WO2016015203A1 - 丁烯基苯酞的用途、其使用方法及将其制备为医药组合物的方法 - Google Patents

丁烯基苯酞的用途、其使用方法及将其制备为医药组合物的方法 Download PDF

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WO2016015203A1
WO2016015203A1 PCT/CN2014/083150 CN2014083150W WO2016015203A1 WO 2016015203 A1 WO2016015203 A1 WO 2016015203A1 CN 2014083150 W CN2014083150 W CN 2014083150W WO 2016015203 A1 WO2016015203 A1 WO 2016015203A1
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butenylquinone
cells
pharmaceutical composition
neurodegenerative disease
hair growth
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PCT/CN2014/083150
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English (en)
French (fr)
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苏鸿麟
张嘉佑
黄效民
卢怀恩
韩鸿志
林欣荣
赖秉杉
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国立中兴大学
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Priority to US15/329,051 priority Critical patent/US10682335B2/en
Priority to PCT/CN2014/083150 priority patent/WO2016015203A1/zh
Priority to EP14898918.9A priority patent/EP3175851B1/en
Priority to JP2017504015A priority patent/JP6371898B2/ja
Publication of WO2016015203A1 publication Critical patent/WO2016015203A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients

Definitions

  • the invention relates to the use of a compound, in particular to the use of butenylquinone, to methods of its use and to a process for preparing it as a pharmaceutical composition.
  • Alzheimer's disease is the most prevalent neurodegenerative disease in the world. Its main pathological feature is the deposition of excessive ⁇ -amyloid protein ( ⁇ ) in the brain to form ⁇ plaques. (plaque) (Glenner and Wong 1984; Masters, CL et al., 1985) and neurofibrillary tangles (NFT) (Gundke-Iqbal, I. et al., 1986; Goedert, M. et Al., 1988).
  • plaque ⁇ -amyloid protein
  • NFT neurofibrillary tangles
  • the ⁇ plaque is composed of amyloid protein precursor (APP) and B- am yl yl in ⁇ ( ⁇ -site amyloid precursor protein cleaving enzyme) (Hussain, I. et al., 1999; Vassar, R. et al. , 1999) and ⁇ -secretase ( ⁇ -secretase) cleavage (Wolfe, MS et al., 1999; Yu, G. et al., 2000), mainly containing ⁇ 40 and ⁇ 42 two forms ( Jarrett, JT Et al., 1993).
  • ⁇ -amyloid protein When ⁇ -amyloid protein is accumulated in large amounts inside and outside the cell, it becomes a major factor in nerve cell death.
  • Down's syndrome is the 21st pair of chromosome meiosis, the chromosomes are unevenly separated, resulting in the extra chromosomes in the cells. Since the gene for amyloid precursor protein is located on chromosome 21 (Rumble, B. et al., 1989; Selkoe, DJ, 1996), most of them believe that excessive expression of amyloid precursor protein will cause early onset in patients with Down's syndrome. Symptoms of sexual cognitive impairment (Burger, PC and FS Vogel, 1973). Many studies have shown that ⁇ plaques can occur in the brains of patients with Down's syndrome (Masters, CL et al., 1985; Beyreuther, K.
  • iPSCs induced pluripotent stem cells
  • Alzheimer's disease Current clinical treatments for Alzheimer's disease include cholinesterase inhibitors (Birks, J., 2006) and NMDA receptor antagonists (McShane, R. et al., 2006), and both drugs are suitable for improving the cognitive status of Alzheimer's patients Function, however, for diseases caused by Alzheimer's disease, such as depression, insomnia, etc., must be treated with other appropriate drugs (Tariot, PN et al., 2004; Feldman, H. et al., 2006; Howard, R. et al., 2012), and the two drugs can only be used to improve symptoms and fail to cure Alzheimer's disease (Farlow, MR et al., 2010).
  • BACE inhibitors BACE inhibitors
  • MK-8931 and ACI-91 Mulllard A., 2012
  • gamma secretase inhibitors such as LY450139 (Siemers, E. et al., 2005) and BMS-708163 (Tong, G. et al. , 2012), or an antibody against ⁇ by an immunization route.
  • vasodilator In addition to changing hair shampoo habits and eating habits to delay hair loss, there are many products that improve hair loss or promote hair growth. There are two main types, one is vasodilator and the other is vasodilator. Prostaglandin related derivatives. Further, the most well-known vasodilator is "Luojian”. Its trade name is Minoxidil ( Messenger AG et a! 2004). The main component is 2,4-diamino acid. -6 2,4-diamino-6-piperidinopyrimidine 3-oxide , however, it does not have a good effect on the sparseness of all hairs, and the effect is only in the use of products. When the product is discontinued, the newly grown hair will fall off again.
  • Prostaglandin F2a and prostaglandin E2 have been reported to be useful for promoting eyelash growth and hair growth (Woodward, DF et a/., 2013), however, The user causes side effects such as redness, pigmentation, and pigmentation, and once stopped, the newly grown hair also falls off.
  • Butylidenephthalide (Bdph) is present in natural plants, such as Umbelliferae or Compositae, and can be extracted from acetone or chloroform.
  • Past studies have indicated that butenylquinone can be used to treat sputum (Ko, WC et al, 1980), antiplatelet aggregation (Teng, CM. et al, 1987), and can inhibit cell growth and promote cancer cell death, for example, through Inhibition of telomerase to inhibit tumor growth (Huang, MH et al., 2014; Tsai, NM et al, 2006), inhibition of inflammatory response by inhibition of NF- ⁇ (Fu, RH et «/., 201 1 ) The effect.
  • Wnt proteins are highly conserved secreted molecules that are important factors in regulating embryonic development and stem cell maintenance. Wnt binds to its receptor on the cell membrane by Frizzled (Frz) and LDL-relevant-related protein (LDP5/6) to form a ternary structure, and acts on the cells of the disordered protein (Dishevelled) , Dsh).
  • Dsh can bind to glycogen synthase kinase-3 (GSK-3), adenomatous polyposis coli (APC) and Axin protein (axis inhibitor protein) to inhibit hepatic glucose synthesis.
  • GSK-3 glycogen synthase kinase-3
  • APC adenomatous polyposis coli
  • Axin protein axis inhibitor protein
  • T cell factor T cell factor
  • Lef lymphoid enhancer factor
  • Siamois Siamois, etc.
  • Wnt can promote hair growth by stimulating the replication of epidermal stem cells (Lim, X. et «/., 2013).
  • Epidermal stem cells are mostly present in the bulge of the hair follicle, which is a label-retaining cell. Usually, the cells are in a dormant state, and they are activated when the epidermis is damaged or the tissue needs to be renewed.
  • the Wnt/p-catenin pathway is an important molecule for maintaining epidermal stem cell self-replication. Activation of Wnt signal allows dormant epidermal stem cells to enter the cell cycle, allowing cells to replicate and differentiate into mature hair cells (Thompson, CC et al, 2006). ).
  • Wnt7a can increase the number of hair follicle regeneration. Similarly, it stabilizes the ⁇ -chain protein without being decomposed by ubiquitination, which increases the concentration of ⁇ -chain protein in the nucleus and promotes the production of new hair follicles (Gat, U. et al , 1998). Conversely, inhibition of Wnt information prevents wound-induced hair follicle formation (Ito, M. et al, 2007).
  • the present invention discloses the use of a butenyl benzoquinone or/and its analogs, which can be used as an active ingredient for a topical composition for promoting hair, or can be used as a pharmaceutical combination for treating or preventing a neurodegenerative disease.
  • the active ingredient of the substance can be used as a topical composition for promoting hair, or can be used as a pharmaceutical combination for treating or preventing a neurodegenerative disease.
  • the main object of the present invention is to provide a topical composition for promoting hair growth, which can be used Reduces side effects on the human body and achieves an effect of effectively promoting hair growth.
  • the present invention discloses the use of butenyl phenylhydrazine or/and its analogs, which uses butenyl phenyl hydrazine, an analog thereof or a combination of the above components for the preparation of a topical composition for promoting hair growth, wherein The butenylquinone has the ability to activate the Wnt signal.
  • butenylquinone is extracted from natural plants, such as Umbelliferae, Compositae, etc., wherein the extraction technique used is well known to those skilled in the art.
  • butenylquinone or an analog thereof is prepared by a chemical synthesis technique, wherein the chemical synthesis technique used is a chemical synthesis method well known in the art and well known to those skilled in the art.
  • Another object of the present invention is to provide a method for promoting hair growth, which comprises applying a topical composition for promoting hair growth to a skin of a body, wherein the external composition comprises an effective amount of butenylbenzene Indole, an analog thereof or a combination of the above ingredients, and a carrier acceptable for pharmaceutical or cosmetic products.
  • the skin is a region having hair follicles.
  • the topical composition for promoting hair growth is applied directly to the skin of the individual.
  • the topical composition for promoting hair growth is sprayed onto the skin of the individual.
  • the concentration of the butenylquinone is 1 ⁇ -1 ⁇ .
  • a second object of the present invention is to provide the use of butenylquinone or its analogs for the prevention or treatment of neurodegenerative diseases such as Alzheimer's disease.
  • the present invention discloses a method for treating a neurodegenerative disease, which comprises administering a pharmaceutical composition containing an effective amount of butenylquinone, an analog thereof or a combination of the above ingredients to one body.
  • the neurodegenerative disease is a symptom of cumulative excess ⁇ -amyloid protein accumulated in the brain.
  • the neurodegenerative disease is Alzheimer's disease.
  • the butenyl phenylhydrazine is extractable from natural plants, such as Umbelliferae, Compositae, etc., wherein the extraction technique used is well known to those skilled in the art.
  • butenylquinone is prepared by chemical synthesis techniques, wherein the chemical synthesis techniques employed are those known in the art and well known to those skilled in the art.
