WO2015161759A1 - 稳定医药组合物 - Google Patents

稳定医药组合物 Download PDF

Info

Publication number
WO2015161759A1
WO2015161759A1 PCT/CN2015/076893 CN2015076893W WO2015161759A1 WO 2015161759 A1 WO2015161759 A1 WO 2015161759A1 CN 2015076893 W CN2015076893 W CN 2015076893W WO 2015161759 A1 WO2015161759 A1 WO 2015161759A1
Authority
WO
WIPO (PCT)
Prior art keywords
oil
component
pharmaceutical composition
polyethylene glycol
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2015/076893
Other languages
English (en)
French (fr)
Inventor
林欣荣
韩鸿志
邱紫文
邱郁涵
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Everfront Biotech Inc
Original Assignee
Everfront Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Everfront Biotech Inc filed Critical Everfront Biotech Inc
Priority to EP15783640.4A priority Critical patent/EP3135284B1/en
Priority to JP2017505699A priority patent/JP6312281B2/ja
Publication of WO2015161759A1 publication Critical patent/WO2015161759A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • 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
    • A61K9/0043—Nose
    • 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 a stable pharmaceutical composition containing a stable presence of n-butylidenephthalide (BP), and more particularly to a stable pharmaceutical composition containing a stable presence of butylenephthalide which can be administered intranasally. It is effective in the treatment of malignant brain tumors.
  • BP n-butylidenephthalide
  • Astrocytoma, anaplastic astrocytoma, and glioblastoma multiforme are common adult malignant brain tumors, of which polymorphic glia Tumors are the most common, with the highest degree of malignancy and the strongest invasiveness.
  • the intranasal administration method is an emerging administration method in drug treatment, which is a non-invasive administration method, which can reduce the risk of infection of a patient, and the medicine can be quickly absorbed through the nasal mucosa after inhalation through the nasal cavity to produce a drug effect without being affected by the liver. Metabolic exclusion of the first-pass effect, in addition Drugs absorbed from the nasal mucosa can bypass the blood-brain barrier and reach the central nervous system, thus reducing the systemic toxicity of the drug, and the effect is better than oral administration or intravenous administration.
  • N-butylidenephthalide is a new potential drug for the treatment of malignant tumors. It is a component derived from Angelica sinensis extract with a boiling point of 139 ° C to 142 ° C (5 mm Hg). Therefore, it is generally an oily liquid.
  • Butylenephthalide is currently known to be useful in the treatment of a variety of malignancies, including polymorphic gliomas and breast malignancies. Other studies have shown that butylene benzoquinone can also be used to inhibit autophagy of motor neurons. For example, US2014/0045765A1 reveals that butylene benzoquinone and its metabolites can be used to inhibit autophagy of motor neurons, and can be used for treatment.
  • Amyotrophic lateral sclerosis ALS
  • butyrene benzoquinone provides efficacy, it is a fat-soluble substance rather than a water-soluble substance, so there are still many limitations in application, including restrictions on oral formulations, injectable formulations, and intranasal formulations. Limitations of clinical application, and limitations in the use of animal studies such as small animals such as rats.
  • the present invention is directed to the research and development results of the foregoing requirements, and is effective for stabilizing butylene benzoquinone by a medium system, thereby more effectively applying butylene benzoquinone.
  • a pharmaceutical composition comprising: (a) a media system comprising a first component, a second component, and a third component, the first component being a phosphate buffer (phosphate) Buffered saline), the second component Selected from the group consisting of vegetable oils, animal oils, fatty acids, and combinations thereof, and the third component is selected from the group consisting of polyethylene glycol, dimethyl sulfoxide (DMSO), ethanol, polypropylene glycol, polysorbate, poly Oxyethylated vegetable oil, ethyl acetate, 2-hydroxyethyl 12-hydroxyoctadecanoate, tocopheryl polyethylene glycol succinate, and a combination thereof; and (b) n-butylidenephthalide (BP) present in the (a) medium system.
  • DMSO dimethyl sulfoxide
  • BP n-butylidenephthalide
  • 1A is an inverted microscope (IM) image of a pharmaceutical composition (400 times; scale bar: 50 microns) in accordance with an embodiment of the present invention
  • 1B is a scanning field emission electron microscope (FE-SEM) image of a pharmaceutical composition according to an embodiment of the present invention (10000 times; scale bar: 1 micron);
  • FE-SEM scanning field emission electron microscope
  • 1C is a transmission electron microscope (TEM) image of a pharmaceutical composition according to an embodiment of the present invention (80000 times; scale bar: 200 nm);
  • TEM transmission electron microscope
  • 2A is a photograph of a pharmaceutical composition and comparative pharmaceutical compositions according to various embodiments of the present invention stored at a low temperature of 4 ° C for a long period of time;
  • 2B is a photograph of a pharmaceutical composition and a comparative pharmaceutical composition according to various embodiments of the present invention stored at a low temperature of 4 ° C for a long period of time;
  • Figure 3 is a graph showing the activity of pharmaceutical compositions (different combinations of media systems) and comparative pharmaceutical compositions according to various embodiments of the present invention, wherein the vertical axis is GBM8401 cells (i.e., human malignant cell lines). Survival rate The composition group, that is, the aspect containing no butylene benzoquinone is 100%), and the horizontal axis is the treatment concentration of the active ingredient butylene benzoquinone;
  • Figure 4 is a graph showing the effect of storage time on the activity of the pharmaceutical composition according to the present invention, wherein the vertical axis is the survival rate of GBM8401 cells (in the control group, ie, the aspect containing no butylene benzoquinone is 100%) ), the horizontal axis is the treated concentration of butylene benzoquinone;
  • Figure 5 is a schematic view of a Franz diffusion device
  • Figure 6 is a graph showing the survival rate of rats administered with different administrations
  • Fig. 7A is a photograph showing the tumor growth condition of the rats after different treatments, after the implantation of the tumor, on the day of death or after the 37th day of treatment, wherein (A) was not treated with the drug (obtained on the day of death of the rat) (B) ) BP stock solution 160 mg / kg (day 37) (C) BP stock solution 320 mg / kg (day 37) (D) pharmaceutical composition 580 mg / kg (day 37) (E) pharmaceutical composition 5160 mg / Kilograms (day 37);
  • Figure 7B shows a graph of tumor weight statistics after the 37th day of treatment after the different treatments were administered to the rats after implantation of the tumor;
  • Figure 7C shows a graph of tumor volume after the 37th day of treatment, after the rats were implanted with different treatments.
  • a common application form is administration in a liquid dosage form containing the active ingredient and a medium which is soluble in the medium or stably present in the medium. More specifically, in combination with different routes of administration (such as oral, somatic cell introduction, or external use), the active ingredient is dissolved in the medium (if it is soluble) or suspended in the medium (if it is insoluble) to form a A solution, suspension or emulsion suitable for administration.
  • routes of administration such as oral, somatic cell introduction, or external use
  • the active ingredient is dissolved in the medium (if it is soluble) or suspended in the medium (if it is insoluble) to form a A solution, suspension or emulsion suitable for administration.
  • the active ingredient is usually dissolved or suspended in a liquid medium, and then the solid dosage form is prepared from the liquid medium in which the active ingredient is dissolved or suspended to accurately control the final form.
  • the concentration (content) of the active ingredient in the product is usually dissolved or suspended in a liquid medium, and then the solid dosage form is prepared from the liquid
  • butylene benzoquinone has the highest solubility for ethanol, and ethanol is an excellent solvent. Among them, the solubility of butylene benzoquinone in ethanol exceeds 222 mg / ml, but the solubility in polyethylene glycol 400, polyethylene glycol 300 or polypropylene glycol is only about 70 to 120 mg / ml.
  • studies have shown that infants and young children are not mature as alcohol dehydrogenase, and there is no way to metabolize ethanol as effectively as adults (see Zuccotti GV, Fabiano V.
  • butylene benzoquinone can be stably present in a specific medium system, and can be stably present in the medium system even after long-term storage (three days storage at room temperature; thirty days storage at 4 ° C). Maintaining activity, the media system can further increase the utilization of butylene benzoquinone in the organism, thereby being relatively low
  • the amount of butyl benzoquinone provides the desired therapeutic effect, slowing or eliminating unnecessary side effects.
  • the present invention provides a stable pharmaceutical composition
  • a stable pharmaceutical composition comprising (a) a media system and (b) butylene benzoquinone stably present in the (a) media system.
  • Butylene phenyl hydrazine is commercially available (for example, available from Changhong Biotech Co., Ltd., Taiwan), chemically synthesized, or extracted by extraction from natural materials (eg, Angelica sinensis). Further, before the extraction of the obtained butylene phenyl hydrazine by extraction, the purification method such as flash column chromatography, high performance liquid chromatography, or crystallization may be further used to enhance the butylene benzoquinone. Purity.
  • the (a) medium system comprises a first component, a second component, and a third component.
  • the first component is phosphate buffered saline
  • the second component is selected from the group consisting of vegetable oil, animal oil, fatty acid, and combinations thereof
  • the third component is selected from the group consisting of polyethylene glycol, Dimethyl sulfoxide, ethanol, polypropylene glycol, polysorbate, polyoxyethylated vegetable oil, ethyl acetate, 2-hydroxyethyl 12-hydroxyoctadecanoate, Tocopheryl polyethylene glycol succinate, and combinations thereof.
  • the phosphate buffer is isotonic and non-toxic to the cells.
  • Any suitable phosphate buffer may be employed in the pharmaceutical compositions of the invention, examples of which include aqueous solutions of NaCl (137 mM) in combination with KCl (2.7 mM) and Na 2 HPO 4 (10 mM), KH 2 PO 4 (1.8 mM), An aqueous solution of a combination of NaCl (137 mM) and KCl (2.7 mM), but is not limited thereto.
  • examples of vegetable oils are, for example, selected from one or more of the following groups: canola oil, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil , safflower seed oil, sesame oil, soybean oil, sunflower oil, almond oil, cashew oil, hazelnut oil, walnut oil, pecan oil, pine nut oil, pistachio oil and castor oil; examples of animal oils, for example From one or more of the following groups: fish oil, cod liver oil, lard, butter, sheep oil, chicken oil, and duck oil; and fatty acids can be saturated or unsaturated fatty acids (such as cis-unsaturated fatty acids, units are not Saturated fatty acids, polyunsaturated fatty acids, trans-unsaturated fatty acids, etc.).
