WO2017117774A1 - 亚丁基苯酞的应用 - Google Patents

亚丁基苯酞的应用 Download PDF

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WO2017117774A1
WO2017117774A1 PCT/CN2016/070376 CN2016070376W WO2017117774A1 WO 2017117774 A1 WO2017117774 A1 WO 2017117774A1 CN 2016070376 W CN2016070376 W CN 2016070376W WO 2017117774 A1 WO2017117774 A1 WO 2017117774A1
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oral
cells
agent
fbmf
use according
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French (fr)
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韩鸿志
邱紫文
林欣荣
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Everfront Biotech Inc
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Everfront Biotech Inc
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Priority to JP2018534811A priority Critical patent/JP6559357B2/ja
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Priority to US16/068,257 priority patent/US10441566B2/en
Priority to PCT/CN2016/070376 priority patent/WO2017117774A1/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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the use of butylene benzoquinone to prevent and/or treat oral submucous fibrosis (OSF).
  • OSF oral submucous fibrosis
  • Oral submucous fibrosis is a pre-cancerous condition of oral cancer, but it is not oral cancer. It is mainly characterized by inflammatory reaction and fibrosis of oral submucosal tissue and deep connective tissue. Treatment is very likely to further evolve into oral cancer.
  • chewing betel nut is the most important cause of oral submucous fibrosis.
  • the lesion is common in the buccal mucosa, followed by the Palatal portion and the posterior caries.
  • the mucosa of the affected part will first turn white, then Repeated ulcers or blisters, and finally the mucosa will lose its elasticity, causing the patient to be unable to open the mouth, seriously affecting daily oral functions such as eating, brushing, oral examination and treatment.
  • the lesion occurs in the soft palate between the oropharynx, it may cause difficulty in swallowing, atrophy or deformation of the uvula.
  • the patient often has a feeling of burning, tingling, and dryness in the mouth. It is extremely sensitive to spicy, hot foods, but the taste is diminished.
  • butylene benzoquinone can effectively inhibit epithelial-mesenchymal transition (EMT) in oral submucosal cells, inhibit the differentiation of oral submucosal cells into myofibroblasts, and inhibit The activation of myofibroblasts inhibits collagen accumulation in the submucosal tissues of the oral cavity, inhibits contraction of interstitial cells in the submucosal tissues of the oral cavity, and provides a drug for preventing and/or treating oral submucous fibrosis.
  • EMT epithelial-mesenchymal transition
  • the active ingredient is selected from the group consisting of butylidenephthalide (BP), a pharmaceutically acceptable salt thereof, And the combination of the foregoing, and the agent is for preventing and/or treating oral submucous fibrosis (OSF).
  • the medicament is an injection, a lozenge, an oral solution, or an smear.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing and/or treating oral submucous fibrosis (OSF) comprising an effective amount of an active ingredient and a pharmaceutically acceptable A carrier wherein the active ingredient is selected from the group consisting of butylidenephthalide (BP), a pharmaceutically acceptable salt thereof, and combinations of the foregoing.
  • the pharmaceutical composition is an injection, a lozenge, an oral solution, or an application.
  • the active ingredient is selected from the group consisting of butylene benzoquinone (BP), a pharmaceutically acceptable salt thereof, or a combination of the foregoing.
  • BP butylene benzoquinone
  • the active ingredient can be administered to the individual in the form of an injection, a lozenge, an oral solution, or a spread.
  • FIG 1 shows buccal mucosal fibroblasts (BMFs; including “BMF-1 (ie, ⁇ )” and “BMF-2 (BMF-1), which are normal buccal mucosa treated with different concentrations of butylenephthalide (BP). That is, ⁇ )”) and fibrotic buccal mucosal fibroblasts (fBMFs; including "fBMF-1 (ie, ) and "fBMF-2 (ie, a plot of relative survival (%);
  • BMFs buccal mucosal fibroblasts
  • BMF-1 ie, ⁇
  • BMF-2 butylenephthalide
  • Figures 2A and 2B show the instantaneous quantitative polymerase chain reaction (Quanititative) Real-time polymerase chain reaction (Q-PCR) analysis of long-term plots of the relative expression (multiples) of Twist, Snail, and ZEB1 genes of fBMF-1 or fBMF-2 treated with different concentrations of butylenephthalide (BP) ,
  • Figure 2A is the result of fBMF-1
  • Figure 2B is the result of fBMF-2;
  • 3A is a photographic diagram showing the transition of BMFs and fBMFs treated with different concentrations of butylene benzoquinone (BP) from the upper tray to the lower tray, wherein the purple stained portion is a cell;
  • BP butylene benzoquinone
  • Figure 3B shows fMFs (including bMF-1 (blue bars), and BMF-2 (red bars)) and fBMFs treated with different concentrations of butylenephthalide (BP) (including fBMF-1 (blue) Bar graph of the relative mobility (%) of strips) and fBMF-2 (red strips);
  • BP butylenephthalide
  • FIGS. 4A and 4B are diagrams showing the ⁇ -SMA, Collal, and S100A4 genes of fBMF-1 or fBMF-2 treated with different concentrations of butylenephthalide (BP) by real-time quantitative polymerase chain reaction (Q-PCR). a bar graph of relative performance (multiple), wherein FIG. 4A is the result of fBMF-1, and FIG. 4B is the result of fBMF-2;
  • BP butylenephthalide
  • 5A is a photographic diagram showing the case where BMFs and fBMFs treated with different concentrations of butylenephthalide (BP) cause colloidal contraction, wherein the range enclosed by the green dotted line is the colloid;
  • BP butylenephthalide
  • Figure 5B shows fBMFs (including fBMF-1) treated with BMFs (including BMF-1 (blue strips), and BMF-2 (red strips)) and different concentrations of butylenephthalide (BP). Long strips of blue strips) and fBMF-2 (red strips) induced relative colloidal area (%) after shrinkage.
  • prevention, preventive, preventive, preventive, and prophylaxix refers to the ability to prevent or minimize the onset or deterioration of a disease or condition before it occurs.
  • Treatment means the eradication, removal, reversal, mitigation, improvement, or control of a disease or condition; the so-called “effective amount” or “therapeutically effective amount” means that at least part of the treatment is effective when administered to an individual.