  • Another object of the present invention is to provide a process for the preparation of the above pharmaceutical composition for reducing the cytotoxicity of the active ingredient in the pharmaceutical composition, thereby improving the safety of the pharmaceutical composition and reducing the side effects.
  • the present invention discloses a method for preparing a pharmaceutical composition, which comprises mixing a butylene-based benzoquinone with a high molecular substance in a weight ratio of 1:1 to 1:2, and adding a polar organic solvent and water. Ding The alkenyl benzoquinone and the high molecular substance are adsorbed by the intermolecular attraction force in the aqueous phase to coat the polymer material with butenyl phenyl hydrazine, and the polar organic solvent is removed.
  • the polar organic solvent is a heterocyclic ether-like compound.
  • the polar organic solvent is tetrahydrofuran.
  • the polymer material is a F127 polymer material.
  • the method of removing the polar organic solvent is a heating method.
  • the butenylquinone or the analog thereof disclosed in the present invention can be used as an active ingredient of a topical composition for promoting hair growth, and can be promoted to promote hair growth by being applied to a specific part of the skin by spraying, smearing, or the like.
  • the effect of improving the appearance of the individual can also achieve the effect of avoiding the side effects of the conventional product on the individual.
  • the preparation method of the pharmaceutical composition disclosed by the invention can use a high molecular substance as a drug carrier to slow down or reduce the cytotoxicity of butenyl phenylhydrazine, and can effectively improve the safety of the pharmaceutical composition.
  • the pharmaceutical composition prepared by the method of the present invention has a function of lowering the concentration of ⁇ -amyloid-like protein in the cells, and therefore, by administering an effective amount of the pharmaceutical composition to the living body, treatment or prevention of neurodegenerative diseases can be achieved. The effect.
  • Fig. 1 shows the results of statistical analysis of the cold-light enzyme expression of each group of cells transfected with Top-flash plastid DNA after different conditions.
  • Fig. 2 shows the results of statistical analysis of the cold-light enzyme expression of each group of cells after the cells transfected with Top-flash plastid DNA or the Fop-flash plastid DNA were cultured under different conditions.
  • Figure 3 shows the appearance changes of mice in each group after different treatments.
  • Fig. 4 shows the results of a cytotoxicity test using butenyl phenyl hydrazine coated with the polymer material F127 and butenyl phenyl hydrazine without coating the high molecular substance F127.
  • Figure 5 is a flow chart showing the differentiation and culture of T21 induced omnipotent stem cells.
  • Figure 6 shows the results of N-cadherin expression by immunofluorescence staining after T21 induced univalent stem cells were cultured in neural differentiation. Among them, red was immunofluorescent stained N-cadherin and blue was DAPI stained nuclei. .
  • Figure 7 shows the results of nestin expression in T21-induced univalent stem cells after immunohistochemical staining. Among them, green is immunofluorescent stained nestin and blue is DAPI-stained nuclei.
  • Fig. 8 shows the results of immunofluorescence staining analysis of Pax-6 protein expression after T21-induced univalent stem cells were subjected to neural differentiation, in which red was immunofluorescent stained Pax-6 protein. Quality, blue is the nucleus stained with DAPI.
  • Fig. 9 shows the results of immunofluorescence staining analysis of the expression of ⁇ -tubular microtubule proteins and neurite outgrowth after T21-induced univalent stem cells were cultured by immunofluorescence staining, in which green is immunofluorescent stained ⁇ microtubule protein, blue The color is the nuclei stained with DAPI.
  • Fig. 10 shows the results of analyzing the expression of ⁇ 40 in each cell by the enzyme-linked immunosorbent assay method for the neural cells differentiated by different cells.
  • Fig. 11 shows the results of statistical analysis of the expression of ⁇ 40 in each cell by enzyme-linked immunosorbent assay after the neural cells differentiated by T21-induced univalent stem cells were cultured under different treatment conditions.
  • the present invention discloses the use of butenylquinone, methods of use thereof, and methods of preparing the pharmaceutical compositions. Based on butenyl phenylhydrazine, it has the ability to promote hair growth and reduce the content of ⁇ -amyloid protein in nerve cells. Therefore, by administering an effective amount of butenyl phenylhydrazine to the living organism, the health and appearance of the individual can be improved.
  • butenyl phenylhydrazine can cause a neurodegenerative disease, such as Alzheimer's disease, in an intracellular accumulation of ⁇ -amyloid-like protein, which can achieve preventive or therapeutic effects, and butenyl phenylhydrazine as The active ingredient of the external composition can effectively promote hair growth at the site of administration.
  • the method for preparing a pharmaceutical composition according to the present invention which comprises preparing the pharmaceutical composition by an organic synthesis reaction, and covalently bonding a polymer substance such as F127 with butenyl phenylhydrazine to coat the polymer substance Butenyl phenylhydrazine achieves a reduction in the cytotoxicity of pharmaceutical compositions against organisms.
  • hair as used in the present invention encompasses hair throughout the individual, including, but not limited to, hair, body hair, eyelashes, and eyebrows.
  • extraction refers to the difference in solubility of substances in different extractants, which will be mixed.
  • the specific component of the compound is transferred from the phase A to the phase B for separation purposes, such as solvent extraction and supercritical extraction.
  • the extractant contains, but is not limited to, acetone, chloroform, carbon dioxide.
  • chemical synthesis technique refers to a series of chemical reactions, such as organic reactions and inorganic reactions, carried out to obtain a specific product.
  • the term "effective amount" as used in the present invention refers to the amount of the compound or active ingredient required to produce the desired effect, which may be expressed as a percentage by weight of the composition. As will be appreciated by those of ordinary skill in the art to which the present invention pertains, the effective amount will vary depending on the mode of administration desired to effect a particular effect. Generally, the active ingredient or compound will be present in the compositions in an amount of from about 1% to about 100%, preferably from about 30% to about 100% by weight of the composition.
  • the term "acceptable carrier in a pharmaceutically or cosmetic product” as used in the present invention encompasses any standard used in a pharmaceutical or cosmetic product, and the carrier may be solid, semi-solid or liquid depending on the form of the composition.
  • the carrier includes, but is not limited to, gelatin, an emulsifier, a mixture of hydrocarbons, water, glycerin, physiological saline, buffered saline, lanolin, paraffin, beeswax, dimethicone, ethanol.
  • analog as used in the present invention encompasses salts of the compounds, esters thereof, structural isomers thereof, such as Z-type structures or E-type structures, or products which undergo structural modification.
  • the high molecular substance F127 disclosed in the invention is a triblock of polyoxyethylene-polyoxypropylene-polyoxyethylene
  • the polymer has the formula: (II): H(OCH 2 CH 2 ) x ⁇ OCH 2 CH) y (OCH 2 CH 2 ) z QH ( ⁇ ) , wherein x, y, and z are each an integer greater than 1. Since the polyoxyethylene at both ends of the polymer substance F127 is hydrophilic, the middle stage polyoxypropylene is hydrophobic, and therefore, the polymer substance F127 is an amphoteric substance.
  • the polymer substance F127 When the polymer substance F127 is present in an aqueous solution, it gradually diffuses toward the interface and adsorbs on the interface, causing the surface tension to decrease, and by controlling the proportion of the hydrophilic-hydrophobic group in the polymer substance, the surface of the polymer substance can be controlled. active.
  • the "intermolecular attraction” disclosed in the present invention includes, but is not limited to, van der Waals force, Coulomb force, hydrogen bond, and hydrophobic bond force.
  • pharmaceutical composition encompasses an effective amount of the desired compound or active ingredient to produce a particular effect, and at least one pharmaceutically acceptable carrier.
  • the form of the pharmaceutical composition may vary depending on the mode of administration desired to cause a particular effect, such as a lozenge, a powder, an injection, etc., and the carrier
  • the form of the pharmaceutical composition can be solid, semi-solid or liquid.
  • the carrier includes, but is not limited to, gelatin, an emulsifier, a mixture of hydrocarbons, water, glycerin, physiological saline, buffered saline, lanolin, paraffin, beeswax, dimethicone, ethanol.
  • polymer material refers to a macromolecule having a relatively high molecular weight by polymerization, and generally, the polymer material is an organic molecule.
  • the number of BHK21 in the hamster kidney fibroblasts was approximately 5 minutes in a six-well culture dish. Each well was plated with 5 ( ⁇ L of Opti-MEM medium (Invitrogen) and 2 L of oligosaccharide 2000 (Lipofectamine 2000). , Invitrogen) mixed in a 1.5 mL microcentrifuge tube for 5 minutes to form a liposome mixture.
  • Top-flash plastid has a wild-type TCF binding site for use as an experimental group; the Fop-flash plastid has a mutant TCF binding site, a control group; The plastids are followed by a sequence of Luciferase.
  • the Top-flash plastid mixture was added to the vesicle mixture to form a mixture of ⁇ , and a total of three groups of the mixture. After the reaction mixture was allowed to stand at room temperature for 20 minutes, the mixture was extracted and added to the culture dish, gently shaken to make it evenly distributed, and then filled up with Opti-MEM medium until the liquid surface just covered the cells.
  • BHK21 was cultured at 37 ° C for 4 hours, and then supplemented with 2 mL of medium volume per well in Opti-MEM medium, and the medium was changed after about 18 to 24 hours, and each group was given different culture conditions, among which, first The group was a blank group, the second group was added with 0.4 ⁇ M of compound hydrazine, and the third group was added with 0.4 ⁇ M of butenylquinone. Each group was further cultured for about 18 to 24 hours, and then BHK21 cells were collected from the culture dishes of each group, and the luminescence activity of the cells was detected. The results are shown in Fig. 1, wherein the * indicates a significant level at 0.05. under.
  • the product of the Top-flash plastid mixture or the Fop-flash plastid mixture is added to the vesicle mixture to form a total volume of 10 (L of Top- Flash mixture or Fop-flash mixture, there are 4 groups of mixed liquid, wherein the first group is a Fop-flash mixture, and the second to fourth groups are Top-flash mixture.