  • modified castor oil is used as the second component, and examples of the modified castor oil include polyethoxylated castor oil and hydrogenated ethoxylated castor oil, but are not limited thereto.
  • Polyethoxylated castor oil also known as glycerol polyethylene glycol colinoleate, is commercially available, for example.
  • EL old name EL; BASF company.
  • EL is a nonionic co-solvent and emulsifier which can be obtained by reacting ethylene oxide with castor oil.
  • Hydrogenated ethoxylated castor oil can be obtained by reacting ethylene oxide with hydrogenated castor oil.
  • Examples of the hydrogenated ethoxylated castor oil include, but are not limited to, polyoxyl 40hydrogenated castor oil and polyoxyl 60 hydrogenated castor oil.
  • the third component of the (a) medium system to which the pharmaceutical composition of the present invention is applied is not limited to the above examples.
  • the medium system is composed of a phosphate buffer (first component), polyethoxylated castor oil (second component), and a component selected from the group consisting of the third component.
  • first component polyethoxylated castor oil
  • second component polyethoxylated castor oil
  • third component a component selected from the group consisting of the third component.
  • the third component is selected from the group consisting of polyethylene glycol, dimethyl sulfoxide, and ethanol
  • the butylene benzoquinone can provide a better stabilizing effect, and the provided pharmaceutical composition can be maintained for a long time.
  • the layering and butylene benzoquinone remain active.
  • the third component is a combination of polyethylene glycol and dimethyl sulfoxide or a combination of polyethylene glycol and ethanol
  • the efficacy of the butylene benzoquinone of the pharmaceutical composition of the present invention can be further improved, wherein
  • the third component is preferably a combination of polyethylene glycol and dimethyl sulfoxide. Therefore, in the (a) medium system of the pharmaceutical composition of the present invention, the third component is preferably selected from the group consisting of polyethylene glycol, dimethyl sulfoxide, ethanol, and combinations thereof, more preferably polyethylene glycol. In combination with dimethyl sulfoxide or a combination of polyethylene glycol and ethanol, a combination of polyethylene glycol and dimethyl sulfoxide is preferred.
  • the concentration of the butylenephthalide as the component (b) is not particularly limited as long as it can be stably present in the (a) medium system. Therefore, when the butylene benzoquinone is dissolved in the (a) medium system, the content of butylene benzoquinone is not higher than the solubility in (a) the medium system; and when the butylene benzoquinone is suspended in ( a) In the medium system, the content of butylene benzoquinone is not stratified
  • the amount of use is the principle. Under this principle, in some embodiments of the present invention, the concentration of butylenephthalide is from 0.001 mg/ml to 1500 mg/ml per ml of the (a) medium system.
  • the first component polyethoxylated castor oil as the second component, and the combination of polyethylene glycol or polyethylene glycol and dimethyl sulfoxide or ethanol as the third component, when (a) the medium system is provided (refer to the implementation) Examples 1, 5 and 6), the amount of butylene phenyl hydrazine as the component (b) can be stably present in the medium of 1,000 mg/ml, so that it can be confirmed that the butylene group is provided in the pharmaceutical composition provided.
  • the phenylhydrazine content can be at least 1,000 mg / ml media system.
  • the amount of the first component, the second component, and the third component selected may be adjusted. proportion.
  • the volume ratio of the first component to the second component may be 1:2 to 20:1, such as 1:1, 2:1, 3:1, 4:1, 5:1, 8:1, 10 : 1, 13: 1, 15: 1, 17: 1, 20: 1, etc.
  • the volume ratio of the first component to the third component may be 1:2 to 30:1, such as 1:1, 2:1 3:1, 4:1, 5:1, 8:1, 10:1, 13:1, 15:1, 17:1, 20:1, 25:1, 30:1, etc.
  • the proportion of each component in the mixture can be selected by the user (e.g., depending on the nature of each component).
  • the mixing ratio is not particularly limited, and ethanol: polyethylene glycol can be used.
  • the volume ratio is, for example, 10:1 to 1:100, preferably 6:1 to 1:50, more preferably 1:1 to 1:10 to mix the two.
  • polyethylene glycol is the third component
  • the volume ratio of the phosphate buffer solution to the polyethoxylated ramie oil can be 3:0.5 to 5 (for example, 3:1)
  • the phosphate buffer solution may be from 3:0.5 to 5 (for example, 3:1).
  • the phosphate buffer solution and the polyethoxylate are used.
  • the volume ratio of castor oil may be 1 to 10:1 (for example, 5:1)
  • the volume ratio of phosphate buffer to polyethylene glycol and dimethyl sulfoxide may be 1 to 5:1 (for example, 3) :1)
  • the ratio of polyethylene glycol to dimethyl sulfoxide may be from 1 to 2:1 to 2 (for example, 1:1).
  • the composition (b) butylene benzoquinone is dissolved in the medium system or stably present in the medium system, thereby allowing the composition to form a solution suitable for the delivery or administration of butylene phenyl hydrazine.
  • An emulsion or suspension allows the pharmaceutical composition to be administered to a subject in need thereof by oral, injection, and/or intranasal administration.
  • the pharmaceutical composition of the present invention can promote animal studies using small animals such as mice or rats weighing only 25 grams. For example, using 25 grams of mice as an animal model for research In experiments for the efficacy of butylphthalide, it is generally necessary to use 2 to 3 microliters ( ⁇ L) of a very small amount of butylenephthalide, however, since this amount is too small, it is quite difficult to measure.
  • ⁇ L microliters
  • the pharmaceutical composition of the present invention since the butylene benzoquinone is stably present in the medium and diluted by the medium, a small amount of the active ingredient, butylene benzoquinone, can be mixed with a large amount of the medium system to A larger volume of the pharmaceutical composition of the present invention provides the desired very small amount of butylene benzoquinone, and as such, it will be relatively easy and precise in metering. Therefore, the pharmaceutical composition of the present invention can also be used for the preparation of a solid dosage form of the active ingredient, which can accurately control the concentration of the active ingredient in the final product.
  • the pharmaceutical composition of the present invention can be prepared by simply mixing (a) a medium system with (b) butylene benzoquinone.
  • a medium system In the case where (b) butylene benzoquinone is soluble in the (a) medium system, the butylene phenyl hydrazine may be directly added to the medium system to be dissolved therein; and (b) the butylene phenyl hydrazine is insoluble.
  • a physical force such as shaking
  • the pharmaceutical compositions of the present invention may optionally contain other additives as long as these additives or other active ingredients do not adversely affect (b) the efficacy of butylenephthalide and its stability and/or solubility in (a) the media system.
  • a suitable amount of preservative, preservative, antibacterial agent, antifungal agent, etc. may be added to further improve the storage property of the prepared pharmaceutical composition; one or more other activities may also be added to the pharmaceutical composition.
  • Ingredients such as antioxidants (such as vitamin E, vitamin C, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA)), chemicals, immunomoduls, etc.
  • antioxidants such as vitamin E, vitamin C, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA)
  • BHA butylated hydroxyanisole
  • the invention is further illustrated by the following examples. Among them, the added butylene phenyl hydrazine was converted by volume, but the density was 1 g/ml, and the corresponding weight was obtained.
  • the following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.
  • the pharmaceutical composition was placed at room temperature for a specified time (three days or seven days), and the appearance was observed for delamination. The stratification was recorded as "V” and the stratification was recorded as "X” to distinguish the appearance stability. Advantages and disadvantages; and the amount of impurities in the pharmaceutical composition was measured by a relative retention time (RRT) using a liquid chromatography mass spectrometer under the following conditions.
  • RRT relative retention time
  • the emulsion of the pharmaceutical composition 2 was prepared in the same manner as in Example 1, except that the proportion of each component was changed as shown in Table 1.
  • An emulsion of the pharmaceutical composition 3 was prepared in the same manner as in Example 1, except that dimethyl sulfoxide (DMSO) was used as the third component, and the proportion of each component was changed as shown in Table 1.
  • DMSO dimethyl sulfoxide
  • the emulsion of the pharmaceutical composition 4 was prepared in the same manner as in Example 1, except that the proportion of each component was changed as shown in Table 1.
  • An emulsion of the pharmaceutical composition 5 was prepared in the same manner as in Example 1, except that a combination of PEG and DMSO was used as the third component, and the proportion of each component was changed as shown in Table 1.
  • An emulsion of the pharmaceutical composition 6 was prepared in the same manner as in Example 1, except that a combination of PEG and ethanol was used as the third component, and the proportion of each component was changed as shown in Table 1.
  • the emulsion of Comparative Pharmaceutical Composition 1 was prepared in the same manner as in Example 1, except that the second component (K-EL) was not added and the PEG was changed.
  • the combination of HS 15 (hereinafter referred to as "HS 15"; BASF Corporation; batch number: 14225516K0; CAS number: 61909-81-7) was used as the third component, as shown in Table 1.
  • Table 3-1 Variation of impurity concentration (volume percentage) of pharmaceutical composition 1
  • the pharmaceutical composition of the present invention can be maintained at room temperature for up to 3 days without delamination, especially in some aspects (Examples 2 to 4) and can be stored for 7 days at room temperature. Without delamination, it can be seen from Table 4 that the pharmaceutical composition of the present invention has a very small change in the concentration of butylene benzoquinone even after long-term storage, and the maintenance ratio is extremely excellent.
  • the pharmaceutical composition of the present invention has excellent stability, and the active ingredient can be stably present in the medium system.
  • the concentration of the active ingredient butylene benzoquinone in the pharmaceutical compositions 1, 5 and 6 per milliliter of the medium system corresponds to about 1,000 mg/ml of the medium system.
  • the pharmaceutical composition emulsion was formulated into a concentration of 200 ⁇ g/ml as a sample, and then a high concentration nanometer particle size and interface potential analyzer (Malvern Nano-ZS) ZEN-3600) analyzes the conductivity of the sample, the interfacial potential, and the average particle size of the emulsified particles.