  • EMT epithelial-mesenchymal transition
  • the genes such as Twist, Snail, and ZEB1 in the cells are highly expressed, and the polarity between the cells and cells is gradually lost, and the cell migration ability is increased and easily Crawling and invading, participating in tissue fibrosis.
  • Myofibroblasts are highly mobile cells that express marker genes such as ⁇ -SMA, Collal, and S100A4, and when activated, secrete excessive extracellular matrix (ECM), such as fibronectin ( Fibronectin), collagen (Collagen), and induces contraction in tissue interstitial cells, thereby promoting Tissue fibrosis. It has also been confirmed that excessive accumulation of extracellular matrix is also closely related to the occurrence of oral submucous fibrosis. For example, the role of epithelial-mesenchymal transition in oral squamous cell carcinoma and oral submucous fibrosis. Clin Chim Acta.
  • Oral submucous fibrosis An update on etiology And pathogenesis-A review.Rama Univ J Dent Sci.Mar;2(1):24-33(2015), Oral submucous fibrosis: Review on aetiology and pathogenesis.Oral Oncol.Jul;42(6):561-568( 2006), and Molecular pathogenesis of oral submucous fibrosis-acollagen metabolic disorder. J Oral Pathol Med. Jul; 34(6): 321-328 (2005), the entire disclosure of which is incorporated herein by reference.
  • EMT epithelial-mesenchymal transition
  • Xianxin can inhibit epithelial-mesenchymal transition (EMT) and inhibit oral submucosal tissue in oral submucosal cells.
  • the differentiation of cells into myofibroblasts and/or inhibition of activation of myofibroblasts can effectively inhibit excessive accumulation of extracellular matrix and inhibit contraction of tissue interstitial cells, and prevent and/or treat oral submucous fibrosis.
  • Betal-drived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.
  • the inventors of the present invention have found that for fibrotic oral submucosal tissue cells, if treated with butylene benzoquinone (BP), cells in the tissue can be effectively inhibited from undergoing epithelial-mesenchymal transition (EMT), particularly inhibiting the cells. It expresses genes such as Twist, Snail, and ZEB1, and inhibits the crawling and invasion of the cells, so that the effect of inhibiting fibrosis of the oral submucosal tissue can be achieved.
  • EMT epithelial-mesenchymal transition
  • the inventors of the present invention have further found that the treatment of fibrotic oral submucosal tissue cells with butylene benzoquinone (BP) can effectively inhibit the differentiation of cells into myofibroblasts and inhibit the activation of myofibroblasts in the tissue, especially inhibiting the inhibition.
  • the tissue cells express genes such as ⁇ -SMA, Collal, and S100A4, and inhibit the contraction of tissue interstitial cells, thereby achieving the effect of inhibiting fibrosis of the oral submucosal tissue.
  • the present invention relates to a medicament, a pharmaceutical composition, and a method for providing prevention and/or treatment of oral submucous fibrosis.
  • the medicament is manufactured using an active ingredient comprising an effective amount of the active ingredient and a pharmaceutically acceptable carrier comprising administering an effective amount of the active ingredient to the individual in need thereof .
  • the active ingredient is selected from the group consisting of butylenebenzaldehyde (BP), a pharmaceutically acceptable salt thereof, or a combination thereof.
  • examples of the pharmaceutically acceptable salt include, but are not limited to, alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts. Salts and strontium salts; transition metal salts such as zinc salts, copper salts, iron salts, cobalt salts, titanium salts, vanadium salts; aluminum salts; tin salts; alkanolamine salts such as diethanolamine salts, 2-amino groups - 2-ethyl-1,3-propanediol salt, and triethanolamine salt; heterocyclic amine salts such as?
  • the active ingredient used in the medicament, pharmaceutical composition, and method of the present invention is butylene benzoquinone (BP).
  • the agent or pharmaceutical composition of the present invention may be in a corresponding convenient dosage form depending on the desired form of administration.
  • the pharmaceutical or pharmaceutical composition can be administered to an individual in need via a route of administration such as intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, dermal, and mucosal.
  • the pharmaceutical or pharmaceutical composition of the present invention can be provided in the form of an injection, but is not limited thereto, as an example of a dosage form suitable for intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or the like.
  • examples of the injection include, but are not limited to, intravenous infusion, emulsion intravenous infusion, dry powder injection, suspension injection, and dry powder suspension injection.
  • the pharmaceutical or pharmaceutical composition can be prepared as a pre-injection solid, the pre-injection solid can be provided in a dosage form or emulsifiable dosage form which is soluble in other solutions or suspensions, and administered to the individual in need thereof. Previously, the pre-injection solid is dissolved in other solutions or suspensions or emulsified to provide the desired injection.
  • the pharmaceutical or pharmaceutical composition provided by the present invention may be, for example, a solid form of a tablet, a pill, a capsule, a granule, a powder, or the like, or an oral solution, a syrup, or an elixir, for example, in a dosage form suitable for oral administration.
  • Liquid type such as spirit), elixir, tincture, etc. are provided, but not limited thereto.
  • the dosage form of the present invention may be, for example, an emulsion, a cream, a gel (hydrogel), a paste (dispersion cream, an ointment), or the like, or a medicinal preparation, which is suitable for administration routes such as skin and mucous membranes. Oral, lotion, spray, or patch (patch)
  • the model is provided, but not limited to this.
  • the pharmaceutical agents and pharmaceutical compositions of the present invention are provided in the form of injections, lozenges, oral solutions, or spreads.
  • a suitable pharmaceutically acceptable carrier may be used to provide the agent of the present invention.
  • a pharmaceutical acceptable composition may be included in the pharmaceutical composition of the present invention.
  • Carrier examples include, but are not limited to, solvents (buffers, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof), oily solvents , diluent, stabilizer, absorption retarder, disintegrating agent, emulsifier, antioxidant, binder, binder, tackifier, solubilizer, dispersant, suspending agent, lubricant, moisture absorbent, solid carrier (eg starch, and bentonite).
  • solvents buffers, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof
  • oily solvents diluent, stabilizer, absorption retarder, disintegrating agent, emulsifier, antioxidant, binder, binder, tackifier, solubilizer, dispersant, suspending agent
  • the pharmaceutical or pharmaceutical composition of the present invention may be encapsulated in a drug delivery system such as a liposome, microparticles, or microcapsules, and then administered to an individual in need thereof to increase the activity in the drug or pharmaceutical composition.