  • the mixture is static at room temperature. The reaction was allowed to stand for 20 minutes, and then extracted and added to the culture dish, gently shaken to make it evenly distributed, and then filled up with Opti-MEM medium until the liquid surface just covered The cells were cultured at 37 ° C for 4 hours, and then supplemented with 2 mL of medium volume per well in Opti-MEM medium.
  • the medium was changed after about 18 to 24 hours, and different culture conditions were given to each group.
  • the first group was added with 4 ⁇ M of butenyl phenylhydrazine
  • the second group was not added with any compound
  • the third group was added with ⁇ ⁇ of butenyl phenyl hydrazine
  • the fourth group was added with 4 ⁇ ⁇ of butenyl phenyl hydrazine.
  • Each group was cultured for another 18 to 24 hours, and then BHK21 cells were collected from the culture dishes of each group, and the luminescence activity was detected. The results are shown in Fig. 2, wherein * indicates a significant level at 0.05. under.
  • the method for detecting the activity of the luminescent enzyme is as follows: First, the culture medium of the cell BHK21 is aspirated, and then washed twice with a phosphate buffer. Add 20 ( ⁇ L IX PLB test U (Passive Lysis Buffer, Promega)) to each well. After shaking for 15 minutes at room temperature, the cells are lysed, and the cell lysate is taken, and then rotated at 12000 rpm at 4 °. After centrifugation for 1 minute at C, the supernatant was collected. Take 2 ( ⁇ L of the supernatant to a 96-well plate, add 10 (L Luciferase Assay Reagent), place the luminometer, measure at a wavelength of 595 nm. The activity of cold light enzymes.
  • the luminescent activity of the luminescent enzyme cannot be detected in the first group. In other words, the luminescence value of the cells is not increased. .
  • the cell luminescence values of the second to fourth groups increased as the amount of butenylquinone added increased. From this, it is known that the activation of the luminescent enzyme is specific and has a dose effect.
  • BIO is a Wnt activator that binds to the TCF binding site in the Top-flash plastid, and when the Wnt signal is activated, it promotes the expression of the luminescent enzyme, and, by Figure 1, As can be seen from the results of Fig. 2, the butenyl phenylhydrazine can significantly increase the activity of the luminescent enzyme as the amount of the additive increases, and accordingly, the butenylquinone of the present invention has the ability to activate Wnt information.
  • C57BL/6 male mice aged 6-8 weeks were selected and divided into three groups, each group of 2 male mice, and the hairless parts of each mouse were the epidermis at the proximal end of the back, with an area of 2 cm ⁇ 2 .
  • the hairless areas were treated daily under different conditions. After 21 days, the hair growth, body weight and appearance of the mice in each group were observed abnormal. Among them, the first group was the control group, and only the hairless area was applied.
  • the second group is the experimental group, the emulsified composition is applied to the hairless area, which contains butenyl phenylhydrazine at a concentration of 10 ⁇ ; the second group is the experimental group, and the emulsified is applied at the hairless place.
  • a composition comprising butenylphenylhydrazine in a concentration of 100 ⁇ M.
  • the result is shown in Figure 3. From the results of Fig. 3, the body weight of each group of mice did not have an abnormal phenomenon. Compared with the first group, the hair in the back of the second group and the third group had obvious hyperplasia, and the hair growth in the back of the third group was more pronounced. It can be seen that the application of the butenylquinone disclosed in the present invention can effectively promote hair proliferation, and the effect of proliferation can be significantly increased as the dose is increased.
  • N-butylidenephthalide (Bdph, W333301) and F127 macromolecular material (Pluronic F127, P2443) were obtained from Sigma-Aldrich. 10 mg of butenyl phenylhydrazine and F127 high molecular substance are mixed at a weight ratio of 1:1 or 1:2, dissolved in 2 ml of tetrahydrofuran, and added to 10 ml of water, followed by rapid heating. The tetrahydrofuran was removed and lyophilized. Thereafter, it is further dissolved in water, and the butenylphthalide particles which are emulsified in the solution have a size of about 30 to 200 nm and a polydispersity index of 0.2 to 0.5.
  • Human pluripotent cells are cultured in serum-free medium Essential 8 TM (Life Technology, USA) and cultured to be bonded matrigel TM Matrigel (Becton-Dickinson, USA). After the culture solution after gettering, the cells were washed with phosphate buffer twice, the enzyme was added Accutase TM (Merck Millipore Corporation, USA) to 2 ⁇ 5 minutes and the culture medium, the cells were washed and broken down into small The pellet was centrifuged at 1000 rpm for 2 minutes, and the supernatant was again aspirated, placed in a new culture dish for about 3 to 5 days, subcultured, and the culture solution was changed every day during the culture.
  • Essential 8 TM Life Technology, USA
  • Matrigel Matrigel
  • the cells After culturing the human omnipotent stem cells to 8-9 minutes, the cells are washed twice with phosphate buffer solution, and then reacted with the enzyme AccutaseTM for 2 to 5 minutes, then the culture medium is added to dilute the enzyme, and then the cells are washed off and dispersed to appropriate After centrifugation at a speed of 800 rpm for about 2 minutes, the cells were placed in DMEM-F12 medium containing 20% replacement serum (Knock out serum replacement, Jane KSR, Life Technology, USA). The dish was subjected to suspension culture for 2 days to obtain an Embryonic body suspension.
  • the suspended embryoid body is placed in a centrifuge tube, and after being allowed to settle naturally, the supernatant is removed, and the suspension culture is carried out for 2 days in a nerve induction medium containing a BiSF small molecule drug, and then the epithelial body of the suspended embryoid body appears.
  • Cell structure wherein the small molecule drug contains 0.5 ⁇ M of BIO (Sigma-Aldrich, USA), 10 ng/mL of fibroblast growth factor-2 (FGF-2, Peprotech, USA) and 10 ⁇ M of SB431542 (Sigma) -Aldrich, USA);
  • BIO Sigma-Aldrich, USA
  • FGF-2 fibroblast growth factor-2
  • SB431542 SB431542
  • the embryoid bodies were sedimented, they were cultured in a neurobasal medium containing 10 ng/mL of fibroblast growth factor-2, and the components of the neurobasal medium were as shown in Table 2 below.
  • the cells After 2 days the suspension, until the embryoid bodies after settling, or an external force to break up the enzyme Accutase TM to small clumps, the cells are placed in a coating for l% matrigel TM Matrigel over 1 hour attachment Petri dish. About 2 to 7 days, cells will attach, grow, and grow nerve structures.
  • the cells When the cells are grown to 7 to 8 minutes, the cells are dialed, and the cells are washed once with phosphate buffer, and then acted as an enzyme solution mixed with phosphate buffer and enzyme Accutase in an equal ratio. After ⁇ 5 minutes, dilute with the neurobasal medium, then use the cell spatula to scrape the cells and break them into small clumps or single cells by mechanical force. Centrifuge at 800 ⁇ 1000rpm for about 2 ⁇ 5 minutes, then aspirate. The supernatant was applied to the plate and 10 ⁇ of Y27632 (Stemgent, USA) was added for 1 day to help the cells attach.
  • the human embryonic stem cell TW1 was cultured in Essential 8TM medium, and subdivided into three groups, and the amount of cells of 1.2 ⁇ 10 5 was placed in a 6-well plate under the following conditions, wherein the first group was simple.
  • the medium was cultured in the medium; the second group was added with a butene-based phenylhydrazine which was not coated with the F127 macromolecular substance at a concentration of 10 ⁇ M; and the third group was added with the coated F127 as in Example 1 in the medium.
  • the high molecular weight butenyl phenylhydrazine has a concentration of 10 ⁇ M.
  • Example 7 T21 induced omnipotent stem cells for neural cell differentiation and culture
  • T21 induced univalent stem cells (T21-iPSCs) were subjected to neural differentiation as described in Example 2, and attached on the 8th day. Immunofluorescence staining was performed on the 12th day after cell attachment to observe the expression of neural stem cell markers, including N-cadherin, Nestin, and Pax-6 proteins. The results are shown in Figures 6-8. The neurite outgrowth of T21-induced univalent stem cells after neuronal differentiation was observed, and analyzed by immunofluorescence staining on the 27th day of culture.
  • Tuj-1 antibody was observed for the expression of mature neural markers such as tubulin (tubulin), and the results are shown in Fig. 9.
  • the procedure of immunofluorescence staining analysis is as follows: when the cells are cultured in a 4-well culture dish of a cover glass, the cells are first aspirated, then washed with phosphate buffer twice, and then added to 4%. Paraformaldehyde (PFA) was applied to the cells for 5 minutes on ice and then removed. After washing with phosphate buffer for 3 times, 0.3% triton (PBST) was added to the ice for 10 minutes to punch holes. After rinsing 3 times with phosphate buffer, blocking with 5% concentration of horse serum for 1 hour, aspirating the liquid and adding a primary antibody at a concentration of 3% horse serum, acting at room temperature 4 Hour or reaction at 4 ° C for 16 hours.
  • PFA Paraformaldehyde
  • PBST 0.3% triton
  • the primary antibody was aspirated and washed 3 times with PBST rinse for 5 minutes each time.
  • the secondary antibody was placed in a phosphate buffer. After the PBST was aspirated, the secondary antibody was added and protected from light for 1 hour, and then washed with PBST rinse for 3 times. The concentration of IgG/mL of nuclear dye DAPI was protected from light at room temperature. After a minute, the cells were washed twice with a PBST rinse solution, and then mixed with a solution of glycerin and phosphate buffer in equal proportions (after soaking), the pieces were picked up, and the fluorescence was observed under an erect fluorescence microscope after the film was mounted.