  • a high concentration nanometer particle size and interface potential analyzer (Malvern Nano-ZS) ZEN-3600) analyzes the conductivity of the sample, the interfacial potential, and the average particle size of the emulsified particles.
  • the medical composition diluted 500 times with pure water was used as a sample, and the microstructure thereof was observed by an inverted microscope (IM), a scanning field emission electron microscope (FE-SEM), and a transmission electron microscope (TEM), respectively.
  • IM inverted microscope
  • FE-SEM scanning field emission electron microscope
  • TEM transmission electron microscope
  • compositions 7 to 11 were prepared in the same manner as in Example 1, except that the composition of the adjustment medium system and the active ingredients were as shown in Table 5.
  • the concentration of the active ingredient butylene phenyl hydrazine in the pharmaceutical compositions 7 to 11 per milliliter of the medium system is about 105 mg / ml medium system (pharmaceutical composition 7), 156 mg / ml medium system (pharmaceutical composition 8 ), 208 mg/ml media system (Pharmaceutical Composition 9), 163 mg/ml media system (Pharmaceutical Composition 10) and 111 mg/ml media system (Pharmaceutical Composition 11).
  • the conductivity, the interface potential, and the average particle size of the emulsified microparticles contained in the comparative pharmaceutical composition 1 were analyzed by the method described in point 2.1.1, the BP stock solution (BP stock solution), the pharmaceutical compositions 1 and 5 to 11 and the comparative pharmaceutical composition 1. And the results are recorded in Table 6 below.
  • the butylene benzoquinone particles contained in the pharmaceutical composition of the present invention are fine particles having a size ranging from 29.89 nm to 1.22 ⁇ m, and the interface potential is between -6.80 and -1.55 mV (mV).
  • the conductivity is between 12.1 and 14.93 millisiemens per centimeter (mS/cm), which is shown to be an oil-in-water (O/W) type emulsion.
  • composition 6 320.75 -6.60 12.7 Pharmaceutical composition 7 80.05 -3.65 14.1 Pharmaceutical composition 8 112.78 -2.41 12.3 Pharmaceutical composition 9 29.89 -2.52 12.1 Pharmaceutical composition 10 143.79 -1.55 12.7 Pharmaceutical composition 11 96.28 -2.02 12.7 Comparative pharmaceutical composition 1 318.31 -6.80 12.6
  • the viscosity of the butylene benzoquinone stock solution, the pharmaceutical compositions 1 and 5 to 11 and the comparative pharmaceutical composition 1 were analyzed by the method described in point 2.1.2, and the average viscosity was calculated. The results are reported in Table 7 below. As shown in Table 7, the viscosity of the butylene benzoquinone stock solution and the pharmaceutical composition all showed a decrease in viscosity with an increase in the shear strain rate, and therefore all belonged to a shear thinning liquid in a non-Newtonian fluid. Further, the viscosity of each pharmaceutical composition is between 0.62 ⁇ 10 -2 and 10.89 ⁇ 10 -2 Pa ⁇ s (Pa.s).
  • composition 10 1.99 ⁇ 0.06
  • Pharmaceutical composition 11 0.62 ⁇ 0.02 Comparative pharmaceutical composition 1 4.49 ⁇ 0.30
  • FIG. 1A is an IM diagram of the pharmaceutical composition 5 (400 times; scale bar: 50 Micron)
  • FIG. 1B is an FE-SEM image of the pharmaceutical composition 5 (10,000 times; scale bar: 1 micrometer)
  • FIG. 1C is a TEM image of the pharmaceutical composition 5 (80,000 times; scale bar: 200 nm).
  • the active ingredient butylidene benzoquinone
  • the active ingredient has a particle size of nanometer to micrometer-sized particles and has a substantially spherical shape.
  • GBM 8401 cells ie, human malignant brain tumor cell lines
  • GBM 8401 cells were cultured in 96-well culture plates at a cell number of 6 ⁇ 10 3 per well, followed by treatment with butylene benzoquinone stock solution or different pharmaceutical compositions for 24 hours.
  • the active ingredient of butylenephthalide was treated at a concentration of 0 ⁇ g/ml (control group), 12.5 ⁇ g/ml, 25 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml, and 200 ⁇ g/ml, respectively.
  • the half-inhibitory concentration of each pharmaceutical composition on the survival of GBM 8401 cells was subsequently analyzed by MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell survival test method (half maximal inhibitory concentration). , IC 50 ) to assess the effect of poisoning.
  • the pharmaceutical composition 5 was stored in batches at 4 ° C for 0, 7, 14, 30 or 60 days, respectively.
  • GBM 8401 cells were cultured in 96-well culture plates at a cell number of 6 ⁇ 10 3 per well, and treated with pharmaceutical composition 5 stored for different time periods, for 24 hours, and then analyzed by MTT cell survival test method.
  • different days storage pharmaceutical composition for 5 GBM 8401 cells surviving half inhibitory concentration (IC 50), to assess the effect of storage time for the toxic effects of the pharmaceutical composition.
  • the active ingredient of butylenephthalide was treated at a concentration of 0 ⁇ g/ml (control group), 12.5 ⁇ g/ml, 25 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml, and 200 ⁇ g/ml, respectively.
  • the ability to penetrate the artificial fiber membrane by using a Franz diffusion device (Sanmei Glass Instrument Line) as shown in FIG. 5 was used to simulate the penetration of the artificial membrane into a nasal mucosa, wherein the device was used.
  • the diffusion surface area is approximately 4.5 square centimeters and the receiving cell has a capacity of approximately 23 milliliters.
  • test methods include: (i) cutting the artificial fiber membrane to an appropriate size and placing it in Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (118 mM NaCl + 1.2 mM MgSO 4 + 4.8 mM KCl) Soaking in +5.5 mM D-glucose + 2.5 mM CaCl 2 + 20 mM Hepes) for 10 minutes; (ii) adding 2 ml of a solution of butylene benzoquinone or a pharmaceutical composition at a concentration of 3 mg/ml to the supply tank; Add Hepes buffer to the receiving tank and stir it by magnetic stirring (rotation speed: 300 rpm); (iv) test at a temperature of 37 ° C, and sample 1 ml of the sampling tube of the receiving tank at intervals of 1 hour.
  • A diffused surface area (square centimeters)
  • RPMI 2650 cells i.e., human squamous cell strains in the nose
  • RPMI 2650 cells were inoculated into a cell insert coated with collagen having a pore size of 0.4 ⁇ m and cultured for 2 days to form cells on the cell membrane.
  • Cell monolayer Next, the cell septum disk was transferred to a 12-well plate to which 1.5 ml of Hepes buffer had been added, and 0.5 ml of a Hebutyl buffer containing a butylene benzoquinone stock solution or a pharmaceutical composition was added to the cell septum tray.
  • the 12-well plate was placed in a 37 ° C, 5% CO 2 incubator, and the buffer in the 12-well plate was collected at intervals of 1 hour, and the absorption at 310 nm was measured by a spectrophotometer.
  • the spectrum was calculated for the concentration of butylenephthalide or the pharmaceutical composition for a total of 6 hours, and the effective penetration (P eff ) and flow rate (J) were calculated by using the above formula 1 and formula 2.
  • the stability of the pharmaceutical composition 1, the comparative pharmaceutical composition 1, the pharmaceutical composition 5, and the pharmaceutical composition 6 at a low temperature was tested by the method set forth in point 3.2.1, and the results are shown in FIG. 2A and FIG. 2B.
  • 2A and 2B are photos before and after storage.
  • the pharmaceutical compositions (pharmaceutical compositions 1, 5 and 6) of the present invention have no significant change in appearance and delamination before and after storage for a long period of time at a low temperature of 4 ° C (up to 30 days).
  • the pharmaceutical composition of the present invention (Comparative Pharmaceutical Composition 1)
  • the foregoing results show that the pharmaceutical composition of the present invention is excellent in stability.
  • composition 5 28.10 ⁇ 2.71
  • Pharmaceutical composition 6 81.97 ⁇ 5.87 Comparative pharmaceutical composition 1 62.34 ⁇ 6.36
  • the poisoning effect stability test of the pharmaceutical composition 5 was carried out by the method described in point 3.2.3, and the results are shown in Fig. 4. As can be seen from Fig. 4, compared with the control group, regardless of the storage time of the pharmaceutical composition 5, the survival rate of the treated group of GBM 8401 cells remained as the dose of the active ingredient butylene benzoquinone increased. law. The foregoing results show that the poisoning effect of the pharmaceutical composition 5 on malignant brain tumor cells is not affected by the storage time, and is a stable formula.
  • the in vitro artificial fiber membrane penetration test was carried out on the butylene benzoquinone stock solution, the pharmaceutical composition 1, the comparative pharmaceutical composition 1, the pharmaceutical composition 5 and the pharmaceutical composition 6 by the method described in point 3.2.4, and the results were recorded in the following table. 9.
  • the effective penetration rate (P eff ) of the pharmaceutical composition 5 and the pharmaceutical composition 6 was the highest, and the effective penetration rate of the butylene benzoquinone stock solution was the lowest.
  • the foregoing results show that the penetrating ability of the pharmaceutical composition of the present invention is remarkably better than that of the butylene benzoquinone stock solution, and the penetrating ability of the pharmaceutical composition 5 and the pharmaceutical composition 6 is optimal.
  • the in vitro human cell strain penetration test was performed on the butylene benzoquinone stock solution, the pharmaceutical composition 1, the comparative pharmaceutical composition 1, the pharmaceutical composition 5, and the pharmaceutical composition 6 by the method described in point 3.2.5, and the results were recorded in the following table. 10.
  • the effective penetration rate (P eff ) of the pharmaceutical composition 6 was the highest, and the effective penetration rate of the butylene benzoquinone stock solution was the lowest.
  • the foregoing results show that the ability of the pharmaceutical composition of the present invention to penetrate cells is significantly better than that of the butylene benzoquinone stock solution, and the ability of the pharmaceutical compositions 5 and 6 to penetrate cells is preferred.
  • the tumor tissue of 9L (ie, rat malignant brain tumor cell line) was intracranially injected into the rat, and a drug treatment test was performed after the tumor was generated intracranially.
  • the test methods included: (i) anesthetizing the rats with 400 mg/kg body weight of chloral hydrate (Sigma-Aldrich), and after coma, shaving the head hair of the rats and keeping the rats Fixed to the Lab Standard TM Stereotaxic Instrument; (ii) cut the scalp with a large scissors to expose the skull, centered on the brain bregma, 3 mm to the right and 5 mm to the left.
  • the two groups of rats were significantly improved after intranasal administration of the solution of butylene benzoquinone (160 mg/kg) and the pharmaceutical composition 5 (80 mg/kg) for 30 days. Similar survival rates, as well as significantly decreased and similar tumor volume and tumor weight. This result shows that the effective dose of butylene benzoquinone in the pharmaceutical composition of the present invention requires only half of the effective dose of the butylene benzoquinone stock solution to achieve the same therapeutic effect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Otolaryngology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