  • a drug delivery system such as a liposome, microparticles, or microcapsules
  • the delivery efficiency of the component is such that the constituents of the drug delivery system do not adversely affect the desired benefit of the active ingredient (i.e., butylene benzoquinone (BP), its pharmaceutically acceptable salt, or a combination of the foregoing).
  • BP butylene benzoquinone
  • a suitable amount of an additive may be further added to the pharmaceutical or pharmaceutical composition provided by the present invention, for example, a flavoring agent (for example, sucrose) which improves the mouthfeel and visual sensation of the pharmaceutical or pharmaceutical composition when administered.
  • a flavoring agent for example, sucrose
  • the pharmaceutical or pharmaceutical composition may optionally contain one or more additional active ingredients (eg, steroids) or may be combined with a drug containing the one or more other active ingredients to further enhance the agent or medical treatment.
  • the medicament or pharmaceutical composition provided by the present invention may be administered at different frequencies, such as once a day, multiple times a day, or once a day, depending on the age, weight, and health condition of the individual to be administered.
  • frequencies such as once a day, multiple times a day, or once a day, depending on the age, weight, and health condition of the individual to be administered.
  • BP butylene benzoquinone
  • / kg body weight preferably about 10 mg / kg body weight to about 120 mg / kg body weight per day, more preferably about 20 mg / kg body weight to about 90 mg / kg body weight per day, wherein the unit "mg / kg body weight" It refers to the amount of drug required per kilogram of body weight. However, for acute patients, the amount can be increased according to actual needs, for example, increased to several times or tens of times.
  • agent or pharmaceutical composition provided by the present invention may be used in combination with one of the following to prevent and/or treat oral submucous fibrosis: surgical treatment, and laser treatment.
  • BP butylene benzoquinone
  • BMF-1 primary fibroblasts
  • BMF-2 BMF-2
  • fBMF-1 The fibrotic buccal mucosal fibroblasts
  • fBMF-2 The fibrotic buccal mucosal fibroblasts
  • BP butylenephthalide
  • Example 2 Analysis of the efficacy of butylene benzoquinone (BP) in inhibiting oral epithelial tissue cells for epithelial-mesenchymal transition (EMT)
  • BP butylene benzoquinone
  • EMT epithelial-mesenchymal transition
  • RNA (1 ⁇ g of each group) provided in [Preparation Example] B was taken for reverse transcription to provide complementary deoxyribonucleic acid (cDNA).
  • cDNA complementary deoxyribonucleic acid
  • Q-PCR quantitative polymerase chain reaction
  • the performance of the isogenic gene in fBMFs (including fBMF-1, fBMF-2) treated with different concentrations (0, 25, or 50 ⁇ g/ml) of butylenephthalide (BP).
  • BP butylenephthalide
  • the relative gene expression amount (multiple) of each group was calculated based on the results of cells treated without butyl benzoquinone (BP), and the results are shown in Fig. 2 .
  • BP butylene benzoquinone
  • This study further uses a penetrating cell migration test system (System, purchased from Corning, UK), with a polycarbonate membrane (polycarbonate membrane, available from Corning, UK) with a pore size of 8 microns, to investigate whether butylenebenzaphthalein (BP) can inhibit the crawling ability of oral submucosal cells. Invasive ability.
  • System purchased from Corning, UK
  • polycarbonate membrane polycarbonate membrane, available from Corning, UK
  • BP butylenebenzaphthalein
  • a culture solution containing 10% FBS fetal calf serum
  • FBS fetal calf serum
  • BMF-1, BMF-2, fBMF-1, and fBMF-2 cells (2 x 10 4 cells each) in [Preparation Example] A were taken, respectively, containing 0, 25, or 50 ⁇ g. /ml of butyl benzoquinone (BP) but serum-free medium (25O microliters) was mixed well, and the mixed solution was injected into the upper tray device.
  • BP butyl benzoquinone
  • serum-free medium 25O microliters
  • the penetrating cell migration test system was taken out, and the cells not transferred from the upper disc membrane surface to the lower disc membrane surface were removed, and the polycarbonate filter membrane was fixed with 4% paraformaldehyde (10 minutes). And stained with 0.1% crystal violet. Finally, the polycarbonate filter was carefully cut and placed on a glass slide, and observed under a microscope at 100 times magnification (5 different fields were observed in each group), photographed, and unp-butylene benzene. Based on the results of BMFs treated with ⁇ (BP), the number of cells on the membrane was converted into the relative mobility (%) of the cells.
  • the foregoing experiment was performed in three repetitions, and the results are shown in Figs. 3A and 3B, in which the portion dyed with purple in Fig. 3A is the cell that migrated from the upper disc to the lower disc, and Fig. 3B shows the result of the three repeated experiments. The average cell-to-migration ability (%) obtained.
  • fibrotic oral submucosal tissue cells have strong crawling ability and invasive ability compared with normal oral submucosal tissue cells, and easy epithelial-mesenchymal transition (EMT), but butylenebenzaphthalein (BP) It can effectively inhibit the crawling ability and invasive ability of oral submucosal tissue cells. That is, butylene benzoquinone (BP) can effectively inhibit epithelial-mesenchymal transition (EMT) in oral submucosal tissue cells, and thus can be used for preventing and/or treating fibrosis of oral submucosal tissues.
  • BP butylene benzoquinone
  • EMT epithelial-mesenchymal transition
  • Example 3 Analysis of the efficacy of butylene benzoquinone (BP) in inhibiting the differentiation of oral submucosal tissue cells into myofibroblasts
  • RNA The total RNA (1 ⁇ g from each group) provided in [Preparation Example] B was subjected to reverse transcription to provide complementary deoxyribonucleic acid (cDNA).
  • the genetic quantification system PRISM ABI7700 Sequence Detecting System, purchased from the United States
  • Applied Biosystems used a primer for specific genes (shown in Table 2 below) for real-time quantitative polymerase chain reaction (Q-PCR) to analyze genes such as ⁇ -SMA, Collal, and S100A4 at different concentrations (0, 25). , or 50 micrograms per milliliter of performance in butylenephthalide (BP) treated fBMFs (including fBMF-1, fBMF-2).
  • BP butylenephthalide
  • fBMFs including fBMF-1, fBMF-2
  • the relative gene expression amount (multiple) of each group was calculated based on the results of cells treated without butyl benzoquinone (BP), and the results are shown in Fig. 4.
