered in an amount of from 50 to 1500 mg per kg per day of the mammal.