一种稳定医药组合物，包含：（a）一介质系统，包含第一成分、第二成分、及第三成分，该第一成分为磷酸盐缓冲液，该第二成分选自以下群组：植物油、动物油、脂肪酸及其组合，且该第三成分选自以下群组：聚乙二醇、二甲亚砜、乙醇、聚丙二醇、聚山梨醇酯、聚氧乙基化蔬菜油、乙酸乙酯、12-羟基硬酯酸羟乙酯、维生素E聚乙二醇琥珀酸酯、及其组合；以及（b）亚丁基苯酞（n-butylidenephthalide，BP）。

Description

稳定医药组合物 技术领域
本发明是关于一种含有稳定存在的亚丁基苯酞(n-butylidenephthalide，BP)的稳定医药组合物，尤其关于一种可经由鼻内投药的含有稳定存在的亚丁基苯酞的稳定医药组合物，其有效于治疗恶性脑瘤。
背景技术
星状细胞瘤(astrocytoma)、退行性星状细胞瘤(anaplastic astrocytoma)、及多型恶性神经胶质瘤(glioblastoma multiforme，GBM)为常见的成人恶性脑瘤，其中又以多型恶性神经胶质瘤最常见、恶性度最高、侵犯能力最强。
恶性脑瘤具有高浸润性及高复发率、高致死率等特性，其治疗至今仍为医学上亟待解决的问题。目前治疗方式包括有外科手术、放射治疗及化学药物治疗等三种。以外科手术切除肿瘤虽可消灭大部分的肿瘤，但会伤害到其他正常脑组织，进而影响病患的行动和语言能力；放射治疗可局部杀死肿瘤，但亦会影响周遭的正常组织细胞；化学药物治疗则常常因血脑障壁(blood brain barrier，BBB)限制、毒性副作用大、或耐药性(drug resistance)等问题，无法获致良好治疗效果。鼻内投药方式为药物治疗中的新兴投药方法，其为非侵入式投药方法，可降低患者感染的风险，且药物经鼻腔吸入后，可经由鼻黏膜快速吸收而产生药效，不会受到肝脏首渡效应(first-pass effect)的代谢排除作用，此外 自鼻粘膜吸收的药物能绕行血脑障壁直达中枢神经系统，因而降低药物全身性毒性，效果优于口服投药或静脉注射投药。
亚丁基苯酞(n-butylidenephthalide，BP)为目前治疗恶性肿瘤的潜力新药，其为一种源自当归(Angelica sinensis)萃取物的成分，沸点为139℃至142℃(5毫米汞柱)，因此在一般情况下为呈油状液体。目前已知亚丁基苯酞可用于治疗多种恶性肿瘤，包括多型恶性神经胶质瘤及乳腺恶性肿瘤。另有研究显示亚丁基苯酞亦可用于抑制运动神经元的自体吞噬(autophagy)，例如US2014/0045765A1即揭露亚丁基苯酞及其代谢物可用于抑制运动神经元的自体吞噬，且可用于治疗肌萎缩侧索硬化症(amyotrophic lateral sclerosis，ALS)。
然而，亚丁基苯酞虽可提供药效，但其为脂溶性物质而非水溶性物质，因此目前在应用上仍多有限制，包括在口服配方、注射配方及鼻内投药配方应用上的限制、临床应用的限制、以及在使用诸如大鼠等小动物的动物研究上的限制。
鉴于此，目前临床上需要一种亚丁基苯酞可稳定存在或溶解于其中的剂型，以提高亚丁基苯酞于生物体的利用率与改善其递送情况，并促进亚丁基苯酞的临床研究。本发明即针对前述需求所为的研发成果，通过一种介质系统以有效稳定亚丁基苯酞，从而更有效应用亚丁基苯酞。
发明内容
因此，本发明的一目的，在于提供一种医药组合物，包含：(a)一介质系统，包含第一成分、第二成分、及第三成分，该第一成分为磷酸盐缓冲液(phosphate buffered saline)，该第二成分 选自以下群组：植物油、动物油、脂肪酸及其组合，且该第三成分选自以下群组：聚乙二醇、二甲亚砜(DMSO)、乙醇、聚丙二醇、聚山梨醇酯、聚氧乙基化蔬菜油(polyoxyethylated vegetable oil)、乙酸乙酯、12-羟基硬酯酸羟乙酯(2-hydroxyethyl12-hydroxyoctadecanoate)、维生素E聚乙二醇琥珀酸酯(tocopheryl polyethylene glycol succinate)、及其组合；以及(b)存在于该(a)介质系统中的亚丁基苯酞(n-butylidenephthalide，BP)。
本发明的详细技术内容及部分实施态样，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1A为根据本发明的一实施态样的医药组合物的倒立式显微镜(IM)图(400倍；比例尺：50微米)；
图1B为根据本发明的一实施态样的医药组合物之扫描式场发射电子显微镜(FE-SEM)图(10000倍；比例尺：1微米)；
图1C为根据本发明的一实施态样的医药组合物的穿透式电子显微镜(TEM)图(80000倍；比例尺：200纳米)；
图2A图为根据本发明的不同实施态样的医药组合物及比较医药组合物于4℃低温下长时间储存前的照片；
图2B为根据本发明的不同实施态样的医药组合物及比较医药组合物于4℃低温下长时间储存后的照片；
图3显示根据本发明的不同实施态样的医药组合物(其中的介质系统组合不同)及比较医药组合物的活性的统计图，其中，纵轴为GBM8401细胞(即，人类恶性肿瘤细胞株)的存活率(以控 制组，即不含亚丁基苯酞的态样为100％计)，横轴为活性成分亚丁基苯酞的处理浓度；
图4显示储存时间对于根据本发明的医药组合物的活性的影响的统计图，其中，纵轴为GBM8401细胞的存活率(以控制组，即不含亚丁基苯酞之态样为100％计)，横轴为亚丁基苯酞的处理浓度；
图5为Franz扩散装置示意图；
图6显示施以不同投药治疗的大鼠存活率统计图；
图7A显示大鼠于种植肿瘤后施以不同治疗，在死亡之日或治疗第37天后的肿瘤生长情形的照片，其中，(A)未经药物处理(大鼠死亡之日所取得)(B)BP原液160毫克/公斤(第37天)(C)BP原液320毫克/公斤(第37天)(D)医药组合物580毫克/公斤(第37天)(E)医药组合物5160毫克/公斤(第37天)；
图7B显示大鼠于种植肿瘤后施以不同治疗，在治疗第37天后的肿瘤重量统计图；以及
图7C显示大鼠于种植肿瘤后施以不同治疗，在治疗第37天后的肿瘤体积统计图。
具体实施方式
以下将具体地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)所使用之「一」、「该」及类似用语应理解为包含单数及复数形式；以及在本说明书中「烃基」是指饱和直链或支链烃基，或具有一或多个π键的不饱和直链或支链烃基。
对于具有治疗效果的活性成分而言，其一通常应用型态为以液体剂型的方式投药，液体剂型中包含活性成分及介质，活性成分可溶于介质中或稳定存在于介质中。更具体言之，配合不同的投药途径需求(如口服、体细胞导入、或外用)，将活性成分溶于介质中(若其可溶)或悬浮于介质中(若其不可溶)而形成一适合投药的溶液、悬浮液或乳化液(emulsion)。此外，就制备含有少量活性成分的固体剂型而言，通常先将活性成分溶解或悬浮于液体介质中，而后再以该溶有或悬浮有活性成分的液体介质进行固体剂型制备，以准确控制最终产物中的活性成分浓度(含量)。
本案发明人研究后发现，亚丁基苯酞对于乙醇之溶解度最高，乙醇为其极佳的溶剂。其中，亚丁基苯酞于乙醇中的溶解度超过222毫克/毫升，但于聚乙二醇400、聚乙二醇300或聚丙二醇中的溶解度则仅为约70至120毫克/毫升。然而，有研究显示，婴幼儿因为醇脱氢酶(alcohol dehydrogenase)尚未成熟，并没有办法如成人般有效的代谢乙醇(可参见Zuccotti GV,Fabiano V.Safety issues with ethanol as an excipient in drugs intended for pediatric use.Expert Opin Drug Saf 2011；10(4):499-502，该文献全文并于此处以供参考)。因此，若能以其他溶剂取代乙醇作为亚丁基苯酞的介质，或者可于介质中减少乙醇的用量，则将可大幅提高亚丁基苯酞的应用范围。
本发明人进一步研究发现，亚丁基苯酞可稳定存在于一特定介质系统中，即使长时间储存(室温下储存三天；4℃下储存三十天)仍可稳定存在于该介质系统中并保持活性，该介质系统进一步还可以提高亚丁基苯酞于生物体中的利用率，从而在相对低的 亚丁基苯酞用量下提供所欲治疗效果，减缓或免除不必要的副作用。
因此，本发明提供一种稳定医药组合物，该医药组合物包含(a)一介质系统以及(b)稳定存在于该(a)介质系统中的亚丁基苯酞。
亚丁基苯酞可由商业上购得(例如可购自长弘生物科技股份有限公司，台湾)、化学合成或由天然材料(例如：当归)中萃取分离而得。此外，于使用萃取分离所得的亚丁基苯酞以前，可进一步通过例如骤沸塔层析法(flash column chromatography)、高效液相层析法、或结晶法的纯化方法，以提高亚丁基苯酞的纯度。
于本发明医药组合物中，(a)介质系统包含第一成分、第二成分、及第三成分。该第一成分为磷酸盐缓冲液(phosphate buffered saline)，该第二成分选自以下群组：植物油、动物油、脂肪酸及其组合，且该第三成分选自以下群组：聚乙二醇、二甲亚砜、乙醇、聚丙二醇、聚山梨醇酯、聚氧乙基化蔬菜油(polyoxyethylated vegetable oil)、乙酸乙酯、12-羟基硬酯酸羟乙酯(2-hydroxyethyl 12-hydroxyoctadecanoate)、维生素E聚乙二醇琥珀酸酯(tocopheryl polyethylene glycol succinate)、及其组合。
作为(a)介质系统的第一成分，磷酸盐缓冲液对细胞而言是等压的且无毒的。可于本发明医药组合物中采用任何合宜的磷酸盐缓冲液，其实例包括NaCl(137mM)与KCl(2.7mM)的组合的水溶液及Na2HPO4(10mM)、KH2PO4(1.8mM)、NaCl(137mM) 与KCl(2.7mM)的组合的水溶液，但不限于此。