  • T21 induced univalent stem cells express N-cadherin, Nestin and Pax-6 neural stem cell specific proteins on the 12th day of neural cell differentiation culture, indicating that T21 induced univalent stem cells are cultured in the above medium. After culture, it can be successfully differentiated into T21 nerve cells. Furthermore, as is clear from the results of Fig. 9, T21-induced univalent stem cells have a large number of neurites after 27 days of culture differentiation, and can express mature neuronal beta-tubular microtubule proteins, indicating that T21-induced univalent stem cells pass through. The 27-day differentiation was cultured into mature T21 nerve cells.
  • Example 8 ⁇ 40 expression of nerve cells
  • the cells differentiated from the cultured T21-induced univalent stem cells were counted, and then plated in a 4-well culture dish, and the culture solution was changed every 48 hours, and the culture solution collected every 48 hours was stored at -20°.
  • C Analysis of the amount of ⁇ 40 expression was carried out by enzyme-linked immunosorbent assay (ELISA), and nerve cells differentiated from karyotype normal human embryonic stem cells TW1 were used as a control group, wherein each group of experiments was repeated 2 or 3 times. The results of the analysis are shown in Figure 10.
  • ELISA enzyme-linked immunosorbent assay
  • the procedure for enzyme-linked immunosorbent assay was as follows: The ⁇ standard protein of the ELISA kit was serially diluted in a working incubation buffer to serve as a standard curve. The collected cell culture solution was diluted with the culture buffer at a ratio of 1:2. Take 10 (L standard solution and sample into a 96-well plate and place at 4 ° C for 16 hours. After aspirating the liquid, rinse the plate with 30 ( ⁇ L and dry. Add 20 ( ⁇ L) The TMB was subjected to a temperature-sensitive reaction at room temperature for about 40 to 45 minutes, and the absorbance at 620 nm was read.
  • the amount of ⁇ 40 expressed in the neural cells differentiated by T21 induced omnipotent stem cells began to be higher than that of the human embryonic stem cell TW1, and began on the 25th day of differentiation, starting from the 20th day of differentiation. There is a difference.
  • the expression of ⁇ 40 in neurons differentiated by T21-induced omnipotent stem cells was 105.38 pg/mL on the 25th day of differentiation, 155.68 pg/mL on the 30th day of differentiation, and 264.47 on the 42nd day of differentiation. Pg/mL.
  • the amount of ⁇ 40 expressed in the neural cells differentiated by T21-induced omnipotent stem cells increases significantly with the increase of differentiation time, and thus can be used as a platform for screening for treatment or prevention of neurodegenerative diseases.
  • the T21-induced univalent stem cells were cultured on the 39th day and divided into three groups. After different culture conditions, the culture was further cultured for 3 days. On the 42nd day, the ⁇ 40 in each group was analyzed by enzyme-linked immunosorbent assay. The concentration, and compared with the T21 induced omnipotent stem cells which were not subjected to neural differentiation culture, the results are shown in Fig. 11, wherein the first group is T21 induced omnipotent stem cells which have not been subjected to neural differentiation culture; the second to fourth groups are nerves. The cultured T21-induced univalent stem cells were differentiated, while the second group did not add any drug during the culture.
  • the third group added the ⁇ -secretin inhibitor DAPT on the 39th day of culture, and the fourth group added the ⁇ package on the 39th day of culture.
  • Each group of experiments was performed 2 to 3 times, and the number of differences was analyzed by single factor analysis and multiple comparison.
  • the ⁇ value was less than 0.05, 95% confidence level, and the mark * indicates a significant level of 0.05.
  • the mark indicates a significant level of 0.01.
  • the concentration of ⁇ 40 in the first group was significantly lower than that in the second group, while the concentration of ⁇ 40 in the third group and the fourth group was lower than that in the second group.
  • Alkenylquinone can significantly reduce the concentration of A ⁇ 40 in nerve cells, and its effect is similar to administration of the ⁇ -secretin inhibitor DAPT. Accordingly, the butenylquinone disclosed in the present invention does have an ability to improve or slow the accumulation of excess ⁇ -amyloid-like protein in cells, and can achieve the effect of treating or preventing a neurodegenerative disease.