  • BP butylene benzoquinone
  • Figs. 5A and 5B show the colloidal contraction caused by each group of cells, and Fig. 5B shows the relative colloidal area (%) of each group.
  • the colloidal area of the "fBMF group” is significantly smaller than that of the "BMFs group", that is, the colloid is significantly contracted.
  • the "fBMFs group” if the cells in the colloid are treated with butylene benzoquinone (BP), the shrinkage of the colloid is significantly improved, and the degree of improvement is with butylenebenzidine (BP).
  • BP butylene benzoquinone
  • the concentration increases and increases.
  • butylene benzoquinone can effectively inhibit epithelial-mesenchymal transition (EMT) in oral submucosal tissue cells, inhibit the differentiation of oral submucosal tissue cells into myofibroblasts, and inhibit the activation of myofibroblasts. It can inhibit the accumulation of collagen in the submucosal tissues of the oral cavity and inhibit the contraction of the interstitial cells in the submucosal tissues of the oral cavity.

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Abstract

一种使用一活性成分于制造一药剂的用途，其中该活性成分是选自以下群组：亚丁基苯酞(butylidenephthalide，BP)、其医药上可接受的盐、及前述的组合，其中该药剂是用于预防及/或治疗口腔粘膜下纤维化症(oral submucous fibrosis，OSF)且可以一针剂、锭剂、口服液、或涂抹剂的型式使用。

Description

亚丁基苯酞的应用 技术领域
本发明是关于使用亚丁基苯酞以预防及/或治疗口腔粘膜下纤维化症(oral submucous fibrosis，OSF)的应用。
背景技术
口腔粘膜下纤维化症为一种口腔癌的癌前病变(pre-cancerous condition)，但并非口腔癌，主要特征在于口腔粘膜下组织和深层结缔组织的发炎反应以及纤维化现象，若是不及时加以治疗，极有可能进一步演变成口腔癌。
流行病学的研究指出，嚼食槟榔是造成口腔粘膜下纤维化症最主要的原因，病灶常见于颊粘膜、其次是腭部(Palatal portion)与臼齿后区，患部黏膜首先会变白，接着反复出现溃疡或水泡，最后粘膜会失去弹性，造成患者无法将口张大，严重影响进食、刷牙、口腔检查与治疗等日常口腔功能。病灶若发生于口咽之间的软腭部位，可能导致吞咽困难、悬雍垂萎缩或变形。患者口内常有灼热、刺痛、干涩的感觉，对辛辣、热等食物极度敏感，但味觉却是减退。
目前并没有可有效预防及/或治疗口腔粘膜下纤维化症的药物，通常系依赖类固醇注射来减少胶原蛋白在口腔黏膜下组织中的含量、或是经由外科手术或雷射来切除病变组织。然而，类固醇注射仅可减缓症状，并不能治愈口腔粘膜下纤维化症，且经常伴随着月亮脸、变胖、水牛肩、骨质疏松、皮肤变薄、水肿、容 易感染、青春痘、长不高、血糖上升、感染率增加、口腔霉菌感染、体毛增多、伤口愈合力变差等副作用。此外，经由外科手术或雷射外科手术及雷射切除较大的病变组织后，伤口愈合时容易收缩而形成疤痕，需要考量张口受限以及美观的问题，于此，口腔外科医师会考虑以植皮的方式补救，但是植皮的部位会有遮掩早期复发病变的隐忧。以外科手术或雷射对年长的患者或病灶太大的患者进行治疗时，更是要考虑手术的伤害性、病人身体抵抗力与生活品质的问题。
有鉴于上述关于口腔粘膜下纤维化症治疗的问题，目前医药界仍致力于开发可预防及/或治疗口腔粘膜下纤维化症的药物。本案发明人研究后发现，亚丁基苯酞可有效抑制口腔粘膜下组织细胞进行上皮间质转换(epithelial-mesenchymal transition，EMT)、抑制口腔粘膜下组织细胞分化成肌纤维母细胞(myofibroblasts)、及抑制肌纤维母细胞的活化，故可抑制口腔粘膜下组织的胶原蛋白累积、抑制口腔粘膜下组织细胞间质内的收缩，用于提供预防及/或治疗口腔粘膜下纤维化症的药物。
发明内容
本发明的一目的，在于提供一种使用一活性成分于制造一药剂的用途，其中该活性成分是选自以下群组：亚丁基苯酞(butylidenephthalide，BP)、其医药上可接受的盐、及前述的组合，且该药剂是用于预防及/或治疗口腔粘膜下纤维化症(oral submucous fibrosis，OSF)。较佳地，该药剂为一针剂、锭剂、口服液、或涂抹剂。
本发明的另一目的，在于提供一种使用预防及/或治疗口腔粘膜下纤维化症(oral submucous fibrosis，OSF)的医药组合物，其包含一有效量的活性成分以及一医药上可接受的载剂，其中该活性成分是选自以下群组：亚丁基苯酞(butylidenephthalide，BP)、其医药上可接受的盐、及前述的组合。较佳地，该医药组合物为一针剂、锭剂、口服液、或涂抹剂。