作为(a)介质系统的第二成分，植物油的实例例如选自以下群组之一或多者：芥花油、椰子油、玉米油、棉籽油、橄榄油、棕榈油、花生油、菜籽油、红花籽油、芝麻油、大豆油、葵花油、杏仁油、腰果油、榛果油、胡桃油、山核桃油、松子油、开心果油及蓖麻油(castor oil)；动物油的实例例如选自以下群组之一或多者：鱼油、鱼肝油、猪油、牛油、羊油、鸡油、及鸭油；而脂肪酸可为饱和脂肪酸或不饱和脂肪酸(如顺式不饱和脂肪酸、单元不饱和脂肪酸、多元不饱和脂肪酸、反式不饱和脂肪酸等)。于本发明的部分实施态样中采用经修饰的蓖麻油作为第二成分，经修饰的蓖麻油的实例包括聚乙氧化蓖麻油(polyethoxylated castor oil)及氢化乙氧化蓖麻油，但不限于此。聚乙氧化蓖麻油亦称为甘油聚乙二醇蓖麻油酸酯(glycerol polyethyleneglycol ricinoleate)，例如是商购可得的
Figure PCTCN2015076893-appb-000001
EL(旧名为
Figure PCTCN2015076893-appb-000002
EL；BASF公司)。
Figure PCTCN2015076893-appb-000003
EL是一种非离子型助溶剂及乳化剂，可通过反应氧化乙烯与蓖麻油而得。氢化乙氧化蓖麻油可通过反应氧化乙烯与氢化蓖麻油而得。氢化乙氧化蓖麻油的实例包括，聚氧乙烯氢化蓖麻油40(polyoxyl 40hydrogenated castor oil)及聚氧乙烯氢化蓖麻油60(polyoxyl 60hydrogenated castor oil)，但不限于此。前者例如是
Figure PCTCN2015076893-appb-000004
RH 40(HLB＝14-16；BASF公司)，后者例如是
Figure PCTCN2015076893-appb-000005
RH 60(HLB＝15-17；BASF公司)。
作为(a)介质系统的第三成分，聚山梨醇酯在市面上以「Tween」的名称销售，其实例包括聚山梨醇酯20、聚山梨醇酯80、聚山梨醇酯85等；聚氧乙基化蔬菜油在市面上以「Emulphor」 的名称销售；12-羟基硬酯酸羟乙酯在市面上的商品包括例如BASF公司的
Figure PCTCN2015076893-appb-000006
HS 15(亦即
Figure PCTCN2015076893-appb-000007
HS 15)；而维生素E聚乙二醇琥珀酸酯的实例则例如是D-α-维生素E聚乙二醇琥珀酸酯1000(D-α-tocopheryl polyethylene glycol 1000succinate；HLB＝13.2)。然而，适用为本发明医药组合物的(a)介质系统的第三成分，并不限于以上所举实例。
于本发明的部分实施态样中，(a)介质系统由磷酸盐缓冲液(第一成分)、聚乙氧化蓖麻油(第二成分)及选自以下群组的成分(第三成分)所组成的混合物：聚乙二醇、二甲亚砜、乙醇、聚丙二醇、及其组合。其中，特别在第三成分选自聚乙二醇、二甲亚砜、及乙醇时，可对亚丁基苯酞提供较佳的稳定效果，所提供的医药组合物于长时间储存仍可维持不分层且亚丁基苯酞仍可保持活性。尤其，在第三成分为聚乙二醇与二甲亚砜的组合或聚乙二醇与乙醇之组合的情况下，可进一步提高本发明医药组合物的亚丁基苯酞的药效，其中又以第三成分为聚乙二醇与二甲亚砜的组合时最佳。因此，于本发明医药组合物的(a)介质系统中，第三成分较佳选自以下群组：聚乙二醇、二甲亚砜、乙醇、及其组合，更佳为聚乙二醇与二甲亚砜的组合或聚乙二醇与乙醇的组合，尤佳为聚乙二醇与二甲亚砜的组合。
于本发明医药组合物中，作为组成(b)的亚丁基苯酞的浓度原则上并无特殊限制，只要可以稳定存在于(a)介质系统中即可。因此，当亚丁基苯酞溶解于(a)介质系统中，则亚丁基苯酞的含量以不高于其在(a)介质系统中的溶解度为原则；而当亚丁基苯酞分悬浮于(a)介质系统中，则亚丁基苯酞的含量以不产生分层 的用量为原则。在此原则下，于本发明的部分实施态样中，以每毫升(a)介质系统计，亚丁基苯酞的浓度为0.001毫克/毫升至1500毫克/毫升。
举例言之，如后附实施例所示，当使用磷酸盐缓冲液([Na2HPO4]＝10mM；[KH2PO4]＝1.8mM；[NaCl]＝137mM与[KCl]＝2.7mM)为第一成分、聚乙氧化蓖麻油为第二成分、以及聚乙二醇或聚乙二醇与二甲亚砜或乙醇的组合为第三成分，提供(a)介质系统时(参照实施例1、5及6)，作为组成(b)之亚丁基苯酞的用量在达到1,000毫克/毫升介质系统时仍可稳定存在于其中，故可确定在所提供的医药组合物中，亚丁基苯酞的含量至少可达1,000毫克/毫升介质系统。又例如，当使用磷酸盐缓冲液([Na2HPO4]＝10mM；[KH2PO4]＝1.8mM；[NaCl]＝137mM与[KCl]＝2.7mM)为第一成分、聚乙氧化蓖麻油为第二成分、以及乙醇与丙二醇的组合为第三成分提供(a)介质系统时，作为组成(b)的亚丁基苯酞的用量在达到208毫克/毫升介质系统时仍可稳定存在于其中，故可确定在所提供的医药组合物中，亚丁基苯酞的含量至少可达208毫克/毫升介质系统。
于本发明医药组合物的(a)介质系统中，本领域技艺人士于观得本案说明书揭露内容后，可视所选用的第一成分、第二成分、及第三成分而调整其等的用量比例。举例言之，第一成分与第二成分的体积比可为1:2至20:1，例如1:1、2:1、3:1、4:1、5:1、8:1、10:1、13:1、15:1、17:1、20:1等；而第一成分与第三成分的体积比可为1：2至30：1，例如1:1、2:1、3:1、4:1、5:1、8:1、10:1、13:1、15:1、17:1、20:1、25:1、30：1等。另外， 在第三成分为多种成分的混合物的情况中，混合物中各构成成分的比例可由使用者(如视各成分的性质)作选择。以采用乙醇与聚乙二醇的混合物作为第三成分为例，由于乙醇与聚乙二醇以任何比例均可均匀混合，故其混合比例并无特殊限制，可采用乙醇：聚乙二醇之体积比为例如10：1至1：100，较佳6：1至1：50，更佳1：1至1：10以混合二者。
举例言之，当以磷酸盐缓冲液([Na2HPO4]＝10mM；[KH2PO4]＝1.8mM；[NaCl]＝137mM与[KCl]＝2.7mM)为第一成分、聚乙氧化蓖麻油为第二成分、聚乙二醇为第三成分，则磷酸盐缓冲液与聚乙氧化蓖麻油脂体积比可为3:0.5～5(例如：3:1)，磷酸盐缓冲液与聚乙二醇的体积比可为3:0.5～5(例如：3:1)。又例如，当以该磷酸盐缓冲液为第一成分、聚乙氧化蓖麻油为第二成分、聚乙二醇与二甲亚砜的组合为第三成分，则磷酸盐缓冲液与聚乙氧化蓖麻油的体积比可为1～10：1(例如：5:1)，磷酸盐缓冲液与聚乙二醇与二甲亚砜之组合的体积比可为1～5：1(例如：3:1)，而聚乙二醇与二甲亚砜的比可为1～2：1～2(例如1:1)。
于本发明的医药组合物中，组成(b)亚丁基苯酞溶于介质系统当中，或稳定存在于介质系统中，因此使得组合物可形成一适合于亚丁基苯酞的输送或投药的溶液、乳化液或悬浮液，例如使得医药组合物可通过口服、注射及/或鼻内投药的方式投药予需要的对象。
此外，由于亚丁基苯酞溶于或稳定存在于介质系统中，本发明医药组合物可促进使用诸如重量仅25克的小鼠或大鼠等小动物的动物研究。举例言之，在使用25克的小鼠作为动物模式研究亚 丁基苯酞的功效的实验中，一般需使用2至3微升(μL)的极微量的亚丁基苯酞，然而由于此量太过于微小，因此计量上相当困难。在此情况下，若使用本发明的医药组合物，则由于亚丁基苯酞稳定存在于介质中而为介质所稀释，故可将少量的活性成分亚丁基苯酞与大量的介质系统混合，以较大体积的本发明医药组合物来提供所欲的极微量亚丁基苯酞，如此，计量上将相对容易且精确。因此，本发明的医药组合物也可用于活性成分的固体剂型的制备，能准确控制最终产品中的活性成分浓度。
本发明医药组合物可通过简单混合(a)介质系统与(b)亚丁基苯酞的方式进行制备。在(b)亚丁基苯酞可溶于(a)介质系统的情况中，可直接将亚丁基苯酞添加至介质系统当中，使其溶解于其中；而在(b)亚丁基苯酞不可溶于(a)介质系统的情况中，则可在将亚丁基苯酞添加至介质系统中后施以一物理力(如振荡)以促进其混合，以提供亚丁基苯酞以微米微滴或纳米微滴的形式存在于其中的一稳定乳化液或悬浮液。
本发明医药组合物可视需要包含其他添加剂，只要这些添加剂或其他活性成分不会不利的影响(b)亚丁基苯酞的功效及其于(a)介质系统中的稳定性及/或溶解度。举例言之，可添加合宜用量的保存剂、防腐剂、抗菌剂、抗真菌剂等，以进一步提高所制得医药组合物的储存性；亦可于医药组合物中添加一或多种其他活性成分，例如抗氧化剂(如维他命E、维他命C、二丁基羟基甲苯(butylated hydroxytoluene，BHT)、丁基羟基甲氧苯(butylated hydroxyanisole，BHA))、化学药剂、免疫调节剂(immune modulators)等，以进一步加强所制得医药组合物的功 效或增加其运用灵活性。
兹以下列实施例进一步例示说明本发明。其中，所添加的亚丁基苯酞以体积计，但以密度为1克/毫升进行换算，得到对应的重量。以下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，本发明保护范围如所附申请专利权利要求所示。
实施例
1.室温稳定性测试
1.1测试方法
将医药组合物置于室温下一指定时间(三天或七天)，观察其外观是否出现分层，未出现分层者记录为「V」，分层者记录为「X」，以区分外观稳定性优劣；以及，以如下条件利用液相层析质谱仪通过相对滞留时间(relative retention time，RRT)量测医药组合物中的不纯物含量。
Figure PCTCN2015076893-appb-000008
1.2医药组合物的制备
实施例1
以表1所示比例，将50微升亚丁基苯酞(BP)(长弘生物科技股份有限公司；批号：F212TR12001)、10微升聚乙二醇(PEG)(SIGMA公司；批号：MKBG2152V；CAS号：25322-68-3)与10微升聚乙氧化蓖麻油(
Figure PCTCN2015076893-appb-000009