  • the butenylquinone of the present invention has the ability to activate Wnt information, and can effectively achieve the effect of promoting hair proliferation. Therefore, the butenylquinone or the like of the present invention can As an active ingredient of a topical composition for promoting hair growth, by administering to a specific part of the skin by spraying or smearing, for example, the effect of promoting hair growth and enhancing the appearance of the individual can be achieved.
  • the product has the effect of having side effects for the individual.
  • the preparation method of the pharmaceutical composition disclosed by the invention can use a high molecular substance as a drug carrier to slow down or reduce the cytotoxicity of butenyl benzoquinone, and can effectively improve the safety of the pharmaceutical composition.
  • the pharmaceutical composition prepared by the above method does have a function of lowering the concentration of ⁇ -amyloid-like protein in the cells, and therefore, by administering an effective amount of the pharmaceutical composition to the living body, treatment or prevention of neurodegenerative diseases can be achieved. efficacy.
  • n-Butylidenephthalide maintains stem cell pluripotency by activating Jak2/Stat3 pathway and increases the efficiency of iPS cells generation.
  • BP n-Butylidenephthalide
  • Gat, U., et al De Novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin. Cell, 1998. 95(5): p. 605-14.. Ito, M., et al " Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding. Nature, 2007. 447(7142): p. 316-20.

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Abstract

公开了丁烯基苯酞的制药用途以及制备医药组合物的方法。基于丁烯基苯酞具有促进毛发生长以及降低神经细胞内β类淀粉样蛋白质含量的能力，因此，通过投予有效量的丁烯基苯酞至生物体中能达到改善个体健康及外观的功效，具体来说，丁烯基苯酞对于过量累积β类淀粉样蛋白质在细胞内会导致神经退化性疾病，如阿兹海默症，能达到预防或治疗的功效，并且丁烯基苯酞作为外用组合物的活性成分，能够有效促进其投予部位毛发生长。还公开了制备医药组合物的方法，其通过有机合成反应制备该医药组合物，通过如F127的高分子物质与丁烯基苯酞间产生共价键结，使高分子物质包覆丁烯基苯酞，达到减少医药组合物对于生物体细胞毒性的功效。

Description

丁烯基苯酞的用途、 其使用方法及将其制备为医药组合物的方法 技术领域
本发明有关于一种化合物的用途， 特别指丁烯基苯酞的用途、 其使用方法 及将其制备为医药组合物的方法。
背景技术
由于现代人的存活年龄增加， 而伴随着年纪老化及生活压力倍增， 越来越 多人面临各种影响身体健康的疾病， 以及外观老化的问题。 具体来说， 阿兹海 默症为全球发生率最高的神经退化性疾病， 其主要的病理特征在于脑内有过量 的 β类淀粉样蛋白质（β-amyloid protein, Αβ )沉积，形成 Αβ斑块（plaque ) ( Glenner and Wong 1984； Masters, C. L. et al.， 1985 ) 以及细胞内缠结的神经纤维 ( neurofibrillary tangles, NFT ) ( Grundke-Iqbal, I. et al.， 1986； Goedert, M. et al.， 1988 ) 。 而 Αβ斑块由淀粉样前驱蛋白质 （amyloid protein precursor, APP ) ， 经 过 BACE酉孝素 ( β-site amyloid precursor protein cleaving enzyme ) ( Hussain, I. et al.， 1999； Vassar, R. et al.,1999 ) 以及 γ分泌酶（γ-secretase )切割后所产生 （Wolfe, M. S. et al.， 1999； Yu, G. et al.， 2000 ) ， 主要包含有 Αβ40以及 Αβ42两种形式 ( Jarrett, J. T. et al., 1993 ) 。 当 β类淀粉样蛋白质在细胞内外大量累积时， 会成 为神经细胞死亡的主要因素。
唐氏症患者为第 21对染色体减数分裂时， 染色体分离不均， 导致细胞内多 带一条染色体所致。 而由于淀粉样前驱蛋白质的基因位于第 21条染色体上 ( Rumble, B. et al., 1989； Selkoe, D. J.， 1996 ) ， 因而大多认为淀粉样前驱蛋白 质过量表现会使唐氏症患者具有早发性认知障碍的症状 （Burger, P. C. and F. S. Vogel, 1973 )。目前许多研究显示， Αβ斑块会出现于唐氏症患者的脑内（Masters, C. L. et al., 1985； Beyreuther, K. et al., 1992； Gyure, K. A. et al., 2001； Mori, C. et al., 2002 ) 。 另有研究指出， 利用来自唐氏症患者的诱导型万能干细胞（induced pluripotent stem cells, iPSCs )所产生的神经细胞， 可用以重现典型阿兹海默症的 病理特征， 例如 Αβ42及 Αβ40的累积、 高度磷酸化的 Tau蛋白质等。 因此， 利用 唐氏症患者的诱导型万能干细胞分化系统可用以作为筛选治疗或预防与 β类淀 粉样蛋白质相关神经退化性疾病的药物平台。
目前临床上治疗阿兹海默症的药物包含胆碱酯酶抑制剂 （ cholinesterase inhibitors )( Birks, J., 2006 )以及 NMDA接受体拮抗剂（NMDA receptor antagonist) ( McShane, R. et al., 2006 ) ， 而该两种药物皆适用以改善阿兹海默症患者认知 功能， 然而， 对于由阿兹海默症所衍生的疾病， 如忧郁、 失眠等， 须通过其他 适合的药物进行治疗（Tariot, P. N. et al.， 2004； Feldman, H. et al.， 2006； Howard, R. et al., 2012 ) ， 并且， 该两种药物仅能用于改善症状， 不能达到治愈阿兹海默 症的功效 （Farlow, M. R. et al.， 2010 ) 。 除此之外， 许多研究针对减低脑内 Αβ 的累积而设计治疗阿兹海默症的新药 （Hong-Qi， Y. et al.， 2012 ) ， 具体来说， 包含有 BACE 抑制剂（BACE inhibitors )，例如 MK-8931以及 ACI-91 ( Mullard A., 2012)，或 γ分泌酶抑制剂，例如 LY450139( Siemers， E. et al.， 2005 )及 BMS-708163 ( Tong, G. et al., 2012 ) ， 或以免疫途径对抗 Αβ的抗体等。
再者， 虽然局部脱发、 掉发或毛发稀疏等问题对于个体的身体健康不会产 生不良影响， 但是， 不可否认地会影响个体的外在观感。 据研究指出， 毛发稀 疏者较为容易导致个体情绪不佳， 无法进行社交活动， 可能造成个体产生社交 障碍、 自信心不足、 自我认同等心理问题。 因此， 毛发稀疏或脱发成为现代人 逐渐重视的问题。
而除通过改变洗发习惯、 饮食习惯延缓毛发掉落外， 目前坊间出现许多改 善毛发脱落或是促进毛发生长的产品， 主要可分为两类， 其中一类为血管舒张 剂， 另一类为前列腺素相关衍生物。 更进一步来说， 血管舒张剂中最为人所熟 知者为 "落健" 其商业名称为米诺地尔 （Minoxidil ) ( Messenger A.G. et a!" 2004 ) ， 主 要 成 分 为 2，4-二胺 基 酸 -6 哌 啶 基 嘧 啶 -3-氧 化 物 ( 2,4-diamino-6-piperidinopyrimidine 3-oxide ) ， 然而， 落健并非对于所有咅分 的毛发稀疏皆具有良好效果， 并且， 效果仅只在有使用产品时， 一旦停止使用 产品， 新生长出的毛发会再次脱落。 而前列腺素 F2a以及前列腺素 E2分别被报 导指出可用以促进睫毛与头发生长 （Woodward, D.F. et a/.，2013 ) ， 不过， 对于 使用者造成如红肿过敏、 色素沉淀等副作用， 并且， 一旦停止使用， 新生长出 的毛发也会脱落。