本发明的又一目的，在于提供一种预防及/或治疗口腔粘膜下纤维化症(oral submucous fibrosis，OSF)的方法，其包含于有需要的个体中投予有效量的活性成分，其中该活性成分是选自以下群组：亚丁基苯酞(BP)、其医药上可接受的盐、或前述的组合。其中，该活性成分可以一针剂、锭剂、口服液、或涂抹剂的型式施用到该个体。
本发明的详细技术内容及部分具体实施态样，将描述于以下内容中，以供本发明所属领域具通常知识者据以明了本发明的特征。
附图说明
图1为显示经不同浓度的亚丁基苯酞(BP)处理的正常口腔颊粘膜的初代纤维母细胞(buccal mucosal fibroblasts，BMFs；包括「BMF-1(即，◆)」及「BMF-2(即，■)」)以及纤维化的口腔颊粘膜的初代纤维母细胞(fibrotic buccal mucosal fibroblasts，fBMFs；包括「fBMF-1(即，
Figure PCTCN2016070376-appb-000001
)」及「fBMF-2(即，
Figure PCTCN2016070376-appb-000002
)」的相对存活率(％)的曲线图；
图2A、2B为显示以即时定量聚合酶链锁反应(Quanititative  real-timepolymerase chain reaction，Q-PCR)分析经不同浓度的亚丁基苯酞(BP)处理的fBMF-1或fBMF-2的Twist、Snail、及ZEB1基因的相对表现量(倍数)的长条图，其中图2A是fBMF-1的结果，图2B则是fBMF-2的结果；
图3A为显示BMFs及经不同浓度的亚丁基苯酞(BP)处理的fBMFs自上层盘移行至下层盘的情形的照片图，其中染有紫色的部分即为细胞；
图3B为显示BMFs(包括BMF-1(蓝色长条)、以及BMF-2(红色长条))以及经不同浓度的亚丁基苯酞(BP)处理的fBMFs(包括fBMF-1(蓝色长条)、以及fBMF-2(红色长条))的相对移行能力(％)的长条图；
图4A、4B为显示以即时定量聚合酶链锁反应(Q-PCR)分析经不同浓度的亚丁基苯酞(BP)处理的fBMF-1或fBMF-2的α-SMA、Collal、及S100A4基因的相对表现量(倍数)的长条图，其中图4A是fBMF-1的结果，图4B则是fBMF-2的结果；
图5A为显示BMFs以及经不同浓度的亚丁基苯酞(BP)处理的fBMFs引起胶体收缩的情形的照片图，其中，绿色虚线所圈选的范围即系该胶体；以及
图5B为显示胶体经由BMFs(包括BMF-1(蓝色长条)、以及BMF-2(红色长条))以及经不同浓度的亚丁基苯酞(BP)处理的fBMFs(包括fBMF-1(蓝色长条)、以及fBMF-2(红色长条))诱导收缩后的相对胶体面积(％)的长条图。
具体实施方式
以下将描述根据本发明的部分具体实施态样；但是，在不背 离本发明精神下，本发明尚可以多种不同形式的态样来实践，不应将本发明保护范围解释为限于说明书所陈述者。此外，除非文中有另外说明，于本说明书中(尤其是在后述专利申请范围中)所使用的「一」、「该」及类似用语应理解为包含单数及复数形式。另，本说明书中所使用的「约」、「大约」或「近乎」等词，实质上代表与所述的数值相差在20％以内者，较佳在10％以内者，且更佳在5％以内者。
于本说明书中，所谓「预防(prevention、prevent、preventing、preventive、及prophylaxix)」，是指将在疾病或病症发病之前，使其发病或恶化得以回避、减至最小、或变得困难的能力；所谓「治疗」，是指包括根除、去除、逆转、缓和、改善、或控制一疾病或病症；所谓「有效量」或「治疗有效量」，是指投予至个体时，可有效至少部分改善怀疑个体的病情的化合物用量；所谓「个体」是指哺乳动物，哺乳动物可为人类或非人动物。
经研究证实，在口腔粘膜下纤维化的过程中，口腔粘膜下的组织细胞会进行上皮间质转换(epithelial-mesenchymal transition，EMT)，并分化成为肌纤维母细胞(myofibroblasts)。其中，当细胞进行上皮间质转换(EMT)时，细胞中的Twist、Snail、及ZEB1等基因会高度表现，同时细胞与细胞之间的极性会渐渐丧失，且细胞的移行能力增加而容易爬行及侵袭，参与组织纤维化。肌纤维母细胞是一种会表现α-SMA、Collal、及S100A4等标记基因且具备高度移动性的细胞，其活化后会分泌过多的胞外基质(extracellular matrix，ECM)，例如纤网蛋白(Fibronectin)、胶原蛋白(Collagen)，并诱导组织细胞间质内的收缩，因而促进 组织纤维化。亦经证实，过多的胞外基质累积亦与口腔粘膜下纤维化症的发生密切相关。前述可参见例如：The role of epithelial-mesenchymal transition in oral squamous cell carcinoma and oral submucous fibrosis.Clin Chim Acta.Aug；383(1-2)：51-56(2007)、Oral submucous fibrosis：An update on etiology and pathogenesis-A review.Rama Univ J Dent Sci.Mar；2(1)：24-33(2015)、Oral submucous fibrosis：Review on aetiology and pathogenesis.Oral Oncol.Jul；42(6)：561-568(2006)、以及Molecular pathogenesis of oral submucous fibrosis-acollagen metabolic disorder.J Oral Pathol Med.Jul；34(6)：321-328(2005)，该等文献的全文并于此处以供参考。