EL，下称『K-EL』；SIGMA公司； 批号：BCBC54780；CAS号：61791-12-6)混合，而后将30微升磷酸盐缓冲液(下称『PBS』；[Na2HPO4]＝10mM；[KH2PO4]＝1.8mM；[NaCl]＝137mM与[KCl]＝2.7mM)滴加至所得混合物中，并快速振荡混合均匀成乳化液，制得医药组合物1。
实施例2
以与实施例1相同的方式制备医药组合物2的乳化液，但是改变各成分之用量比例，如表1所示。
实施例3
以与实施例1相同的方式制备医药组合物3的乳化液，但是改以二甲亚砜(DMSO)作为第三成分，并改变各成分的用量比例，如表1所示。
实施例4
以与实施例1相同的方式制备医药组合物4的乳化液，但是改变各成分的用量比例，如表1所示。
实施例5
与实施例1相同的方式制备医药组合物5的乳化液，但是改以PEG与DMSO的组合作为第三成分，并改变各成分的用量比例，如表1所示。
实施例6
以与实施例1相同的方式制备医药组合物6的乳化液，但是改以PEG与乙醇之组合作为第三成分，并改变各成分的用量比例，如表1所示。
比较例1
以与实施例1相同的方式制备比较医药组合物1的乳化液，但 是不添加第二成分(K-EL)并改以PEG与
Figure PCTCN2015076893-appb-000010
HS 15(下称『HS 15』；BASF公司；批号：14225516K0；CAS号：61909-81-7)的组合作为第三成分，如表1所示。
表1：不同医药组合物的组成体积百分比
Figure PCTCN2015076893-appb-000011
1.3稳定性测试结果
以第1.1点所载方法观察医药组合物1至6及比较医药组合物1乳化液的外观并量测不纯物含量，以评估组合物的稳定性，结果记录于下表2(乳化液外观)及表3-1至3-7与表4，其中表2显示乳化液外观，表3-1至3-7分别显示医药组合物1至6及比较医药组合物1中的不纯物浓度(体积百分比)随储存时间的变化情形，表4则为由此计算而得的各组合物的亚丁基苯酞浓度维持率(体积百分比)变化情形。
表2：室温下储存前后的外观
医药组合物 1 2 3 4 5 6 比较1
配置完成时 V V V V V V V
室温储存3天后 V V V V V V X
室温储存7天后 X V V V X X X
表3-1：医药组合物1的不纯物浓度(体积百分比)变化情形
RRT RRT 0.7 RRT 0.86 RRT 0.89 RRT 0.9 总合
配置完成时 0.32 ND ND ND 0.32
室温储存3天后 0.36 ND ND 0.3 0.66
室温储存7天后 0.43 0.25 0.08 0.84 1.60
*ND代表仪器未检出。
表3-2：医药组合物2的不纯物浓度(体积百分比)变化情形
RRT RRT 0.7 RRT 0.86 RRT 0.89 RRT 0.9 总合
配置完成时 0.25 ND ND <0.05 0.25
室温储存3天后 0.73 ND ND 1.24 1.97
室温储存7天后 0.43 0.23 0.10 0.71 1.47
表3-3：医药组合物3的不纯物浓度(体积百分比)变化情形
RRT RRT 0.7 RRT 0.8 RRT 0.86 总合
配置完成时 0.11 <0.05 <0.05 0.11
室温储存3天后 0.20 0.12 <0.05 0.32
室温储存7天后 0.42 0.15 0.11 0.68
表3-4：医药组合物4的不纯物浓度(体积百分比)变化情形
RRT RRT 0.7 RRT 0.8 RRT 0.86 总合
配置完成时 0.09 ND <0.05 0.09
室温储存3天后 0.11 0.11 <0.05 0.22
室温储存7天后 0.19 0.16 0.07 0.42
表3-5：医药组合物5的不纯物浓度(体积百分比)变化情形
RRT RRT 0.7 RRT 0.86 RRT 0.89 RRT 0.9 总合
配置完成时 0.04 0.01 0.07 0.02 0.14
室温储存3天后 0.02 0.33 0.02 0.03 0.40
室温储存7天后 0.02 0.67 ND 0.02 0.71
表3-6：医药组合物6的不纯物浓度(体积百分比)变化情形
Figure PCTCN2015076893-appb-000012
Figure PCTCN2015076893-appb-000013
表3-7：比较医药组合物1的不纯物浓度(体积百分比)变化情形
RRT RRT 0.7 RRT 0.86 RRT 0.9 总合
配置完成时 0.28 ND ND 0.28
室温储存3天后 0.26 ND 0.2 0.46
室温储存7天后 0.32 0.21 0.36 0.89
表4：活性成分(亚丁基苯酞)浓度维持率(体积百分比)变化情形
医药组合物 1 2 3 4 5 6 比较1
配置完成时 99.68 99.75 99.89 99.91 99.22 99.25 99.72
室温储存3天后 99.34 98.03 99.68 99.78 98.73 98.77 99.54
室温储存7天后 98.40 98.53 99.32 99.58 98.30 98.48 99.11
由表2可知，本发明医药组合物在外观上可于室温下保持长达3天仍不会发生分层，尤其在部分态样中(实施例2至4)甚至可于室温下保存7天而不发生分层；而由表4可知，本发明医药组合物即使经长时间储存，亚丁基苯酞浓度的变化极小，维持率非常优异。
上述结果显示，于本发明医药组合物具有优异的稳定性，活性成分可稳定存在于介质系统当中。其中，以每毫升介质系统计，医药组合物1、5及6中的活性成分亚丁基苯酞之浓度相当于约1,000毫克/毫升介质系统。
2.医药组合物物理性质分析
2.1分析方法
2.1.1粒径、导电度及界面电位分析
将医药组合物乳化液配制成浓度200微克/毫升作为样品，再以高浓度纳米粒径及界面电位分析仪(Malvern Nano-ZS  ZEN-3600)分析样品的导电度、界面电位及所含乳化微粒的平均粒径大小。
2.1.2黏度分析
采用40毫米4度的角锥-平板模具，取约1毫升的样品置入平行板间，使用TA仪器公司之AR2000ex系统(应力控制)的流变仪分析样品的粘度随应变率的变化关系与流变特性，以判断样品系牛顿流体(粘度不会随着剪应变率的增加而增加或减少)或非牛顿流体(粘度随着剪应变率的增加而增加或减少)。
2.1.3显微结构分析
以经纯水稀释500倍的医药组合物作为样品，分别以倒立式显微镜(IM)、扫描式场发射电子显微镜(FE-SEM)及穿透式电子显微镜(TEM)观察其显微结构。
2.2医药组合物的制备
实施例7至11
以与实施例1相同的方式制备医药组合物7至11，但是调整介质系统及活性成分的组成如表5所示。其中以每毫升介质系统计，医药组合物7至11中活性成分亚丁基苯酞的浓度分别约为105毫克/毫升介质系统(医药组合物7)、156毫克/毫升介质系统(医药组合物8)、208毫克/毫升介质系统(医药组合物9)、163毫克/毫升介质系统(医药组合物10)及111毫克/毫升介质系统(医药组合物11)。
表5：不同医药组合物的组成体积百分比
Figure PCTCN2015076893-appb-000014
Figure PCTCN2015076893-appb-000015
2.3分析结果
2.3.1粒径、界面电位及导电度分析结果
以第2.1.1点所载方法分析亚丁基苯酞原液(BP原液)、医药组合物1及5至11与比较医药组合物1的导电度、界面电位及所含乳化微粒之平均粒径大小，并将结果记录于下表6。如表6所示，本发明医药组合物中所含之亚丁基苯酞微粒系尺寸介于29.89纳米至1.22微米之间的细小微粒，且界面电位介于-6.80至-1.55毫伏(mV)之间，导电度介于12.1至14.93毫西门子/公分(mS/cm)，此显示其为水包油(O/W)型式的乳化液。表6
配方 尺寸(纳米) 界面电位(毫伏) 导电度(毫西门子/公分)
BP原液 - 0.38 0.02
医药组合物1 1223.21 -4.41 12.5
医药组合物5 518.70 -5.65 12.7
医药组合物6 320.75 -6.60 12.7
医药组合物7 80.05 -3.65 14.1
医药组合物8 112.78 -2.41 12.3
医药组合物9 29.89 -2.52 12.1
医药组合物10 143.79 -1.55 12.7
医药组合物11 96.28 -2.02 12.7
比较医药组合物1 318.31 -6.80 12.6
2.3.2粘度分析结果
以第2.1.2点所载方法分析亚丁基苯酞原液、医药组合物1及5至11与比较医药组合物1的粘度，并计算其平均黏度，结果记录于下表7。如表7所示，亚丁基苯酞原液及医药组合物的粘度均呈现粘度随着剪应变率的增加而减少的现象，因此均属非牛顿流体中的剪切变稀流体(shear thinning liquid)；此外，各医药组合物的粘度介于0.62×10-2至10.89×10-2帕·秒(Pa.s)之间。
表7
配方 平均黏度(×10-2帕·秒)±标准差(SD)
BP原液 1.57±0.01
医药组合物1 10.89±0.59
医药组合物5 6.96+0.69
医药组合物6 4.56±0.37
医药组合物7 0.99±0.02
医药组合物8 8.69±0.13
医药组合物9 9.58±0.23
医药组合物10 1.99±0.06
医药组合物11 0.62±0.02
比较医药组合物1 4.49±0.30
2.3.3显微结构分析结果
以第2.1.3点所载方法分析医药组合物5之显微结构，结果如图1A、图1B及图1C所示，其中图1A为医药组合物5的IM图(400倍；比例尺：50微米)，图1B为医药组合物5的FE-SEM图(10,000倍；比例尺：1微米)，且图1C为医药组合物5的TEM图(80,000倍；比例尺：200纳米)。由图1A至图1C的结果可知，于本发明医药组合物中，活性成分(亚丁基苯酞)的颗粒尺寸为纳米级至微米级的微粒，且具有大致为球形的型态。
3.医药组合物的体外试验
3.1分析对象
实验组：医药组合物1、医药组合物5及医药组合物6控制组：比较医药组合物1