丁烯基苯酞 （butylidenephthalide , Bdph ) 存在于天然植物中， 例如伞形科 或菊科的植物， 可由丙酮或氯仿萃取而得。 过去研究指出丁烯基苯酞可用于治 疗痉挛 (Ko， W.C. et al , 1980)、 抗血小板凝集 (Teng, CM. et al , 1987)， 并且能抑 制细胞生长、 促进癌细胞死亡， 例如， 通过抑制端粒酶达到抑制肿瘤生长的功 效 (Huang, M.H. et al., 2014; Tsai, N.M. et al , 2006)、 通过抑制 NF-κΒ达到抑制发 炎反应 （Fu，R.H. et «/.，201 1 ) 的功效。 再者， 近期研究也发现， 丁烯基苯酞得 通过活化 Jak2/stat3信息路径， 维持胚胎干细胞的生长， 以及促进诱导型干细胞 的形成 （Liu, S.P. et a/.， 2012 ) 。 Wnt蛋白质是高度保守 （highly conserved) 的分泌性分子， 为调控胚胎发育 与干细胞维持的重要因子。 Wnt得通过和其在细胞膜上的受体 Frizzled ( Frz ) 及 LDL受体相关蛋白质 （LDL-receptor-related protein, LRP5/6 ) 结合形成三元体 结构， 并且作用于细胞内的蓬乱蛋白质 （Dishevelled, Dsh) 。 Dsh能与肝醣合 成酶激酶 -3β ( glycogen synthase kinase-3 , GSK-3 ) 、 腺瘤性结肠息肉蛋白质 ( adenomatous polyposis coli, APC ) 以及 Axin蛋白质 ( axis inhibitor protein ) 结 合， 以抑制肝醣合成酶激酶 -3β的活性， 进而抑制 β-链蛋白质 （β-catenin ) 磷酸 化， 以及 β-链蛋白质经由泛素化作用 （ubiquitination) 分解的途径。 活化 Wnt信 息得引发细胞核内 β-链蛋白质的累积。 β-链蛋白质进入细胞核后， 可启动其他特 殊转录因子， 如 Τ细胞因子 （T cell factor, Tcf) ， 以及淋巴增强因子 （lymphoid enhancer factor, Lef) ， 与 Siamois等， 藉此以调控细胞的成长与个体的发育。 活化细胞内 wnt信息太多或不及， 均会导致生物体的损害， 造成早期胚胎发育上 的缺陷， 或导致晚期成体中产生肿瘤或功能不全 （Fodde，R. et a/.，2007 ) 。
目前许多研究均已证实， 活化 Wnt可以通过刺激表皮干细胞的复制，达到促 进毛发生长的功效 （Lim，X. et «/.， 2013 ) 。 表皮干细胞多存在于毛囊隆起区域 ( bulge ) ， 为一标志残留细胞 （label-retaining cells ) ， 平时细胞多处于休眠状 态， 在表皮受损或组织需要更新时， 才会活化。 Wnt/p-catenin 路径为维持表皮 干细胞自我复制的重要分子， 活化 Wnt signal可使休眠状态的表皮干细胞进入细 胞周期， 使细胞进行复制， 并分化为成熟的毛发细胞 （Thompson, C.C. et al , 2006 ) 。 表现 Wnt7a则可以增加毛囊再生的数目， 相同地， 稳定 β-链蛋白质而不 受泛素化作用分解， 得提高 β-链蛋白质在细胞核的浓度， 促使新生毛囊的产生 ( Gat, U. et al , 1998 )。相反地，抑制 Wnt信息可以防止创伤引起的毛囊形成（Ito， M. et al , 2007 ) 。
由上可知， 先前技术中无法提供一种有效且无副作用的组合物， 用以治疗 或预防如阿兹海默症的神经退化性疾病， 以及改善脱发或促进毛发生长。 因此， 开发一种新颖并且能够有效改善或治疗上述症状的组合物为目前最重要的研究 课题。
发明内容
因此， 本发明揭露一种丁烯基苯酞或 /及其类似物的用途， 其可用于作为促 进毛发的外用组合物的活性成分， 或是可用以作为治疗或预防神经退化性疾病 的医药组合物的活性成分。
本发明的主要目的在于提供一种促进毛发生长的外用组合物， 其能够用以 减少对于人体副作用， 并且达到有效促进毛发生长的功效。
为了达成上述目的， 本发明揭露丁烯基苯酞或 /及其类似物的用途， 其将丁 烯基苯酞、 其类似物或上述成分的组合用于制备促进毛发生长的外用组合物， 其中， 该丁烯基苯酞具有活化 Wnt信号的能力。
较佳地， 丁烯基苯酞可由天然植物中萃取而得， 如伞形科植物、 菊科植物 等， 其中， 所使用的萃取技术为该技术领域且具通常知识者周知。
较佳地， 丁烯基苯酞或其类似物得由化学合成技术所制备而得， 其中， 所 使用的化学合成技术为该技术领域且具通常知识者周知的化学合成方法。
本发明的另一目的在于提供一种促进毛发增生的方法， 其将一促进毛发生 长的外用组合物， 涂抹在一个体的皮肤上， 其中， 该外用组合物包含一有效量 的丁烯基苯酞、 其类似物或上述成分的组合， 以及一药学或美容产品上能接受 的载体。
较佳地， 该皮肤为具有毛囊的区域。
较佳地， 该促进毛发生长的外用组合物直接涂抹在该个体的皮肤上。
较佳地， 该促进毛发生长的外用组合物喷洒在该个体的皮肤上。
较佳地， 该丁烯基苯酞的浓度为 1μΜ-1ηιΜ。
本发明的次一目的在于提供丁烯基苯酞或 /及其类似物的用途， 其用于预防 或治疗神经退化性疾病， 如阿兹海默症。
为能达成上述目的， 本发明揭露一种治疗神经退化性疾病的方法， 其投予 一含有有效量的丁烯基苯酞、 其类似物或上述成分的组合的医药组合物至一个 体。
较佳地， 该神经退化性疾病为脑内累积过量 β类淀粉样蛋白质的病征。 较佳地， 该神经退化性疾病为阿兹海默症。
较佳地， 丁烯基苯酞可自天然植物中萃取而得， 例如伞形科植物、 菊科植 物等， 其中， 所使用的萃取技术为该技术领域且具通常知识者周知。
较佳地， 丁烯基苯酞可由化学合成技术所制备而得， 其中， 所使用的化学 合成技术为该技术领域且具通常知识者周知的化学合成方法。
本发明的又一目的在于提供一种上述医药组合物的制备方法， 其用以降低 医药组合物内活性成分的细胞毒性， 达到提升医药组合物安全性及减少副作用 的功效。
为能达成该目的， 本发明揭露一种医药组合物的制备方法， 其将丁烯基苯 酞与高分子物质以重量比 1 : 1〜1： 2混合后， 加入一极性有机溶剂及水， 使丁 烯基苯酞与高分子物质在水相通过分子间引力进行吸附作用而使该高分子物质 包覆丁烯基苯酞， 再去除该极性有机溶剂。
较佳地， 该极性有机溶剂为类杂环醚类化合物。
较佳地， 该极性有机溶剂为四氢呋喃 （tetrahydrofuran) 。
较佳地， 该高分子物质为 F127高分子物质。
较佳地， 去除该极性有机溶剂的方法为加热法。
本发明的有益效果为：
本发明所揭丁烯基苯酞或其类似物能作为于促进毛发生长的外用组合物的 活性成分， 而通过以如喷洒、 涂抹等方式投予于个体特定部位的皮肤上， 达到 促进毛发增生， 同时提升个体外观的功效， 也能达到避免习知产品对于个体具 有副作用的功效。 再者， 本发明所揭医药组合物制备方法能以高分子物质作为 药物载体， 减缓或降低丁烯基苯酞的细胞毒性， 能有效提升医药组合物的安全 性。而通过本发明的方法所制得的医药组合物具有降低细胞内 β类淀粉样蛋白质 浓度的功能， 因此， 通过投予有效量的医药组合物至生物体， 能达到治疗或预 防神经退化性疾病的功效。
附图说明
图 1为各组转染 Top-flash质体 DNA的细胞经不同条件培养后， 侦测各组 细胞的冷光酶表现并且经统计分析出的结果。
图 2为转染 Top-flash质体 DNA或该 Fop-flash质体 DNA的细胞经不同条 件培养后， 侦测各组细胞的冷光酶表现并且经统计分析出的结果。
图 3为各组小鼠分别经不同处理后的外观变化。
图 4为以包覆高分子物质 F127的丁烯基苯酞及未包覆高分子物质 F127的 丁烯基苯酞进行细胞毒性试验的结果。
图 5为 T21诱导性万能干细胞进行神经分化培养的流程图。
图 6为 T21诱导性万能干细胞进行神经分化培养后， 以免疫荧光染色分析 观察其内 N-cadherin表现的结果， 其中， 红色为经免疫荧光染色的 N-cadherin， 蓝色为以 DAPI染色的细胞核。
图 7为 T21诱导性万能干细胞进行神经分化培养后， 以免疫荧光染色分析 观察其内 nestin表现的结果， 其中， 绿色为经免疫荧光染色的 nestin, 蓝色为以 DAPI染色的细胞核。
图 8为 T21诱导性万能干细胞进行神经分化培养后， 以免疫荧光染色分析 观察其内 Pax-6蛋白质表现的结果， 其中， 红色为经免疫荧光染色的 Pax-6蛋白 质， 蓝色为以 DAPI染色的细胞核。
图 9为 T21诱导性万能干细胞进行神经分化培养后， 以免疫荧光染色分析 观察其内 βΠΙ微管蛋白质表现及神经突起生长情形的结果， 其中， 绿色为经免 疫荧光染色的 βΠΙ微管蛋白质， 蓝色为以 DAPI染色的细胞核。
图 10为经不同细胞所分化的神经细胞，分别以酵素连结免疫吸附分析法统 计分析各细胞内 Αβ40表现量的结果。
图 11为 T21诱导性万能干细胞所分化的神经细胞经不同处理条件进行培养 后， 以酵素连结免疫吸附分析法统计分析各细胞内 Αβ40表现量的结果。
具体实施方式
本发明揭露丁烯基苯酞的用途、 其使用方法及将的制备为医药组合物的方 法。基于丁烯基苯酞具有促进毛发生长以及降低神经细胞内 β类淀粉样蛋白质含 量的能力， 因此， 通过投予有效量的丁烯基苯酞至生物体中能达到改善个体健 康及外观的功效， 具体来说， 丁烯基苯酞对于过量累积 β类淀粉样蛋白质在细胞 内会导致神经退化性疾病， 如阿兹海默症， 能达到预防或治疗的功效， 并且丁 烯基苯酞作为外用组合物的活性成分， 能够有效促进其投予部位毛发生长。 此 外， 本发明所揭制备医药组合物的方法， 其通过有机合成反应制备该医药组合 物， 通过如 F127的高分子物质与丁烯基苯酞间产生共价键结， 使高分子物质包 覆丁烯基苯酞， 达到减少医药组合物对于生物体细胞毒性的功效。
除非另有定义， 本发明的说明书及权利要求书所使用的技术及科学名词的 意义， 其与本发明所属技术领域且具通常知识者的一般理解相同。 若有矛盾的 情形， 以本发明内容为准。
Figure imgf000008_0001
本发明所揭丁烯基苯酞的结构式为如式 （I) ： ( I) ， 其 制备或取得方式为该所属技术领域且具通常知识者的一般知识， 并且非本发明 的技术特征， 故于本发明不加以赘述。 举例来说， 中国专利申请， 申请号为 20091006666.6中揭露丁烯基苯酞可以有机溶剂自伞形科或菊科植物萃取而得； 中国专利公告第 1041725C号揭露丁烯基苯酞以化学合成方式合成。
本发明所揭 "毛发" 一词包含个体身上各处的毛发， 包含有， 但不限于， 头发、 体毛、 睫毛、 眉毛。
本发明所揭 "萃取" 一词指利用物质在不同萃取剂中溶解度的差异， 将混 合物中特定成分由甲相转移到乙相中， 达到分离的目的， 例如溶剂萃取、 超临 界萃取。 一般来说， 萃取剂包含有， 但不限于， 丙酮、 氯仿、 二氧化碳。
本发明所揭 "化学合成技术" 一词指为得到特定产物而进行的一系列化学 反应， 如有机反应、 无机反应。
本发明所揭 "有效量" 一词指欲产生所求特定效果所需化合物或活性成分 的量， 可以其在组合物中所占重量百分比表示。 如同本发明所属技术领域中具 通常知识者所了解， 该有效量会因为欲引起特定效果的投予方式而有所不同。 一般来说， 活性成分或化合物在组合物中的量可占该组合物重量的约 1 %至约 100%， 较佳者为约 30%至约 100%。
本发明所揭 "药学或美容产品上能接受的载体" 一词包含任何标准在医药 或美容产品上所使用的载体， 而该载体依据组合物的型态， 得为固态、 半固态 或液态。 举例来说， 载体包含， 但不限于， 明胶、 乳化剂、 烃类混合物、 水、 甘油、 生理食盐水、 缓冲生理盐水、 羊毛脂、 石蜡、 蜂蜡、 二甲基硅油、 乙醇。
本发明所揭 "类似物"一词包含有化合物的盐类、 其酯类、 其结构异构物， 如 Z型结构或 E型结构， 或进行结构修饰后的产物。
本发明所揭高分子物质 F127， 其为聚氧乙烯-聚氧丙烯 -聚氧乙烯的三嵌段
CH3
聚合物， 化学式如式 （II) ： H(OCH2CH2 )x{OCH2CH)y (OCH2CH2)z QH ( π ) ， 其中， x、 y、 z分别为大于 1的整数。 由于高分子物质 F127两端的聚氧乙烯具有 亲水性， 中段的聚氧丙烯属于疏水性， 因此， 高分子物质 F127为两性物质。 当 高分子物质 F127存在于水溶液中时， 其会逐渐向界面扩散并吸附在界面上， 使 表面张力下降， 并且， 通过控制高分子物质内亲疏水基的比例， 能达到控制高 分子物质的表面活性。
本发明所揭 "分子间引力" 包含有， 但不限于， 凡得瓦力、 库伦作用力、 氢键、 疏水键力。
本发明所揭 "医药组合物" 一词包含一有效量的欲产生特定效果的所需化 合物或活性成分， 以及至少一药学上能接受的载体。 如同本发明所属技术领域 中具通常知识者所了解， 医药组合物的型态可随着欲引起特定效果的投予方式 有所不同， 如锭剂、 粉剂、 针剂等， 并且， 该载体也随着医药组合物的型态而 可为固态、 半固态或液态。 举例来说， 载体包含， 但不限于， 明胶、 乳化剂、 烃类混合物、 水、 甘油、 生理食盐水、 缓冲生理盐水、 羊毛脂、 石蜡、 蜂蜡、 二甲基硅油、 乙醇。 本发明所揭 "高分子物质" 一词指通过聚合反应而生成具有相当高分子量 的大分子， 一般来说， 高分子物质皆为有机分子。
以下， 为能更进一步说明本发明的功效， 将列举若干实施例作详细说明， 然而， 该等实施例为用以解说的例示， 其中所使用的任何词汇并不限制本发明 说明书及权利要求的范围及意义。
另 须加 以 说 明 的 是 ， 下列 实施例 中 所使用 的丁烯基苯酞
( n-butylidenephthalide， Bdph， W333301 ) 由美国西格玛奧瑞奇集团 ( Sigma-Aldrich) 所取得。
实施例 1 : Wnt活性测试
于六孔培养皿准备幼仓鼠肾脏纤维母细胞 BHK21数量约 5分满，每孔培养皿 分别以 5(^L的 Opti-MEM培养基 （ Invitrogen公司） 与 2 L的微脂粒 2000 ( Lipofectamine 2000 , Invitrogen公司） 于 1.5mL微量离心管混合 5分钟， 形成一 微脂粒混合液。
分别将 50μ 的 Opti-MEM培养基与 9.6 g的 Top-flash质体 DN A或 Fop-flash质 体 DNA混合， 形成一 Top-flash质体混合液及一 Fop-flash质体混合液， 其中， Top-flash质体具有原生型 TCF结合点 （wild-type TCF binding site ) ， 用以作为实 验组； Fop-flash质体具有突变 TCF结合点 （ mutant TCF binding site ) ， 控制组； 而该两种质体均接有冷光酶 （Luciferase ) 的序列。
于第一测试组中， 将 Top-flash质体混合液加入该微脂粒混合液中， 使其成 为一总体积为 ΙΟΟμΙ^的混合液， 共有 3组混合液。 将各组混合液于室温静置反应 20分钟后， 抽出各组该混合液， 分别加入培养皿中， 轻摇使其均匀分布， 再以 Opti-MEM培养基补满到液面刚好覆盖过细胞 BHK21 , 于 37°C培养 4小时， 而后 再以 Opti-MEM培养基补至每孔 2mL的培养基体积， 约 18〜24小时后更换培养 基， 并且给予各组不同培养条件， 其中， 第一组为空白组， 第二组加入 0.4μΜ 的化合物 ΒΙΟ， 第三组加入 0.4μΜ的丁烯基苯酞。 将各组再进行培养约 18〜24小 时， 而后分别自各组的该培养皿中收集细胞 BHK21 , 侦测其冷光酶活性， 结果 如图 1所示， 其中， *号表示在 0.05的显著水准之下。
于第二测试组中， 将该 Top-flash质体混合液或该 Fop-flash质体混合液的产 物加入该微脂粒混合液中，分别使其成为一总体积为 10( L的 Top-flash混合液或 Fop-flash混合液， 而共有 4组混合液， 其中， 第一组为 Fop-flash混合液， 第二至 四组皆为 Top-flash混合液。 将各组混合液于室温静置反应 20分钟， 并且抽出而 加入培养皿中， 轻摇使其均匀分布， 再以 Opti-MEM培养基补满到液面刚好覆盖 过细胞 BHK21 , 于 37°C培养 4小时， 而后再以 Opti-MEM培养基补至每孔 2mL的 培养基体积， 约 18〜24小时后更换培养基， 并且给予各组不同培养条件， 其中， 第一组加入 4μΜ的丁烯基苯酞， 第二组未添加任何化合物， 第三组加入 Ι μΜ的 丁烯基苯酞， 第四组加入 4μΜ的丁烯基苯酞。 将各组再进行培养约 18〜24小时， 而后分别自各组的该培养皿中收集细胞 BHK21 , 侦测其冷光酶活性， 结果如图 2 所示， 其中， *号表示在 0.05的显著水准之下。
而侦测冷光酶活性的方法如下： 先吸除细胞 BHK21的培养液， 再以磷酸盐 缓冲液清洗 2次。 每孔培养皿加入 20(^L的 IX PLB试齐 U ( Passive Lysis Buffer, Promega公司） ， 于室温下震荡 15分钟后， 待细胞裂解， 取其细胞溶解物， 再以 转速 12000rpm， 于 4°C下离心 1分钟后， 收集其上清液。 取 2(^L的上清液至 96孔 盘， 加入 10( L冷光酶分析试剂 （Luciferase Assay Reagent) ， 置入冷光仪， 以 波长 595nm测量冷光酶的活性。
由图 1的结果显示， 通过未经化合物处理的第一组， 其冷光值可作为实验的 参考值。 相对于第一组， 当细胞处理 0.4μΜ的化合物 BIO或丁烯基苯酞时 （第二 组及第三组） ， 有转染 Top-flash质体 DNA的细胞冷光值显著升高， 表示细胞中 TCF启动子被活化， 而使细胞冷光酶的活性及表现被侦测到。
由图 2的结果显示， 由于第一组具有突变的 Fop-Flash报导基因， 因此， 即使 加入丁烯基苯酞， 第一组中也无法侦测到冷光酶活性， 换言之， 细胞冷光值未 增加。 第二组至第四组的细胞冷光值随着丁烯基苯酞添加剂量增加而提升。 由 此可知， 冷光酶的活化具有专一性并且具有剂量效应。
如同过去文献中所指出， BIO为一种 Wnt活化剂，能与 Top-flash质体中的 TCF 结合点结合， 而于细胞 Wnt信号被活化下， 会促进冷光酶的表现， 并且， 由图 1 及图 2的结果可知， 丁烯基苯酞能随着添加剂量增加， 而能显著提高冷光酶的活 性， 据此， 本发明所揭丁烯基苯酞具有活化 Wnt信息的能力。
实施例 2: 动物试验
选取 6〜8周龄的 C57BL/6小公鼠， 分为三组， 每组 2只小公鼠， 而各该小鼠 的无毛处分别为背部近尾端处表皮， 面积为 2平方公分， 每日分别以不同条件予 以处理其无毛处， 经过 21天后， 观察各该组小鼠的毛发生长、 体重、 外观有无 异常， 其中， 第一组为对照组， 在无毛处仅涂抹磷酸生理食盐水； 第二组为实 验组， 在无毛处涂抹经乳化的组合物， 其含有丁烯基苯酞， 浓度为 10μΜ; 第二 组为实验组，在无毛处涂抹经乳化的组合物，其含有丁烯基苯酞，浓度为 100μΜ。 结果如图 3所示。 由图 3的结果显示， 各该组小鼠的体重不具有异常现象。 相较于第一组， 第 二组及第三组小鼠背部无毛处的毛发有明显增生， 并且， 第三组小鼠背部无毛 处的毛发增生现象更为显著。 由此可知， 涂抹本发明所揭丁烯基苯酞能有效促 进毛发增生， 并且增生的效果能随着剂量提升而显著增加。
实施例 3 : 合成水相丁烯基苯酞
取 自美国西格玛奧瑞奇集团 （ Sigma-Aldrich ) 取得丁烯基苯酞 ( n-butylidenephthalide , Bdph, W333301 ) 及 F127高分子物质 ( Pluronic F127 , P2443 ) 。 将 10毫克丁烯基苯酞与 F127高分子物质以重量比 1 : 1或 1 : 2的比例混 合后， 溶于 2毫升的四氢呋喃 （tetrahydrofuran) ， 再将之加入 10毫升的水中， 而后快速加热去除四氢呋喃， 进行冷冻干燥。 之后， 再回溶于水， 于溶液中乳 化后的丁烯基苯酞微粒大小约为 30〜200nm， 多分散指数（polydispersity index) 为 0.2-0.5。
实施例 4 : 细胞培养
将人类万能干细胞培养于无血清培养基 Essential 8™ ( Life Technology公司， 美国） 中， 并且以 matrigel TM基质胶 （ Becton-Dickinson公司， 美国） 进行贴附 培养。 将培养液吸除， 以磷酸盐缓冲液冲洗细胞 2次后， 加入酵素 Accutase TM ( Merck Millipore公司， 美国）反应 2〜5分钟后以培养液中和， 并且将细胞冲刷 下来后打散为小团块， 进行离心 1000rpm、 2分钟， 再吸除上清液， 放入新培养 皿中进行培养约 3〜5天， 进行继代培养， 且在培养期间须每天更换培养液。
实施例 5 : 神经细胞分化
将人类万能干细胞培养至 8-9分满， 以磷酸盐缓冲液冲洗细胞 2次后， 以酵 素 Accutase™作用 2〜5分钟后， 加入培养液稀释酵素， 再将细胞冲洗下来， 打 散至适当大小后以 800rpm的速度进行离心约 2分钟，再将细胞置于含有 20 %替代 血清 (knock out serum replacement, 简禾尔 KSR， Life Technology公司， 美国)的 DMEM-F12培养基， 以无盖培养皿进行悬浮培养 2天， 获得类胚体 （Embryonic body) 悬浮液。
将悬浮的类胚体置于离心管， 待自然沉降后， 去除上清液， 以含有 BiSF小 分子药物的神经诱导培养基进行悬浮培养 2天， 而后， 悬浮的类胚体会出现环状 的上皮细胞结构， 其中， 小分子药物包含有 0.5μΜ的 BIO ( Sigma-Aldrich公司， 美国） 、 10ng/mL的纤维母细胞生长因子 -2 ( FGF-2, Peprotech公司， 美国） 以 及 10μΜ的 SB431542 ( Sigma-Aldrich公司， 美国） ； 神经诱导培养液的成分如下 表 1所示。 表 1 神经诱导培养液的成分
Figure imgf000013_0001
再将类胚体进行沉降后， 放入含有 10ng/mL的纤维母细胞生长因子 -2的神 经基础培养基中进行培养， 其中， 神经基础培养基的成分如下表 2所示。 悬浮 2天后， 待类胚体沉降后， 以外力或酵素 Accutase™将之打散至小团块， 置于 已涂布 l % matrigel TM基质胶超过 1 小时的培养皿中进行细胞贴附。 大约 2〜7 天细胞会贴附、生长并且长出神经构造。而于细胞生长至 7〜8分满时进行分盘， 其中， 细胞分盘时使用磷酸盐缓冲液冲洗 1 次后， 以磷酸盐缓冲液与酵素 Accutase 以等比例混合的酵素溶液进行作用约 2〜5分钟，再以神经基础培养 基稀释， 而后使用细胞刮勺将细胞刮起并以机械力打散成为小团块或单细胞， 进行 800〜1000rpm的离心约 2〜5分钟， 再吸除上清液进行贴盘， 并且贴盘时 须加入 10μΜ的 Y27632 ( Stemgent公司， 美国） 1天， 以帮助细胞贴附。
表 2 神经基础培养基的成分
Figure imgf000013_0002
实施例 6: 细胞毒性试验
将人类胚胎干细胞 TW1培养于 Essential 8™培养基中， 并且在继代时分为 三组， 分别于 6孔盘放入 1.2xl 05的细胞量以下述条件进行培养， 其中， 第一组 为单纯以培养基进行培养；第二组在培养基中添加未包覆 F127高分子物质的丁 烯基苯酞， 浓度为 10μΜ; 第三组在培养基中添加如实施例 1所制得包覆 F127 高分子物质的丁烯基苯酞， 浓度为 10μΜ。 在培养过程中观察各组细胞型态， 并 且于培养 4〜5天， 进行细胞计数， 每组重复 1〜3次， 结果如图 4所示， 其中， 图 4的结果经单因子变异数分析（one-way ANOVA)及多重比较测定（Tukey's Multiple Comparison Test 分析） 而得， P值小于 0.05， 95 %信心水准， 记号 * 表示在 0.05的显著水准之下， 记号 * *表示在 0.01的显著水准之下。
由图 4的结果可知， 在培养 5天后， 第二组的细胞数量显著少于第一组或 第三组的细胞数量。 由此可知， 丁烯基苯酞会对于细胞具有较高的细胞毒性， 而通过包覆高分子物质 F127得有效减少丁烯基苯酞的细胞毒性。
实施例 7: T21诱导性万能干细胞进行神经细胞分化培养
请参阅实施例 4及图 5， 将 T21诱导性万能干细胞 （T21-iPSCs ) 依据实施 例 2所述进行神经分化， 并且在第 8天进行贴附。 在细胞贴附后第 12天进行免 疫荧光染色分析，用以观察神经干细胞标的，包含 N-cadherin、 Nestin以及 Pax-6 蛋白质的表现， 结果如图 6至图 8所示。 另观察 T21诱导性万能干细胞进行神 经分化培养后的神经突起生长状况， 在培养第 27天以免疫荧光染色分析， 通过
Tuj-1抗体观察其内如 βΠΙ微管蛋白质 （tubulin) 的成熟神经标记的表现， 结果 如图 9所示。
而免疫荧光染色分析的操作流程如下：将细胞培养于盖玻片的 4孔培养皿， 进行染色时， 先将细胞培养液吸除后， 以磷酸盐缓冲液冲洗 2次后， 加入 4%的 三聚甲醛 （paraformaldehyde, PFA) 于冰上作用 5分钟进行细胞固定后移除， 再以磷酸盐缓冲液冲洗 3次后， 加入 0.3%的 triton ( PBST)于冰上作用 10分钟 进行打洞， 再以磷酸盐缓冲液冲洗 3 次后， 加入浓度为 5%的马血清进行阻断 (blocking) 1 小时， 吸除液体并加入配置于为浓度 3%马血清的一级抗体， 室 温下作用 4小时或于 4°C反应 16小时。 之后， 吸除一级抗体并以 PBST冲洗液 冲洗 3次， 每次 5分钟。 二级抗体配置于磷酸盐缓冲中， 吸除 PBST后加入二 级抗体并避光作用 1小时后再以 PBST冲洗液冲洗 3次，加入浓度为 l g/mL的 细胞核染剂 DAPI室温避光作用 10分钟， 而后以 PBST冲洗液冲洗 2次， 再以 甘油与磷酸盐缓冲液以等比例混合成的溶液 20(^L浸湿后， 进行挑片， 封片后 在正立荧光显微镜下观察荧光。
由图 6至图 8的结果可知， T21诱导性万能干细胞于神经细胞分化培养第 12天时会表现 N-cadherin、 Nestin及 Pax-6神经干细胞特殊蛋白质， 显示 T21 诱导性万能干细胞以上述培养基进行培养后能成功分化成为 T21神经细胞。 再 者， 由图 9的结果可知， T21诱导性万能干细胞于经过 27天的培养分化后， 其 具有大量神经突起， 并且能表现为成熟神经标的的 βΠΙ微管蛋白质， 显示 T21 诱导性万能干细胞经过 27天的分化培养化成为成熟的 T21神经细胞。
实施例 8: 神经细胞的 Αβ40表现量 将贴附培养的 T21诱导性万能干细胞所分化的神经细胞进行细胞计数后， 贴盘于 4孔培养皿中， 每 48小时更换一次培养液， 将每 48小时收集的培养液 保存于 -20°C， 通过酵素连结免疫吸附分析法 （ELISA) 进行 Αβ40表现量的分 析， 并且使用核型正常的人类胚胎干细胞 TW1分化的神经细胞作为控制组， 其 中， 每组试验重复 2或 3次。 分析结果如图 10所示。
而酵素连结免疫吸附分析法的操作流程如下： 以培养缓冲液 （working incubation buffer) 将 ELISA套组的 Αβ标准蛋白进行序列稀释， 用以作为标准 曲线。 将收取的细胞培养液与该培养缓冲液以 1 : 2 的比例进行稀释。 分别取 10( L的标准品溶液及样本放入 96孔盘中， 置于 4°C、 16小时。 吸除液体后， 以 30(^L冲洗孔盘，并且晾干。加入 20(^L的 TMB受质，室温避光反应约 40〜 45分钟后， 读取 620nm的吸光值。
由图 10的结果可知， 于分化第 20天开始， 由 T21诱导性万能干细胞所分 化的神经细胞内 Αβ40表现量开始高于由人类胚胎干细胞 TW1所分化的神经细 胞， 并且在分化第 25天开始出现差异。 详言之， 由 T21诱导性万能干细胞所分 化的神经细胞内 Αβ40表现量， 在分化第 25天时为 105.38pg/mL， 在分化第 30 天时为 155.68pg/mL， 并在分化第 42天时为 264.47pg/mL。
据此可知， 由 T21诱导性万能干细胞所分化的神经细胞内 Αβ40表现量会 随着分化时间增加而显著上升， 因而能作为用以筛选治疗或预防神经退化性疾 病的平台。
实施例 9: 药物筛选
将 T21诱导性万能干细胞进行神经分化培养至第 39天时分为三组，分别给 予不同培养条件后， 再进行培养 3天， 即在第 42天时， 以酵素连结免疫吸附分 析各该组内的 Αβ40浓度， 并且与未进行神经分化培养的 T21诱导性万能干细 胞比较， 结果如图 11所示， 其中， 第一组为未进行神经分化培养的 T21诱导性 万能干细胞； 第二至四组为进行神经分化培养的 T21诱导性万能干细胞， 而第 二组在培养过程中未添加任何药剂，第三组在培养第 39天时添加 γ分泌素抑制 剂 DAPT, 第四组在培养第 39天时添加 ΙΟμΜ的包覆 F127高分子物质的丁烯 基苯酞。 每组试验进行 2〜3次， 并且以单因子变异数分析及多重比较测定分析 数值是否存在显数差异， 其中， Ρ值小于 0.05， 95 %信心水准， 记号 *表示在 0.05的显著水准之下， 记号 表示在 0.01的显著水准之下。
由图 11 的结果可知， 第一组的 Αβ40浓度显著低于第二组的 Αβ40浓度， 而第三组及第四组的 Αβ40 浓度分别较第二组下降。 由此可知， 本发明所揭丁 烯基苯酞能显著降低神经细胞内的 Αβ40浓度，并且其效果类似于投予 γ分泌素 抑制剂 DAPT。 据此， 本发明所揭丁烯基苯酞确实具有改善或减缓细胞内累积 过量 β类淀粉样蛋白质的能力， 而能达到治疗或预防神经退化性疾病的功效。
通过上述实验例结果可知， 本发明所揭丁烯基苯酞具有活化 Wnt信息的能 力， 而能确实地达到促进毛发增生的功效， 因此， 本发明所揭丁烯基苯酞或其 类似物能作为于促进毛发生长的外用组合物的活性成分， 而通过以如喷洒、 涂 抹等方式投予于个体特定部位的皮肤上， 达到促进毛发增生， 同时提升个体外 观的功效， 也能达到避免习知产品对于个体具有副作用的功效。 再者， 本发明 所揭医药组合物制备方法能以高分子物质作为药物载体， 减缓或降低丁烯基苯 酞的细胞毒性， 确实能有效提升医药组合物的安全性。 而通过上述方法所制得 的医药组合物确实具有降低细胞内 β类淀粉样蛋白质浓度的功能， 因此， 通过 投予有效量的医药组合物至生物体，能达到治疗或预防神经退化性疾病的功效。
以上仅是通过各该实施例详细说明本发明， 熟知该技术领域者于不脱离本 发明精神下， 而对于说明书中的实施例所做的任何简单修改或是变化， 均应为 本案权利要求书所涵摄。
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Claims

权利要求书
1、 一种将丁烯基苯酞、 其类似物或上述成分的组合用于制备促进毛发生长 的外用组合物的用途。
2、 根据权利要求 1所述的用途， 其特征在于， 丁烯基苯酞萃取自伞形科植 物。
3、根据权利要求 1所述的用途，其特征在于，丁烯基苯酞萃取自菊科植物。
4、 根据权利要求 1所述的用途， 其特征在于， 丁烯基苯酞或其类似物由化 学合成技术所制备而得。
5、 根据权利要求 1 所述的用途， 其特征在于， 丁烯基苯酞具有活化 Wnt 信号的能力。
6、 一种促进毛发增生的方法， 其将一促进毛发生长的外用组合物， 投予至 一个体的皮肤上， 其特征在于， 该外用组合物包含一有效量的丁烯基苯酞或其 类似物， 以及一药学或美容产品上能接受的载体。
7、根据权利要求 6所述的方法，其特征在于，所述皮肤为具有毛囊的区域。
8、 根据权利要求 6所述的方法， 其特征在于， 所述促进毛发生长的外用组 合物直接涂抹在所述个体的皮肤上。
9、 根据权利要求 6所述的方法， 其特征在于， 所述促进毛发生长的外用组 合物喷洒在所述个体的皮肤上。
10、 根据权利要求 6所述的方法， 其特征在于， 所述式 （I) 化合物的浓度 为 1 μΜ-1ηιΜ。
11、 一种治疗神经退化性疾病的方法， 其将一含有有效量的丁烯基苯酞、 其类似物或上述成分的组合的医药组合物投予至一个体。
12、 根据权利要求 11所述的治疗神经退化性疾病的方法， 其特征在于， 所 述神经退化性疾病为脑内累积过量 β类淀粉样蛋白质的病征。
13、 根据权利要求 11所述的治疗神经退化性疾病的方法， 其特征在于， 所 述神经退化性疾病为阿兹海默症。
14、 根据权利要求 11所述的治疗神经退化性疾病的方法， 其特征在于， 丁 烯基苯酞萃取自伞形科植物。
15、 根据权利要求 11所述的治疗神经退化性疾病的方法， 其特征在于， 丁 烯基苯酞萃取自菊科植物。
16、 根据权利要求 11所述的治疗神经退化性疾病的方法， 其特征在于， 丁 烯基苯酞或其类似物由化学合成技术所制备而得。
17、 一种医药组合物的制造方法， 其将丁烯基苯酞与高分子物质以重量比 1： 1〜1： 2混合后， 依序加入一极性有机溶剂及水， 使丁烯基苯酞与高分子物 质在水相通过分子间引力进行吸附作用而使该高分子物质包覆丁烯基苯酞， 再 去除该极性有机溶剂。
18、 根据权利要求 17所述的制造方法， 其特征在于， 所述极性有机溶剂为 类杂环醚类化合物。
19、 根据权利要求 18所述的制造方法， 其特征在于， 所述极性有机溶剂为 四氢呋喃。
20、 根据权利要求 17 所述的制造方法， 其特征在于， 所述高分子物质为
F127高分子物质。
21、 根据权利要求 17所述的制造方法， 其特征在于， 去除所述极性有机溶 剂的方法为加热法。
PCT/CN2014/083150 2014-07-28 2014-07-28 丁烯基苯酞的用途、其使用方法及将其制备为医药组合物的方法 WO2016015203A1 (zh)

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