由于组织细胞的上皮间质转换(EMT)及肌纤维母细胞的活化与口腔粘膜下纤维化密切相关，咸信若能抑制口腔粘膜下组织细胞进行上皮间质转换(EMT)、抑制口腔粘膜下组织细胞分化成肌纤维母细胞、及/或抑制肌纤维母细胞的活化，即可有效抑制胞外基质过度累积、抑制组织细胞间质内的收缩，而可预防及/或治疗口腔粘膜下纤维化症。前述可参见例如：Betal-drived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.J Pathol.Feb；223(3)：366-377(2011)、Arecoline-inducedmyofibroblast transdifferentiation from human buccal mucosal fibroblasts is mediated by ZEB1.J Cell Mol Med.Apr；18(4)：698-708(2014)、以及Elevation of S100A4 expression in buccal mucosal fibroblasts by arecoline：involvement in  the pathogenesisoforal submucousfibrosis.PLoS One.8(1)：e55122(2013)，该等文献之全文并于此处以供参考。
本案发明人研究发现，对于纤维化的口腔粘膜下组织细胞，若以亚丁基苯酞(BP)进行处理，可有效抑制该组织中的细胞进行上皮间质转换(EMT)，尤其可抑制该细胞表现Twist、Snail、及ZEB1等基因、及抑制该细胞的爬行与侵袭，故可达到抑制口腔粘膜下组织纤维化的效果。
本案发明人另发现，以亚丁基苯酞(BP)对纤维化的口腔粘膜下组织细胞进行处理，可有效抑制该组织中细胞分化成肌纤维母细胞、及抑制肌纤维母细胞活化，尤其可抑制该组织细胞表现α-SMA、Collal、及S100A4等基因、及抑制组织细胞间质内的收缩，而可达到抑制口腔粘膜下组织纤维化的效果。
因此，本发明是关于提供预防及/或治疗口腔粘膜下纤维化症的药剂、医药组合物、及方法。其中，该药剂的制造是使用一活性成分，该医药组合物包含一有效量的活性成分以及一医药上可接受的载剂，该方法包含将有效量的活性成分投予至有需要的个体中。根据本发明的药剂、医药组合物、及方法，该活性成分是选自以下群组：亚丁基苯酞(BP)、其医药上可接受的盐、或前述的组合。
于本发明的药剂、医药组合物、及方法中，该医药上可接受的盐的例子包含但不限于：碱金属盐类，如钠盐及钾盐；碱土金属盐类，如钙盐、镁盐及钡盐；过渡金属盐类，如锌盐、铜盐、铁盐、钴盐、钛盐、钒盐；铝盐；锡盐；烷醇胺盐类如二乙醇胺盐、2-胺基-2-乙基-1，3-丙二醇盐、及三乙醇胺盐；杂环胺盐类如吗 福林盐(morpholine salts)、哌嗪盐(piperazine salts)、及哌啶盐(piperidine salts)；以及碱胺盐类如铵盐、精胺酸盐、离胺酸盐、及组胺酸盐等。较佳地，于本发明的药剂、医药组合物、及方法中所使用的活性成分是亚丁基苯酞(BP)。
当使用本发明的药剂或医药组合物以预防及/或治疗口腔粘膜下纤维化症时，该药剂或医药组合物可视所欲的投药形式而呈对应的合宜剂型。举例言之，但不以此为限，该药剂或医药组合物可经由皮内、肌肉、腹膜内、静脉、皮下、口服、皮肤、以及黏膜等投药途径而投予至有需要的个体。
以适于皮内、肌肉、腹膜内、静脉、皮下等投药途径的剂型为例，本发明的药剂或医药组合物可以针剂的型式提供，但不以此为限。举例言之，该针剂的例子是包括静脉输注液、乳剂静脉输注液、干粉注射剂、悬液注射剂、以及干粉悬液注射剂，但不以此为限。此外，可将该药剂或医药组合物制备成一注射前固体，以可溶于其他溶液或悬浮液中的剂型、或可乳化的剂型提供该注射前固体，并于投予至该有需要的个体之前，将该注射前固体溶于其他溶液或悬浮液中、或将其乳化，提供所欲的注射剂。
以适于口服投药的剂型为例，本发明所提供的药剂或医药组合物可以例如锭剂、丸剂、胶囊剂、颗粒剂、散剂等固体型式、或是例如口服液、糖浆剂、醑剂(spirit)、酏剂(elixir)、酊剂(tincture)等液体型式提供，但不以此为限。
以适于皮肤、黏膜等投药途径的剂型为例，本发明药剂或医药组合物可以例如乳液、乳霜、凝胶(水凝胶)、膏状物(分散膏、软膏)等涂抹剂、漱口剂、洗剂、喷雾剂、或是贴剂(贴片)的 型式提供，但不以此为限。
较佳地，本发明的药剂及医药组合物是以针剂、锭剂、口服液、或涂抹剂的型式提供。
于本发明中，视投药途径及/或投药形式而定，可选用合宜的医药上可接受的载剂以提供本发明药剂，另外，亦可于本发明医药组合物中包含一医药上可接受的载剂。举例言之，该医药上可接受的载剂的例子包含但不限于：溶剂(缓冲液、水、食盐水、葡萄糖(dextrose)、甘油、乙醇或其类似物、及前述的组合)、油性溶剂、稀释剂、安定剂、吸收延迟剂、崩散剂、乳化剂、抗氧化剂、粘合剂、结合剂、增粘剂、增溶剂、分散剂、悬浮化剂、润滑剂、吸湿剂、固体载剂(例如淀粉、及皂土(bentonite))。
视需要地，可将本发明的药物或医药组合物封装于微脂体、微粒子、或微胶囊等药物输送系统中，再投予至有需要的个体，以提高药物或医药组合物中的活性成分的输送效率，只要构成该药物输送系统的成分对该活性成分(即，亚丁基苯酞(BP)、其医药上可接受的盐、或前述的组合)的所欲效益没有不利的影响即可。
视需要地，亦可于本发明所提供的药剂或医药组合物中另含有合宜用量的添加剂，例如可提高该药剂或医药组合物于服用时的口适感及视觉感受的调味剂(例如蔗糖)、调色剂、着色剂等，以及可改善该药剂或医药组合物的稳定性及储存性的缓冲剂、保存剂、防腐剂、抗菌剂、抗真菌剂等。此外，该药剂或医药组合物可视需要另含一或多种其他活性成分(例如类固醇)，或者与含该一或多种其他活性成分的药物并用，以进一步加强该药剂或医 药组合物的功效或增加制剂配方的运用灵活性与调配度，只要该其他活性成分对本发明的活性成分(即，亚丁基苯酞(BP)、其医药上可接受的盐、或前述的组合)的所欲效益没有不利的影响即可。
可以一日一次、一日多次、或数日一次等不同频率施用本发明所提供的药剂或医药组合物，端视投予个体的年龄、体重、及健康况状而异。举例言之，当以口服方式施用至一个体以预防及/或治疗口腔粘膜下纤维化症时，以亚丁基苯酞(BP)计，其用量为每天约5毫克/公斤体重至约500毫克/公斤体重，较佳为每天约10毫克/公斤体重至约120毫克/公斤体重，更佳为每天约20毫克/公斤体重至约90毫克/公斤体重，其中，该单位『毫克/公斤体重』是指每公斤体重个体所须的投药量。但是，对于急性患者而言，其用量可视实际需要而酌增，例如增加至数倍或数十倍。
此外，亦可将本发明所提供的药剂或医药组合物与以下之一者并用，以预防及/或治疗口腔粘膜下纤维化症：外科手术治疗、及雷射治疗。
根据本发明的预防及/或治疗口腔粘膜下纤维化症的方法中，有关该活性成分(即，亚丁基苯酞(BP)、其医药上可接受的盐、或前述的组合)的投予途径、投予形式、适用剂量、以及相关治疗的应用，均如上述的说明。
兹以下列实施例进一步例示说明本发明。其中该等实施例仅提供作为说明，而非用以限制本发明的保护范围。本发明保护范围如权利要求书所限定的范围所示。
实施例
[制备实施例]：
A.细胞的培养
将二组取自正常口腔颊粘膜的初代纤维母细胞(buccal mucosal fibroblasts；以下简称为「BMF-1」及「BMF-2」；或统称为「BMFs」)、以及二组取自纤维化的口腔颊粘膜的初代纤维母细胞(fibrotic buccal mucosal fibroblasts；以下简称为「fBMF-1」及「fBMF-2」；或统称为「fBMFs」)，分别进行以下处理：以每孔2x104的细胞数分别接种(seed)于24孔盘中，并培养至8分满(即，达到80％confluence)，再分别以不同浓度(0、12.5、25、50、100、或200微克/毫升)的亚丁基苯酞(BP)进行处理，历时48小时。
B.细胞总RNA(Total RNA)的萃取
以胰蛋白酶(trypsin)使[制备实施例]A所提供的各组细胞悬浮后，分别进行以下步骤：(i)离心(1000rpm、5分钟)后去除上清液；(ii)加入1毫升的TRIzol试剂(购自Invitrogen Life Technologies公司)，混合均匀后，置于室温下静置5分钟；(iii)加入100微升的BCP(bromochloropropane)并上下摇晃使其混和均匀，再置于室温下静置5分钟；(iv)离心(Eppendorf离心机、F45-30-11转子、4℃、12000rpm、15分钟)后将上层液体移至新的离心小管，并于其中加入异丙酮(isopropanol)混和均匀，再置于室温下静置5分钟；(v)离心(Eppendorf离心机、F45-30-11转子、4℃、12000rpm、10分钟)后去除上清液，再以500微升的75％酒精清洗沈淀于管底的RNA；(vi)离心(室温、12000rpm、 5分钟)后去除酒精层，并置于抽风柜中风干；以及(vii)以20微升的经焦碳酸二乙酯(diethyl pyrocarbonate，DEPC)处理的水回溶RNA沉淀物，以测量其于260奈米波长下的吸光值，并计算RNA浓度。
实施例1：评估亚丁基苯酞(BP)的细胞毒性
取[制备实施例]A所提供之各组细胞，分别进行以下步骤：(i)去除24孔盘中的上清液，再于各孔中加入500微升的3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐(3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazoliumbromide，简称MTT)缓冲液(最终浓度为0.5毫克/毫升)；(ii)将孔盘放置于37℃、5％CO2的培养箱中作用，历时3个小时；(iii)去除上清液后，分别于各孔中加入1000微升的异丙醇，并将孔盘放置于震荡器(shaker)上摇晃，历时10分钟；以及(iv)从各孔取200微升的混合溶液至96孔盘中，以分光光度计测定其于570奈米波长下的吸光值，并换算成各组细胞的相对存活率(％)，结果示于图1。
由图1可知，于培养液中添加浓度为50微克/毫升以下的亚丁基苯酞(BP)，对正常颊黏膜的初代纤维母细胞(BMFs)以及纤维化颊黏膜的初代纤维母细胞(fBMFs)的生长皆不会有影响。因此，后续的实验是以浓度为0至50微克/毫升的亚丁基苯酞(BP)进行。
实施例2：分析亚丁基苯酞(BP)于抑制口腔粘膜下组织细胞进行上皮间质转换(epithelial-mesenchymal transition，EMT)的效益
A.上皮间质转换(EMT)的标记基因的表现量
已知细胞在进行上皮间质转换(EMT)的过程中，Twist、Snail、及ZEB1等基因的表现量会增加，故该等基因被视为上皮间质转换(EMT)的标记基因。本实验是通过即时定量聚合酶链锁反应(Quanititative real-time polymerase chain reaction，Q-PCR)，探讨亚丁基苯酞(BP)是否会影响Twist、Snail、及ZEBl等基因在口腔颊粘膜组织细胞中的表现。
首先，取[制备实施例]B所提供的总RNA(各组取1微克)，进行反转录作用，以提供互补去氧核醣核酸(cDNA)。接着，以基因定量系统(PRISMABI7700SequenceDetectingSystem，购自美国Applied Biosystems公司)搭配特定基因的引子(如下表1所示)进行即时定量聚合酶链锁反应(Q-PCR)，以分析Twist、Snail、及ZEB1等基因在经不同浓度(0、25、或50微克/毫升)的亚丁基苯酞(BP)处理的fBMFs(包括fBMF-1、fBMF-2)中的表现。最后，以未经亚丁基苯酞(BP)处理的细胞的结果为基准，计算各组的相对基因表现量(倍数)，结果示于图2。
表1
Figure PCTCN2016070376-appb-000003
由图2的结果可知，不论是fBMF-1或fBMF-2，Twist、Snail、 及ZEB1等基因的表现皆明显随着亚丁基苯酞(BP)的浓度提高而下降。前述结果显示，亚丁基苯酞(BP)可有效抑制口腔粘膜下组织细胞进行上皮间质转换(EMT)，故可用于预防及/或治疗口腔粘膜下组织的纤维化。
B.口腔粘膜下组织细胞的爬行能力与侵袭能力
本研究进一步以穿透式细胞移行试验系统(
Figure PCTCN2016070376-appb-000004
system，购自英国Corning公司)搭配孔径大小为8微米的聚碳酸酯滤膜(polycarbonatemembrane，购自英国Corning公司)，探讨亚丁基苯酞(BP)是否可抑制口腔粘膜下组织细胞的爬行能力与侵袭能力。
首先，于下层盘(lower chamber)加入含有10％FBS(胎牛血清)的培养液，并将该聚碳酸酯滤膜装置置于一细胞培养盘的底部，此称为上层盘(upper chamber)。另一方面，取[制备实施例]A中的BMF-1、BMF-2、fBMF-1、fBMF-2细胞(各取2×104个细胞)，分别与含有0、25、或50微克/毫升之亚丁基苯酞(BP)但不含血清的培养液(25O微升)混合均匀，再将混合溶液注入上层盘装置中。接着，将该穿透式细胞移行试验系统放置于培养箱中培养，诱使细胞移行，历时24小时。将该穿透式细胞移行试验系统取出，将未自上层盘膜面移行至下层盘膜面的细胞移除后，以4％的聚甲醛(paraformaldehyde)固定该聚碳酸酯滤膜(10分钟)，并以0.1％结晶紫(crystal violet)进行染色。最后，将该聚碳酸酯滤膜小心切下放置于载玻片上，于显微镜下以100倍的放大倍率进行观察(各组皆观察5个不同视野)、拍照纪录，并以未经亚丁基苯酞(BP)进行处理的BMFs的结果为基准，将该膜上的 细胞数目换算成细胞的相对移行能力(％)。前述实验是进行三重复，且结果是示于图3A、3B，其中，图3A中染有紫色的部分即是自上层盘移行至下层盘的细胞，图3B则是将该三重复实验的结果平均而得到的细胞相对移行能力(％)。
由图3A、3B的结果可知，相较于「BMFs组」，「fBMFs组」自上层盘移行至下层盘的细胞明显较多。然而，在「fBMFs组」组中，若细胞是先经亚丁基苯酞(BP)处理，则自上层盘移行至下层盘的细胞是明显随着亚丁基苯酞(BP)的浓度提高而减少。前述结果显示，相较于正常的口腔粘膜下组织细胞，纤维化的口腔粘膜下组织细胞具有较强的爬行能力与侵袭能力，容易进行上皮间质转换(EMT)，而亚丁基苯酞(BP)可有效抑制口腔粘膜下组织细胞的爬行能力与侵袭能力。此即，亚丁基苯酞(BP)可有效抑制口腔粘膜下组织细胞进行上皮间质转换(EMT)，故可用于预防及/或治疗口腔粘膜下组织的纤维化。
实施例3：分析亚丁基苯酞(BP)于抑制口腔粘膜下组织细胞分化成肌纤维母细胞的效益
已知在组织发生纤维化的过程中，组织中的细胞会分化、转变成肌纤维母细胞，而α-SMA、Collal、及S100A4等基因系肌纤维母细胞的标记基因。因此，本实验是通过即时定量聚合酶链锁反应(Q-PCR)，探讨亚丁基苯酞(BP)是否会抑制α-SMA、Collal、及S100A4等基因在纤维化口腔颊粘膜组织的细胞中的表现。
取[制备实施例]B所提供的总RNA(各组取1微克)，进行反转录作用，以提供互补去氧核醣核酸(cDNA)。接着，以基因定量系统(PRISM ABI7700 Sequence Detecting System，购自美国 Applied Biosystems公司)搭配特定基因的引子(如下表2所示)进行即时定量聚合酶链锁反应(Q-PCR)，以分析α-SMA、Collal、及S100A4等基因在经不同浓度(0、25、或50微克/毫升)的亚丁基苯酞(BP)处理的fBMFs(包括fBMF-1、fBMF-2)中的表现。最后，以未经亚丁基苯酞(BP)处理的细胞的结果为基准，计算各组的相对基因表现量(倍数)，结果示于图4。
表2
Figure PCTCN2016070376-appb-000005
由图4的结果可知，不论是fBMF-1或fBMF-2，α-SMA、Collal、及S100A4等基因的表现皆明显随着亚丁基苯酞(BP)的浓度提高而下降。前述结果显示，亚丁基苯酞(BP)具有抑制口腔粘膜下组织细胞分化成肌纤维母细胞的效益，故可抑制口腔粘膜下组织的胶原蛋白累积，有效预防及/或治疗口腔粘膜下组织的纤维化。
实施例4：分析亚丁基苯酞(BP)于抑制口腔黏膜下组织细胞间质内收缩的效益
取[制备实施例]A中的BMF-1、BMF-2、fBMF-1、fBMF-2细胞，分别将前述细胞溶于0.5毫升的浓度为2毫克/毫升的胶原蛋白溶液(购自Sigma-Aldrich)中，再将各该混合溶液移至24孔盘，并置于37℃、5％CO2的培养箱中作用2小时，使胶原蛋白胶体凝结， 所获得的胶体分别称为「BMFs组」、以及「fBMFs组」。接着，将凝结的胶体脱离培养盘，加入0.5毫升的含有不同浓度(0、25、或50微克/毫升)的亚丁基苯酞(BP)的细胞培养液进行培养，历时48小时后，观察各组胶体收缩的情形、拍照记录，并使用影像分析软体ImageJ(美国，国立卫生研究院)，以未经亚丁基苯酞(BP)处理的「BMFs组」的胶体面积为基准，计算各组的相对胶体面积(％)。结果示于图5A、5B，其中，图5A显示各组细胞所引起的胶体收缩的情形，而图5B是显示各组的相对胶体面积(％)。
由图5A、5B可知，相较于「BMFs组」，「fBMF组」的胶体面积明显较小，此即，胶体明显有收缩的现象。然而，在「fBMFs组」当中，若胶体中的细胞是先经亚丁基苯酞(BP)处理，则该胶体收缩的现象会明显改善，且改善的程度是随着亚丁基苯酞(BP)的浓度上升而增加。前述结果显示，亚丁基苯酞(BP)可有效抑制肌纤维母细胞的活化，而抑制肌纤维母细胞诱导的组织细胞间质内收缩。
由以上实验结果可知，亚丁基苯酞(BP)可有效抑制口腔粘膜下组织细胞进行上皮间质转换(EMT)、抑制口腔粘膜下组织细胞分化成肌纤维母细胞、抑制肌纤维母细胞的活化，故可抑制口腔粘膜下组织的胶原蛋白累积、抑制口腔粘膜下组织细胞间质内的收缩。

Claims (8)

  1. 一种使用一活性成分于制造一药剂的用途，其中该活性成分是选自以下群组：亚丁基苯酞(butylidenephthalide，BP)、其医药上可接受的盐、及前述的组合，且该药剂是用于预防及/或治疗口腔粘膜下纤维化症(oral submucous fibrosis，OSF)。
  2. 如权利要求1所述的用途，其中该药剂是用于抑制口腔粘膜下组织细胞进行上皮间质转换(epithelial-mesenchymal transition，EMT)。
  3. 如权利要求2所述的用途，其中该药剂是用于抑制口腔粘膜下组织细胞的爬行能力与侵袭能力。
  4. 如权利要求1所述的用途，其中该药剂是用于抑制口腔粘膜下组织细胞分化成肌纤维母细胞。
  5. 如权利要求1所述的用途，其中该药剂是用于抑制肌纤维母细胞的活化。
  6. 如权利要求1至5中任一项所述的用途，其中该药剂是用于抑制口腔粘膜下组织细胞间质内的收缩。
  7. 如权利要求6所述的用途，其中该药剂为一针剂、锭剂、口服液、或涂抹剂。
  8. 如权利要求1至5中任一项所述的用途，其中该药剂为一针剂、锭剂、口服液、或涂抹剂。
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