3.2分析方法
3.2.1低温稳定性测试
将医药组合物置于4℃冰箱储存30天后，拍照并观察是否出现分层，并与刚制备完成时的医药组合物作外观比较。
3.2.2毒杀试验(IC50值)
将GBM 8401细胞(即，人类恶性脑瘤细胞株)以每孔6×103的细胞数目培养在96孔培养盘中，接着以亚丁基苯酞原液或不同医药组合物进行处理，历时24小时。其中，活性成分亚丁基苯酞的处理浓度分别为0微克/毫升(控制组)、12.5微克/毫升、25 微克/毫升、50微克/毫升、100微克/毫升及200微克/毫升。随后以MTT(3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)细胞存活测试方法分析各医药组合物对于GBM 8401细胞的存活的半抑制浓度(half maximal inhibitory concentration，IC50)，以评估毒杀效果。
3.2.3毒杀效果稳定度试验
将医药组合物5分批分别于4℃下储存0、7、14、30或60天。将GBM 8401细胞以每孔6×103的细胞数目培养在96孔培养盘中，并以经储存不同时间的医药组合物5进行处理，历时24小时，随后以MTT细胞存活测试方法分析该经不同天数储存的医药组合物5对于GBM 8401细胞的存活的半抑制浓度(IC50)，以评估储存时间对于医药组合物的毒杀效果的影响。其中，活性成分亚丁基苯酞的处理浓度分别为0微克/毫升(控制组)、12.5微克/毫升、25微克/毫升、50微克/毫升、100微克/毫升及200微克/毫升。
3.2.4 体外人工纤维膜穿透试验
利用如图5所示的Franz扩散装置(三美玻璃仪器行)估计亚丁基苯酞原液及不同医药组合物穿透人工纤维膜的能力，以模拟其穿透鼻腔粘膜的情形，其中，此装置之扩散表面积大约有4.5平方公分，且接受池的容量约23毫升。
具体试验方法包括：(i)将人工纤维膜裁切成适当大小，并置于Hepes(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)缓冲液(118mM NaCl+1.2mM MgSO4+4.8mM KCl+5.5mM D-葡萄糖+2.5mM CaCl2+20mM Hepes)中浸泡10分钟；(ii) 于供给池中加入2毫升的亚丁基苯酞原液或浓度为3毫克/毫升的医药组合物；(iii)于接受池中加入Hepes缓冲液，并采用磁力搅拌(转速：300rpm)方式搅拌之；(iv)在温度37℃下进行试验，并每间隔1小时由接受池的取样管取样1毫升，同时补回已加温的Hepes缓冲液1毫升；(v)以分光光度计测量取样所得的溶液于310纳米波长下的吸收光谱，并计算亚丁基苯酞的浓度，共测量10小时；以及(vi)分别利用以下公式1及2计算求得有效穿透值(Permeability coefficient，Peff)与流量(Flux，J)。
Figure PCTCN2015076893-appb-000016
  Karasulu et al.,2008)公式1
Figure PCTCN2015076893-appb-000017
  公式2
V：接受池的容量(毫升)
C0：初始供给的药物浓度(微克/毫升)
Peff：有效穿透率(公分/秒)
J：流量(微克/平方公分·秒)
A：扩散表面积(平方公分)
Figure PCTCN2015076893-appb-000018
稳定状态下，浓度随时间的变化(微克/毫升秒)
3.2.5体外人体细胞株穿透试验
将RPMI 2650细胞(即，人类鼻中膈鳞状细胞株)接种于涂布有胶原蛋白之孔径为0.4微米的细胞隔膜盘(cell insert)中，并培养2天，使细胞在细胞隔膜上形成细胞单层膜(monolayer)。接着，将细胞隔膜盘转移到已加入1.5毫升Hepes缓冲液的12孔盘中，再于细胞隔膜盘中加入0.5毫升的含亚丁基苯酞原液或医药组合物的Hepes缓冲液。然后，将12孔盘置于37℃、5％CO2的 培养箱中培养，每间隔1小时，分别收集12孔盘内的缓冲液，并以分光光度计测量其于310纳米波长下的吸收光谱，计算亚丁基苯酞或医药组合物浓度，共测量6小时，并利用前述公式1及公式2，计算求得有效穿透(Peff)与流量(J)。
3.3试验结果
3.3.1低温稳定性测试结果
以第3.2.1点所载方法测试医药组合物1、比较医药组合物1、医药组合物5及医药组合物6于低温下的稳定性，结果如图2A图及图2B所示，其中图2A图及图2B分别为储存前及储存后的照片。由图2A及2B图可知，本发明医药组合物(医药组合物1、5及6)在4℃低温下长时间储存(长达30天)前后外观不会有显著变化且不会分层，而非本发明医药组合物(比较医药组合物1)在4℃低温下储存后则有明显分层的现象。前述结果显示，本发明医药组合物的稳定性优异。
3.3.2毒杀试验结果：以第3.2.2点所载方法对亚丁基苯酞原液、医药组合物1、比较医药组合物1、医药组合物5及医药组合物6进行毒杀试验，结果如图3及表8所示。由图3可知，相较于控制组，GBM 8401细胞的存活率系随着活性成分亚丁基苯酞的剂量提高而下降，且各医药组合物中，以医药组合物5的毒杀效果最好，其抑制GBM 8401细胞的存活的IC50为28.20微克/毫升。
表8
配方 IC50(微克/毫升)±SD
BP原液 85.32±1.70
医药组合物1 95.29±6.73
医药组合物5 28.10±2.71
医药组合物6 81.97±5.87
比较医药组合物1 62.34±6.36
3.3.3毒杀效果稳定度试验结果
以第3.2.3点所载方法对医药组合物5进行毒杀效果稳定度试验，结果如图4所示。由图4可知，相较于控制组，不论医药组合物5的储存时间如何，经处理后的各组GBM 8401细胞的存活率仍保持着随着活性成分亚丁基苯酞的剂量提高而下降的规律。前述结果显示，医药组合物5对恶性脑瘤细胞的毒杀效果并不会受到储存时间的影响，为一稳定的配方。
3.3.4体外人工纤维膜穿透试验结果
以第3.2.4点所载方法对亚丁基苯酞原液、医药组合物1、比较医药组合物1、医药组合物5及医药组合物6进行体外人工纤维膜穿透试验，结果记录于下表9。由表9可知，医药组合物5和医药组合物6的有效穿透率(Peff)最高，而亚丁基苯酞原液的有效穿透率最低。前述结果显示，相较于亚丁基苯酞原液，本发明医药组合物的穿透能力明显较佳，其中又以医药组合物5和医药组合物6的穿透能力为最佳。
表9
Figure PCTCN2015076893-appb-000019
Figure PCTCN2015076893-appb-000020
3.3.5体外人体细胞株穿透试验结果
以第3.2.5点所载方法对亚丁基苯酞原液、医药组合物1、比较医药组合物1、医药组合物5及医药组合物6进行体外人体细胞株穿透试验，结果记录于下表10。由表10可知，医药组合物6的有效穿透率(Peff)最高，而亚丁基苯酞原液的有效穿透率最低。前述结果显示，相较于亚丁基苯酞原液，本发明医药组合物穿透细胞的能力明显较佳，其中又以医药组合物5及6的穿透细胞的能力为佳。
表10
Figure PCTCN2015076893-appb-000021
4.医药组合物的动物实验
4.1实验方法
于大鼠颅内植入9L(即，大鼠恶性脑瘤细胞株)肿瘤组织，待颅内生成肿瘤后再进行药物治疗试验。试验方法包括：(i)利用400毫克/公斤体重的水合氯醛(chloral hydrate)(Sigma-Aldrich公司)麻醉大鼠，待其昏迷后，将大鼠头部毛发剃除，并将大鼠保持固定于鼠脑定位仪(Lab StandardTM Stereotaxic Instrument)上；(ii)以大剪将头皮剪开以露出头盖骨，以脑部囱门(bregma)为中心，向右3毫米，向下5毫 米，到定位钻孔处，利用钻孔器将头盖骨磨出5毫米直径的空洞；(iii)用镊子夹取预先准备好的9L肿瘤组织(1×1×1立方毫米)并植入脑内3毫米深处，静置1分钟待血液凝固后取出镍子，并于空洞处涂上骨腊，最后将大鼠头皮缝合，并给予抗生素；(iv)待植入肿瘤组织后第7天，随机分组如下表11，并利用微量吸管吸取药物(亚丁基苯酞原液或医药组合物5)滴进大鼠鼻腔中，进行鼻内投药治疗，连续投药30天，并于期间记录大鼠体重及死亡只数；以及(v)取下肿瘤，利用电子式游标卡尺来测量肿瘤的长和宽，并且用下列公式3计算肿瘤之总体积，同时测量肿瘤重量，将测得之数值绘制成长条图与曲线图，以判断治疗成效。
表11
Figure PCTCN2015076893-appb-000022
*「n」为各组大鼠只数
Figure PCTCN2015076893-appb-000023
  公式3
4.2动物实验结果
以第4.1点所载方法进行动物实验，实验结果如图6及图7A至7C所示。由图6及图7A至7C可知，相较于控制组，亚丁基苯酞原液(160毫克/公斤)及亚丁基苯酞原液(320毫克/公斤)皆具有抑制肿瘤生长的效果。前述结果显示，经由鼻内投予BP原液，确实可提供治疗恶性肿瘤的效果。
由图6及图7A至7C亦可知，经鼻内投予亚丁基苯酞原液(160毫克/公斤)与医药组合物5(80毫克/公斤)30天后，该二组大鼠具有明显提升且相似的存活率、以及明显下降且相似的肿瘤体积及肿瘤重量。此结果显示，本发明医药组合物中的亚丁基苯酞的有效剂量，只需要亚丁基苯酞原液的有效剂量的一半，即可达到相同的治疗效果。
由图7B、7C可知，投予医药组合物5(160毫克/公斤)30天后，该组肿瘤重量可减少约92％(图7B)，而肿瘤体积可减少约94％(图7C)。前述结果显示，本发明医药组合物5(160毫克/公斤)具有优异的治疗恶性肿瘤的效果。
以上体外及动物实验结果皆显示，将亚丁基苯酞制成如本发明的医药组合物，可提高鼻内投药后活性成分穿透鼻腔粘膜的效率，加乘其治疗效果，尤其是治疗恶性脑瘤的功效。
上述实施例仅用以例示说明本发明的原理及功效，而非用于限制本发明。任何熟于此项技艺之人士均可在不违背本发明的技术原理及精神的情况下，对上述实施例进行修改及变化。因此，本发明的权利保护范围应如所附申请专利权利要求所界定。

Claims (9)

  1. 一种稳定医药组合物，包含：
    (a)一介质系统，包含第一成分、第二成分、及第三成分，该第一成分为磷酸盐缓冲液(phosphate buffered saline)，该第二成分选自以下群组：植物油、动物油、脂肪酸、及其组合，且该第三成分选自以下群组：聚乙二醇、二甲亚砜、乙醇、聚丙二醇、聚山梨醇酯、聚氧乙基化蔬菜油(polyoxyethylated vegetable oil)、乙酸乙酯、12-羟基硬酯酸羟乙酯(2-hydroxyethyl 12-hydroxyoctadecanoate)、维生素E聚乙二醇琥珀酸酯(tocopheryl polyethylene glycol succinate)、及其组合；以及
    (b)亚丁基苯酞(n-butylidenephthalide，BP)。
  2. 如权利要求1所述的组合物，其中该植物油选自以下群组：芥花油、椰子油、玉米油、棉籽油、橄榄油、棕榈油、花生油、菜籽油、红花籽油、芝麻油、大豆油、葵花油、杏仁油、腰果油、榛果油、胡桃油、山核桃油、松子油、开心果油、蓖麻油(castor oil)及其组合；且该动物油选自以下群组：鱼油、鱼肝油、猪油、牛油、羊油、鸡油、鸭油及其组合。
  3. 如权利要求1所述的组合物，其中该第二成分为经修饰的蓖麻油(modified castor oil)。
  4. 如权利要求3所述的组合物，其中该经修饰的蓖麻油为聚乙氧化蓖麻油(polyethoxylated castor oil)，且该第三成分选自以下 群组：聚乙二醇、二甲亚砜、乙醇、聚丙二醇、及其组合。
  5. 如权利要求4所述的组合物，其中该第三成分选自以下群组：聚乙二醇、二甲亚砜、乙醇、及其组合。
  6. 如权利要求5所述的组合物，其中该第三成分为聚乙二醇与二甲亚砜的组合或聚乙二醇与乙醇的组合。
  7. 如权利要求6所述的组合物，其中该第三成分为聚乙二醇与二甲亚砜的组合。
  8. 如权利要求1至7中任一项所述的组合物，其中以每毫升(a)介质系统计，(b)亚丁基苯酞的浓度为0.001毫克/毫升至1500毫克/毫升。
  9. 如权利要求1至7中任一项所述的组合物，其中于(a)介质系统中，第一成分与第二成分的体积比为1:2至20:1，且第一成分与第三成分的体积比为1：2至30：1。
PCT/CN2015/076893 2014-04-24 2015-04-17 稳定医药组合物 Ceased WO2015161759A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP15783640.4A EP3135284B1 (en) 2014-04-24 2015-04-17 Stable pharmaceutical composition
JP2017505699A JP6312281B2 (ja) 2014-04-24 2015-04-17 安定的な医薬組成物

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201461983619P 2014-04-24 2014-04-24
US61/983,619 2014-04-24
US201462023205P 2014-07-11 2014-07-11
US62/023,205 2014-07-11

Publications (1)

Publication Number Publication Date
WO2015161759A1 true WO2015161759A1 (zh) 2015-10-29

Family

ID=54331739

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/076893 Ceased WO2015161759A1 (zh) 2014-04-24 2015-04-17 稳定医药组合物

Country Status (4)

Country Link
EP (1) EP3135284B1 (zh)
JP (1) JP6312281B2 (zh)
TW (1) TWI547280B (zh)
WO (1) WO2015161759A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1943606A (zh) * 2005-10-08 2007-04-11 财团法人佛教慈济综合医院 用于治疗癌症的当归萃取物
CN103565797A (zh) * 2012-08-10 2014-02-12 中国医药大学 用于抑制运动神经元自体吞噬的医药组合物及其应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070082947A1 (en) * 2003-05-14 2007-04-12 D Orazio Daniel Use of phthalide derivatives for the treatment and prevention of diabetes mellitus
CN100361656C (zh) * 2004-08-27 2008-01-16 石药集团中奇制药技术（石家庄）有限公司 丁苯酞自乳化释药体系及其制备方法和应用
SG121980A1 (en) * 2004-10-08 2006-05-26 Buddhist Tzu Chi General Hospi Angelicae sinensis extracts useful for treatment of cancers
CN100367951C (zh) * 2005-12-16 2008-02-13 石药集团恩必普药业有限公司 丁苯酞静脉乳剂及其应用
JP2011195457A (ja) * 2010-03-17 2011-10-06 Denki Kagaku Kogyo Kk ヒアルロン酸及び／又はその塩の含有液の脱泡方法
TWI386203B (zh) * 2011-01-07 2013-02-21 Univ China Medical 治療腦癌或用以降低腦癌細胞對替莫唑胺之抗藥性之醫藥組合物及其應用
US8927601B2 (en) * 2011-12-20 2015-01-06 National Dong Hwa University Uses of N-butylidenephthalide in treating a liver injury and improving liver function
JP6125610B2 (ja) * 2012-04-12 2017-05-10 ヴァージニア コモンウェルス ユニバーシティ 代謝障害、高脂血症、糖尿病、脂肪性肝疾患及びアテローム性動脈硬化症の治療のための新規コレステロール代謝物、５−コレステン−３β，２５−ジオールジスルファート（２５ＨＣＤＳ）

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1943606A (zh) * 2005-10-08 2007-04-11 财团法人佛教慈济综合医院 用于治疗癌症的当归萃取物
CN103565797A (zh) * 2012-08-10 2014-02-12 中国医药大学 用于抑制运动神经元自体吞噬的医药组合物及其应用

Also Published As

Publication number Publication date
JP2017511384A (ja) 2017-04-20
JP6312281B2 (ja) 2018-04-18
EP3135284B1 (en) 2018-12-05
EP3135284A1 (en) 2017-03-01
TWI547280B (zh) 2016-09-01
EP3135284A4 (en) 2017-11-29
TW201540298A (zh) 2015-11-01

Similar Documents

Publication Publication Date Title
CN113952460B (zh) 一种用于治疗斑秃的复方外用制剂及其制备方法
JP5135441B2 (ja) タクロリムス外用剤
KR101651448B1 (ko) 체지방을 국소적으로 증가시키는 방법 및 조성물
JPH07508976A (ja) 親油性薬剤投与のための無水調剤
Zhang et al. Co-delivery of evodiamine and rutaecarpine in a microemulsion-based hyaluronic acid hydrogel for enhanced analgesic effects on mouse pain models
CN102196821A (zh) 包含至少两种渗透增强剂组合的局部组合物
EA020569B1 (ru) Применение рофлумиласта и его производных и фармацевтический препарат для местного применения
CN105030760B (zh) 稳定医药组合物
Yong-Tai et al. Solid lipid nanoparticles formulated for transdermal aconitine administration and evaluated in vitro and in vivo
CN102327214A (zh) 一种百里香酚纳米药物及其制备方法
CN101396344A (zh) 丹皮酚微乳制剂及其制备方法
AU2577500A (en) A transdermal composition of an antivomiting agent and a preparation containing the same
CN1286529C (zh) 具有皮肤靶向性的药物组合物及其制备方法和用途
WO2023016583A1 (zh) 一种芦可替尼组合物及其用途
CN114452255B (zh) 一种阿戈美拉汀微乳、微乳凝胶及其制备方法
CN102552137A (zh) 雷公藤内酯醇脂肪乳注射剂及其制备方法
CN101933902A (zh) 盐酸格拉司琼微乳凝胶剂及其制备方法
JP2016175884A (ja) びまん性神経線維腫用の外用薬
CN104958257A (zh) 一种隐丹参酮皮肤角质类脂体制剂及其制备方法
TWI547280B (zh) 穩定醫藥組合物
CN102871989B (zh) 和厚朴酚或其药学上可接受的盐的新用途
CN101926764B (zh) 多拉菌素微乳透皮剂及其制备方法
CN106560175A (zh) 一种薄荷脑-樟脑低共熔物纳米乳原位凝胶制剂
WO2022044005A1 (en) Topical roflumilast compositions and uses thereof
CN101836955A (zh) 一种羟甲香豆素微乳药物及其制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15783640

Country of ref document: EP

Kind code of ref document: A1

REEP Request for entry into the european phase

Ref document number: 2015783640

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2015783640

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2017505699

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE