Compositions and methods for increasing bioavailability of active ingredients

Abstract

A composition comprising an oily medium system and an active ingredient, and a method for improving the bioavailability of the active ingredient, wherein the method comprises incorporating the active ingredient into an oily medium system, wherein the oily medium system contains vegetable oil and / or vegetable oil components, but does not contain the following substances: phosphate buffer, polyethylene glycol, dimethyl sulfoxide, ethanol, polypropylene glycol, polysorbate, ethyl acetate, hydroxyethyl 12-hydroxystearate, and tocopheryl polyethylene glycol succinate; the active ingredient is selected from the group consisting of: compounds of formula (I), pharmaceutically acceptable salts thereof, and combinations thereof, wherein T1 is a C6 cyclic hydrocarbon, and R1 is a C1-C8 aliphatic hydrocarbon group.

Description

Technical Field

[0001] The present invention relates to a method for improving the bioavailability (BA) of an active ingredient, which comprises incorporating the active ingredient into an oily medium system. The present invention also relates to a composition comprising the oily medium system and the active ingredient. In the foregoing method and composition, the oily medium system involved contains vegetable oil and / or components of vegetable oil, but does not contain the following substances: phosphate buffer, polyethylene glycol, dimethyl sulfoxide, ethanol, polypropylene glycol, polysorbate, ethyl acetate, hydroxyethyl 12-hydroxystearate, and tocopheryl polyethylene glycol succinate; in addition, the active ingredient involved is selected from the following group: compounds of formula (I), pharmaceutically acceptable salts thereof, and combinations of the foregoing, (I), wherein, T1 is a C6 cycloalkane, and R1 is a C1-C8 aliphatic hydrocarbon group. Prior Art

[0002] Both brain cancer and pancreatic cancer are cancers with high invasiveness, high recurrence rate, high fatality rate and poor prognosis. Brain cancer can occur at all ages. For common malignant brain tumors such as anaplastic astrocytoma and glioblastoma multiforme (GBM), although traditional surgical treatment can remove most of the tumor mass, it cannot effectively eradicate all cancer cells and must be supplemented with other treatment methods (such as chemotherapy and radiotherapy). As for pancreatic cancer, since pancreatic cancer cells often migrate, invade, and metastasize to surrounding nerve or vascular tissues, most patients with pancreatic cancer are unable to undergo surgical treatment when discovered and must use chemotherapy as the preferred treatment method.

[0003] However, currently, the chemical drugs used clinically for brain cancer or pancreatic cancer are still quite limited, and most of the treatment effects are relatively low. Therefore, it is still necessary to continuously develop drugs or methods that can effectively treat brain cancer or pancreatic cancer.

[0004] Studies have shown that butylphthalide can be used not only to delay or treat neurological diseases such as amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia, and Alzheimer's disease, but also to treat cerebrovascular diseases such as stroke, improve diabetes, and combat liver and lung fibrosis. Moreover, it has excellent effects in killing brain cancer and pancreatic cancer cells and can be applied in the field of cancer treatment.

[0005] Although butylphthalide has the aforementioned pharmacological effects, its application is still limited. The main reason is that the liver's metabolic elimination effect reduces the bioavailability of butylphthalide, often requiring higher doses to achieve the desired therapeutic effect, which leads to an increased risk of systemic side effects and exacerbates the burden on patients. The problems caused by the need to increase the dosage due to bioavailability are particularly severe in the case of oral administration. Therefore, if the bioavailability of the active ingredient can be effectively improved, unnecessary dosage increases can be avoided, the limitations in application can be solved, and a more effective treatment method can be provided for patients. Summary of the Invention

[0006] The inventors of this case have found through research that the active ingredient of the present invention (i.e., the compound of formula (I) and / or its pharmaceutically acceptable salt) has good solubility in the oily medium system of the present invention and can stably exist in the oily medium system for a long time (for example, stored at room temperature for up to two years). Further research has found that when the active ingredient of the present invention is administered in the form of being incorporated into the oily medium system, the bioavailability of the active ingredient can be improved, thereby avoiding the problem of having to increase the dosage to provide the desired therapeutic benefit and reducing or eliminating unnecessary side effects.

[0007] Therefore, an object of the present invention is to provide a composition comprising: (1) An oily medium system containing vegetable oil and / or components of vegetable oil, but not containing the following substances: phosphate buffer, polyethylene glycol, dimethyl sulfoxide, ethanol, polypropylene glycol, polysorbate, ethyl acetate, hydroxyethyl 12-hydroxystearate, and tocopheryl polyethylene glycol succinate; and (2) An active ingredient selected from the group consisting of: the compound of formula (I), its pharmaceutically acceptable salt, and combinations thereof, (I), wherein, T1 is a C6 cycloalkane, and R1 is a C1-C8 aliphatic hydrocarbon group. Preferably, the composition is used to improve the bioavailability of the active ingredient. The composition is preferably a pharmaceutical composition, a food composition, or a food additive composition. Among them, when the composition is a pharmaceutical composition, it is in the form of oral or sublingual administration; or, when the combination is a food composition, it is a health food, a dietary supplement, a functional food, a nutritional supplement food or a special nutritional food.

[0008] Another object of the present invention is to provide a method for improving the bioavailability of an active ingredient, which comprises incorporating the active ingredient into an oily medium system, wherein: (1) The oily medium system contains vegetable oil and / or components of vegetable oil, but does not contain the following substances: phosphate buffer, polyethylene glycol, dimethyl sulfoxide, ethanol, polypropylene glycol, polysorbate, ethyl acetate, hydroxyethyl 12-hydroxy stearate, and tocopheryl polyethylene glycol succinate; and (2) The active ingredient is selected from the group consisting of a compound of formula (I), a pharmaceutically acceptable salt thereof, and a combination of the foregoing, (I), wherein, T1 is a C6 cycloalkane, and R1 is a C1-C8 aliphatic hydrocarbon group. Preferably, the combined oily medium system and the active ingredient are in the form of a pharmaceutical composition, a food composition, or a food additive composition. Preferably, when the combined oily medium system and the active ingredient are in the form of a pharmaceutical composition, it is used for oral or sublingual administration; when the combined oily medium system and the active ingredient are in the form of a food composition, it is a health food, a dietary supplement, a functional food, a nutritional supplement food or a special nutritional food.

[0009] In the above-mentioned composition or method according to the present invention, the vegetable oil involved is preferably selected from the group consisting of sesame oil, perilla oil, inca nut oil, brown rice oil, soybean oil, seabuckthorn fruit oil, coconut oil, corn oil, palm oil, peanut oil, grape seed oil, safflower oil, pumpkin seed oil, almond oil, cashew nut oil, hazelnut oil, walnut oil, black bean oil, pine nut oil, black cumin oil, evening primrose oil, sesame leaf essential oil, canola oil, cottonseed oil, olive oil, rapeseed oil, sunflower oil, pecan oil, pistachio oil, castor oil, and a combination of the foregoing. More preferably, the vegetable oil is selected from the group consisting of sesame oil, soybean oil, canola oil, and a combination of the foregoing.

[0010] In the above composition or method according to the present invention, the active ingredient involved is preferably selected from the group consisting of: a compound of formula (II), a pharmaceutically acceptable salt thereof, a compound of formula (III), a pharmaceutically acceptable salt thereof, and a combination of the foregoing. (II), (III), wherein, R1 is a C1-C6 aliphatic hydrocarbon group (preferably a C4 alkyl or alkenyl group).

[0011] In the above composition or method according to the present invention, based on the volume of each milliliter of the oily medium system, the concentration of the active ingredient is preferably 0.001 mg / mL to 1,500 mg / mL; in addition, the volume ratio of the oily medium system to the active ingredient is preferably 1:15 to 30:1. Brief Description of the Drawings

[0012] Figure 1 is a photograph of the appearance of the composition according to different embodiments of the present invention after being configured.

[0013] Figure 2 is a photograph of the appearance of the composition according to an embodiment of the present invention after being made into soft capsules. Figure 3A is an HPLC analysis chromatogram of the content when the soft capsules are made. Figure 3B is an HPLC analysis chromatogram of the content of the soft capsules after being stored for one year. Figure 3C is an HPLC analysis chromatogram of ACN (i.e., the blank group).

[0014] Figure 4 is a photograph of the appearance of the composition according to different embodiments of the present invention after being stored for two years.

[0015] Figure 5 is a line graph showing the concentration of the active ingredient in the serum extracted at different time points after the composition according to an embodiment of the present invention is administered to rats.

[0016] Figure 6 is a line graph showing the concentration of the active ingredient in the serum extracted at different time points after the composition according to an embodiment of the present invention is administered to a human individual.

[0017] Figure 7 is a bar graph showing the relative survival rates of the brain cancer cell line 1XM (or the pancreatic cancer cell line Mia-PaCa2) treated differently compared to the blank group brain cancer cell line 1XM (or the blank group pancreatic cancer cell line Mia-PaCa2), wherein, "***" indicates that the p-value between the two groups is <0.001.

[0018] Figure 8 shows a bar graph of the relative survival rates of the brain cancer cell line 1XM treated differently compared to the blank group of the brain cancer cell line 1XM. Among them, "***" indicates that the p-value between the two groups is <0.001, "**" indicates that the p-value between the two groups is <0.01, and "*" indicates that the p-value between the two groups is <0.05. Embodiments

[0019] The following will describe the detailed technical content and some specific implementation aspects of the present invention for those of ordinary skill in the art to understand the features of the present invention; however, without departing from the spirit of the present invention, the present invention can also be practiced in different forms, and the protection scope of the present invention should not be construed as limited to what is specifically stated in the specification.

[0020] Unless otherwise stated in the text, the terms "a", "the", and similar terms used in this specification (especially in the following patent claims) should be understood to include both singular and plural forms; the numerical ranges used (such as 5 to 100) should be understood to also include all rational numbers within that range and the ranges composed of any rational numbers within that range, so it includes all possible combinations of numerical values between the listed minimum and maximum values; "mg / kg body weight" refers to the dosage used per kilogram of the individual's body weight; the so-called "individual" refers to mammals, including humans or non-human animals; Bioavailability (BA) is an indicator of the rate and extent of the absorption of the active ingredient (also known as the active component) of a drug from the formulation into the systemic blood circulation or the site of action.

[0021] The bioavailability (BA) of a known active ingredient can be known through the information provided by the graph of the change in the concentration of the active ingredient in the serum of the organism over time after administering the active ingredient to the organism. Briefly, first administer the active ingredient to the organism, continuously collect serum samples from the organism at specific time intervals after administration, then analyze the concentration of the active ingredient in the samples and plot a graph of concentration versus time, and then calculate the area under the curve (Area Under Curve, AUC). The value of AUC is positively correlated with the bioavailability. The higher the AUC, the higher the bioavailability of the active ingredient, and vice versa, the lower the AUC indicates the lower the bioavailability of the active ingredient. The foregoing can be referred to, for example, in Drug Bioavailability (National library of medicine. Gary Price; Deven A. Patel. July 30, 2023), the full text of which is incorporated herein for reference.

[0022] It is known that the compound of formula (I) of the present invention has certain therapeutic benefits in neurological diseases, cerebrovascular diseases, and / or cancers, but its application is still limited. The main reason is its low bioavailability (AUC < 300 ng×time / mL), and often higher doses need to be administered to achieve the desired therapeutic effect, which leads to an increased risk of systemic side effects and exacerbates the burden on patients. The problems caused by the need to increase the dosage due to bioavailability are particularly serious in the case of oral administration. See, for example, Curr Drug Metab. 2012 Jun 1;13(5):524-34. and Drug Metab Dispos.2008 Feb;36(2):400-8., the full texts of the foregoing literatures are incorporated herein for reference.

[0023] In view of the foregoing limitations in application, the inventors of the present case have found through research that the compound of formula (I) and / or its pharmaceutically acceptable salt have good solubility in the oily medium system of the present invention and can stably exist in the oily medium system for a long time (for example, stored at room temperature for up to two years). Further research has found that when the active ingredient of the present invention is administered in the form incorporated into the oily medium system, the bioavailability of the active ingredient can be improved, thereby avoiding the problem of having to increase the dosage to provide the desired therapeutic benefit and reducing or eliminating unnecessary side effects.

[0024] Therefore, the present invention relates to a composition comprising: (1) An oily medium system containing vegetable oil and / or components of vegetable oil, but not containing the following substances: phosphate buffer, polyethylene glycol, dimethyl sulfoxide, ethanol, polypropylene glycol, polysorbate, ethyl acetate, hydroxyethyl 12-hydroxystearate, and tocopheryl polyethylene glycol succinate; and (2) An active ingredient selected from the group consisting of: the compound of formula (I), its pharmaceutically acceptable salt, and combinations thereof, (I), wherein, T1 is a C6 cycloalkane, and R1 is a C1-C8 aliphatic hydrocarbon group.

[0025] In some embodiments according to the present invention, the composition is used to improve the bioavailability of the active ingredient.

[0026] The present invention also relates to a method for improving the bioavailability of an active ingredient, which comprises incorporating the active ingredient into an oily medium system, wherein: (1) The oily medium system contains vegetable oil and / or components of vegetable oil, but does not contain the following substances: phosphate buffer, polyethylene glycol, dimethyl sulfoxide, ethanol, polypropylene glycol, polysorbate, ethyl acetate, hydroxyethyl 12-hydroxy stearate, and tocopheryl polyethylene glycol succinate; and (2) The active ingredient is selected from the group consisting of a compound of formula (I), a pharmaceutically acceptable salt thereof, and a combination of the foregoing, (I), wherein, T1 is a C6 cycloalkane, and R1 is a C1-C8 aliphatic hydrocarbon group.

[0027] In the composition or method according to the present invention, the vegetable oil involved is preferably selected from the group consisting of sesame oil, perilla oil, inca inchi oil, brown rice oil, soybean oil, seabuckthorn fruit oil, coconut oil, corn oil, palm oil, peanut oil, grape seed oil, safflower oil, pumpkin seed oil, almond oil, cashew nut oil, hazelnut oil, walnut oil, black bean oil, pine nut oil, black cumin oil, evening primrose oil, sesame leaf essential oil, canola oil, cottonseed oil, olive oil, rapeseed oil, sunflower oil, pecan oil, pistachio oil, castor oil, and a combination of the foregoing; more preferably selected from the group consisting of sesame oil, soybean oil, canola oil, and a combination of the foregoing.

[0028] In some embodiments of the composition or method according to the present invention, the oily medium system consists of the vegetable oil.

[0029] In the composition or method according to the present invention, the active ingredient involved is preferably selected from the group consisting of a compound of formula (II), a pharmaceutically acceptable salt thereof, a compound of formula (III), a pharmaceutically acceptable salt thereof, and a combination of the foregoing, (II), (III), wherein, R1 is a C1-C6 aliphatic hydrocarbon group (preferably a C4 alkyl or alkenyl group).

[0030] In the composition or method according to the present invention, the compound of formula (I) and its pharmaceutically acceptable salt can be commercially available, prepared by synthetic methods known in the art of the present invention, or isolated from natural extracts, but not limited thereto.

[0031] In the composition or method according to the present invention, the concentration of the active ingredient in the oily medium system is based on the principle that no delamination occurs after mixing the two. Preferably, based on the weight of each gram of the oily medium system, the concentration of the active ingredient can be 50 mg / g to 500 mg / g; based on the volume of each milliliter of the oily medium system, the concentration of the active ingredient can be 0.001 mg / mL to 1,500 mg / mL. In some embodiments of the present invention, the volume ratio of the oily medium system to the active ingredient can be 1:15 to 30:1, such as 1:13, 1:11.5, 1:10, 1:8, 1:6, 1:4, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 8:1, 10:1, 13:1, 15:1, 17:1, 20:1, 25:1. In some embodiments of the present invention, when the oily medium system involved contains sesame oil, when the concentration of the compound of formula (I) reaches 920 mg / mL based on the volume of each milliliter of the oily medium system, it can still stably exist in the oily medium system.

[0032] The composition of the present invention can be prepared by simply mixing the oily medium system and the active ingredient. In the case where the active ingredient is soluble in the oily medium system, the active ingredient can be directly added to the oily medium system and dissolved therein; while in the case where the active ingredient is insoluble in the oily medium system, a physical force (such as shaking, magnetic stirring) can be applied after adding the active ingredient to the oily medium system to provide a stable suspension in which the active ingredient exists in the form of micron droplets or nano droplets.

[0033] The composition provided according to the present invention can be a pharmaceutical composition, a food composition, or a food additive composition. In addition, in the method according to the present invention, the combination of the active ingredient and the oily medium system can be in the form of a pharmaceutical composition, a food composition, or a food additive composition. In some embodiments of the present invention, the combination of the active ingredient and the oily medium system is provided as an oral pharmaceutical composition.

[0034] The pharmaceutical composition provided by the present invention can be used for systemic administration or local administration, and can be delivered through various drug delivery systems (DDS). For example, but not limited thereto, the pharmaceutical composition can be delivered by an oral drug delivery system, a transmucosal drug delivery system, a transdermal drug delivery system, and / or an injectable drug delivery system. In addition, the pharmaceutical composition can be administered to an individual in need in one or more of the following administration methods: oral, sublingual, subcutaneous injection, intramuscular injection, intraperitoneal injection, and intravenous injection (including drip infusion and rapid injection), nasal administration, transdermal administration, subcutaneous implantation, and interstitial implantation.

[0035] Depending on the form of use and application, any pharmaceutically acceptable excipient can be selected to provide the pharmaceutical composition, as long as the excipient does not adversely affect the desired benefits of the compound of formula (I) of the present invention and / or its pharmaceutically acceptable salt and its solubility in the oily medium system. The pharmaceutically acceptable excipients can be those known in the art and can be used, including, but not limited to: diluents, surfactants, glidants, disintegrants, binders, buffers, colorants, flavoring agents, antioxidants, preservatives, film-forming agents, etc.

[0036] The food composition provided by the present invention can be a health food, a dietary supplement, a functional food, a nutritional supplement food, or a special nutritional food. For example, but not limited thereto, the food composition can be provided in the form of an oil product. For example: provided in the form of a capsule containing oil droplets.

[0037] Depending on the form of use and application, the food additive composition provided by the present invention can be in any suitable form without special limitation, as long as it is a form convenient for addition during the food manufacturing process. For example, but not limited thereto, the food additive composition can be provided in the form of an oily food additive. For example: provided in the form of an edible essential oil.

[0038] The present invention is further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention is as shown in the appended claims.

[0039] [Examples]

[0040] In the following examples, the materials and equipment used are as follows: 1. Black sesame oil: Purchased from Yi Mei Food Company, product name: Yi Mei 100% Pure Black Sesame Oil. 2. White sesame oil: Purchased from Seitenbacher Company, product name: Premium Sesame oil. 3. Canola oil: Purchased from TTET UNION Company. 4. Soybean oil: Purchased from TTET UNION Company. 5. 2,6-Di-tert-butyl-p-cresol, also known as 2,6-Di-tert-butylhydroxytoluene (Butylated hydroxytoluene, abbreviated as BHT): Purchased from Alfa Aesar Company. 6. n-Butylidenephthalide (BP): Purchased from Penta Manufacturing Company; purity ≥ 97%. 7. (Z)-n-Butylidenephthalide (Z-BP): Provided by Changhong Biotechnology Co., Ltd.; batch number: F212TR12001; purity 99.8%. 8. Ligustilide (LG): Purchased from Sigma-Aldrich; purity ≥ 96%. 9. Butylphthalide: Purchased from Toronto Research Chemicals Company; purity ≥ 98%. 10. 1XM cells (a human glioblastoma cell line): Obtained from the research team of Professor Han Hongzhi of Hualien Tzu Chi Hospital (website: hlm.tzuchi.com.tw / ). 11. Mia-PaCa2 cells (a human pancreatic cancer cell line): Obtained from the Bioresource Collection and Research Center (Bioresource Collection and Research Center, BCRC; website: www.bcrc.firdi.org.tw / ); deposit number: BCRC 60139. 12. 1XM cell culture medium: DMEM medium (purchased from Hyclone) supplemented with 10% fetal bovine serum (FBS; purchased from Hyclone) and 1% penicillin and streptomycin (P / S; purchased from Hyclone). 13. Mia-PaCa2 cell culture medium: DMEM / High Glucose medium (purchased from Hyclone) supplemented with 10% fetal bovine serum (FBS; purchased from Hyclone), 2.5% horse serum (purchased from Gibco), and 1% penicillin and streptomycin (P / S; purchased from Hyclone). 14. MTT assay kit: purchased from Sigma-Aldrich. 15. ELISA reader (immunoenzymatic analyzer): purchased from PerkinElmer (USA).

[0041] [Examples] [1] [: Preparation of the Composition of the Present Invention]

[0042] [1-1.] [Preparation of the Composition]

[0043] In a glass bottle, the components were mixed in the dosage ratios shown in Table 1 and rapidly stirred with a magnet for 20 minutes to form a homogeneous liquid, and Compositions 1 to 4 were respectively prepared. After removing the magnet, the glass bottle was tightly closed, and then its appearance was observed and photographed. The results are shown in Figure 1.

[0044] Table 1: Composition volume percentages of Compositions 1 to 4 Composition 1 2 3 4 BP 50 83.3 50 83.3 Black sesame oil 50 16.7 White sesame oil 50 16.7

[0045] As shown in Figure 1, no layering occurred in Compositions 1 to 4, indicating good solubility of BP in sesame oil. In addition, Composition 3 was further made into soft capsules, and a photo of the soft capsules is shown in Figure 2.

[0046] [1-2.] [Component Analysis]

[0047] The components of the contents of the soft capsules provided in Example 1-1 were analyzed at different times. This included preparing samples with a concentration of 1 mg / mL of the contents with acetonitrile (ACN) at the time when the soft capsules were made and after storing them in an environment without sunlight at room temperature for one year, and analyzing the components in the samples by high performance liquid chromatography (HPLC) under the conditions shown in Table 2. The results are shown in Figures 3A and 3B, and Figure 3C is the HPLC chromatogram of ACN. As can be seen from Figures 3A to 3C, even after storing the soft capsules at room temperature for up to one year, the contents still contained Z-BP and E-BP, indicating that the active ingredients can be stably present in the oily medium system of the present invention for a long time.

[0048] Table 2 Analytical Equipment Waters series LC system Column Sharpsil-U C18 (s-5 μm, 100 Å, 4.6 x 250 mm) with additional guard column C18 (5 μm, 4.6 x 20 mm) Mobile Phase Conditions 0.1% H3PO4 ACN / H2O (30 / 70): 0.1% H3PO4 ACN = 100:0 gradient to 5:95 Temperature 45 °C Flow Rate 1 mL / min UV Detection Wavelength 261 nanometers (nm)

[0049] [Examples] [2] [Stability of the Composition of the Present Invention]

[0050] [2-1.] [Preparation of the Composition]

[0051] In a glass bottle, the components were mixed in the dosage ratios shown in Table 3 and rapidly stirred with a magnet for 20 minutes to form a homogeneous liquid, and Compositions 5 to 11 were respectively prepared. After removing the magnet, the glass bottle was tightly closed.

[0052] Table 3: Composition Weight Percentages of Compositions 5 to 11 Composition 5 6 7 8 9 10 11 12 Schema Label F10319 F10700 F10600 F10900 F10300 F1900 F1300 D2018-2 BP 8 12 15 30 8 30 8 8 Black Sesame Oil 80 87 85 70 92 Canola Oil 11.9 70 92 Soybean oil 92 BHT 0.1

[0053] [2-2.] [Stability test]

[0054] The compositions 5 to 10 provided in Example 2-1 were placed in an environment without sunlight at room temperature, and their appearance was observed and photographed after two years. The results are shown in Figure 4.

[0055] As can be seen from Figure 4, the compositions 5 to 10 did not stratify even after being stored at room temperature for up to two years. The above results again show that even after long-term storage, the active ingredients contained in the compositions of the present invention can still stably exist in the oily medium system, having quite excellent stability.

[0056] [Examples] [3] [: Bioavailability test (experimental animals)]

[0057] [3-1.] [Preparation of serum samples]

[0058] After fasting the rats for 12 hours, 0.3 ml of blood was drawn from the tail vein of the rats and injected into a blood collection tube containing Potassium (K2) EDTA. Then, the blood collection tube was centrifuged for 10 minutes (2000 xg, 2-8 °C), and the supernatant (i.e., the serum of the blank group) was frozen at -70 °C for subsequent analysis.

[0059] Afterwards, the rats were randomly divided into five groups (2 rats in each group) and treated under the following conditions respectively: 1. Group F10900: The composition 8 provided in Example 2-1 was administered to the rats by oral gavage at a dosage of 300 mg (BP) / kg body weight. 2. Group F10300: Treated in the same way as Group F10900, but the composition 8 was replaced with composition 9. 3. Group F1900: Treated in the same way as Group F10900, but the composition 8 was replaced with composition 10. 4. Group F1300: Treated in the same way as Group F10900, but the composition 8 was replaced with composition 11. 5. Group D2018-2: Treated in the same way as Group F10900, but the composition 8 was replaced with composition 12.

[0060] At 1, 2, 4, 6, 8, and 24 hours after the feeding administration, 0.3 mL of blood was drawn from the tail vein of the rats in each group and injected into a blood collection tube containing dipotassium ethylenediaminetetraacetate (EDTA K2), and then placed on ice. Afterwards, each blood collection tube was centrifuged for 10 minutes (2000 xg, 2-8 °C), and the supernatant was taken. The obtained sera were the sera of Group F10300, Group F1900, Group F1300, Group F1900, and Group D2018-2 respectively. Each group of sera was stored frozen at -70 °C for subsequent analysis.

[0061] [3-2.] [Preparation of Analytical Standard]

[0062] 50 μL of the standard solution (containing BP in the concentration range of 0 to 0.5 mg / mL) was mixed with 150 μL of the blank group serum provided in Example 3-1, and then sonicated for 3 minutes. Then, 400 μL of acetonitrile (ACN) was added, and after mixing, it was sonicated for another 3 minutes. Then, the mixture was placed in an environment below 0 °C for 10 minutes and then taken out. After returning to room temperature for 5 minutes, it was filtered through a 0.2 μm PTFE filter membrane. The obtained filtrate was the analytical standard.

[0063] [3-3.] [Preparation of Analytical Sample]

[0064] Mix 150 μL of the F10300 group serum, F1900 group serum, F1300 group serum, F1900 group serum, or D2018-2 group serum provided in Example 3-1 with 450 μL of ACN, and then mix the mixture by ultrasonic oscillation for 3 minutes. Next, place the mixture in an environment below 0 °C for 10 minutes and then take it out. After returning to room temperature for 5 minutes, filter it through a 0.2-μm PTFE filter membrane. The resulting filtrate is the analysis sample.

[0065] [3-4. HPLC] [Pharmacokinetic analysis]

[0066] Perform HPLC analysis on the analysis standards and analysis samples provided in Examples 3-2 and 3-3 under the conditions shown in Table 4, and quantify BP in the serum samples at each sampling time point. The results are shown in Figure 5.

[0067] Table 4 Analysis equipment Waters series LC system Column Sharpsil-U C18 (s-5 μm, 100 Å, 4.6 x 250 mm) with additional guard column C18 (5 μm, 4.6 x 20 mm) Mobile phase conditions 0.1% H3PO4 ACN / H2O (30 / 70): 0.1% H3PO4 ACN = 100:0 gradient to 9:91 Temperature 40 °C Flow rate 1 mL / min UV detection wavelength 261 nanometers (nm)

[0068] After inputting the obtained BP concentration values at each sampling time point into Pksolver software, calculate the parameters such as Tmax, Cmax, AUC, T1 / 2, etc., and the bioavailability (BA) using the NCA IV Bolus mode and the linear log Trapezoidal method. The results are shown in Table 5.

[0069] Table 5 Group Tmax (hour) Cmax (ng / mL) AUC (ng×time / mL) T 1 / 2 BA(%) F10900 3 1506 8368 6.9 13.5 F10300 3 2902 19645 4.4 31.7 F1900 2 1891 5270 7.9 9 F1300 0.5 950 8895 6 14.3 D2018-2 1 1386 2768 6.3 4.5

[0070] As can be seen from Figure 5 and Table 5, BP can be detected in the sera taken within 24 hours after the rats in groups F10300, F1300, F10900, and F1900 orally received the composition of the present invention. Moreover, the BP contents in the sera of groups F10300, F1300, F10900, F1900, and D2018-2 reached their peaks within 3 hours after the rats orally received the composition of the present invention. In addition, the AUC and BA of group F10300 were higher than those of group F10900, and the AUC of group F1300 was higher than that of group F1900. The above results indicate that the oral absorption rate and absorption extent of the composition of the present invention in rats are quite excellent, and the bioavailability of the active ingredient contained in the composition of the present invention increases with the increase in the proportion of vegetable oil in the composition.

[0071] The results of this example show that incorporating the compound of formula (I) of the present invention into the oily medium system of the present invention and then administering it to an individual animal can improve the bioavailability of the compound of formula (I).

[0072] [Examples] [4] [: Bioavailability Test (Human)]

[0073] [4-1.] [Preparation of Serum Samples] 1. Blank group: Before the subject (body weight: 70 kg) orally received the soft capsule provided in Example 1-1, 5 mL of blood was first drawn and injected into a blood collection tube containing EDTA K2. Then, the blood collection tube was centrifuged for 10 minutes (1900 xg, 2-8 °C), and the supernatant (i.e., blank group serum) was frozen at -70 °C for subsequent analysis. 2. "Experiment (Sesame Oil)" group: At the 1st, 2nd, 4th, 8th, and 24th hours after the subject orally received the soft capsule provided in Example 1-1 (dosage: 4.285 mg / kg body weight based on the weight of BP), 5 mL of blood was drawn each time and injected into a blood collection tube containing EDTA K2, and it was placed on ice. Then, each blood collection tube was centrifuged for 10 minutes (1900 xg, 2-8 °C), and the supernatant (i.e., serum) was frozen at -70 °C for subsequent analysis.

[0074] [4-2.] [Preparation of Analytical Standards]

[0075] Performed by referring to Example 3-2, but replacing the blank group serum provided in Example 3-1 with the blank group serum provided in Example 4-1 to provide an analytical standard.

[0076] [4-3.] [Preparation of analytical samples]

[0077] Performed by referring to Example 3-3, but replacing the F10300 group serum / F1900 group serum / F1300 group serum / F1900 group serum with the "Experiment (sesame oil)" group serum provided in Example 4-1 to provide an analytical sample.

[0078] [4-4. HPLC] [Analysis]

[0079] According to the method and conditions of Example 3-4, the analytical standards and analytical samples provided in Examples 4-2 and 4-3 were analyzed, and BP in the serum samples at each sampling time point was quantified. The results are shown in Figure 6.

[0080] As can be seen from Figure 6, BP can be detected in the serum within 24 hours after the subjects orally administered the composition of the present invention, and the BP content in the serum reached the peak approximately 2 hours after orally administering the composition of the present invention. The above results show that the absorption rate and absorption degree of the composition of the present invention for human oral administration are also quite excellent.

[0081] [Example] [5] [: Cytotoxicity test against cancer cells] [I]

[0082] This example uses the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to study the effect of the serum obtained after administering the composition of the present invention on killing cancer cells.

[0083] The cell cultures in this example were all carried out in an environment of 95% relative humidity, 5% CO2, and 37°C.

[0084] [5-1.] [Cell culture]

[0085] The brain cancer cell line 1XM (or the pancreatic cancer cell line Mia-PaCa2) was grouped and cultured in a 6-well cell culture dish with the following culture media for 48 hours: 1. Blank group: 1XM cell culture medium (or Mia-PaCa2 cell culture medium). 2. "BP(50)" group or "BP(100)" group: 1XM cell culture medium (or Mia-PaCa2 cell culture medium) supplemented with BP (50 or 100 μg / mL). 3. "Blank serum (low)", "blank serum (medium)", "blank serum (high)" groups: 1XM cell culture medium (or Mia-PaCa2 cell culture medium) supplemented with the blank group serum provided in Example 4-1 (at a content of 6.25% v / v, 12.5% v / v, or 25% v / v in the culture medium). 4. "Experimental serum (low)", "experimental serum (medium)", "experimental serum (high)" groups: 1XM cell culture medium (or Mia-PaCa2 cell culture medium) supplemented with the experimental group serum taken at the 1st, 2nd, 4th, 8th, or 24th hour after taking the oral soft capsule in Example 4-1 (at a content of 6.25% v / v, 12.5% v / v, or 25% v / v in the culture medium).

[0086] [5-2. MTT] [Analysis method]

[0087] Remove the culture medium from each well of the cell culture plate and add MTT reagent (to make the final concentration of MTT in the culture medium of each well 0.5 mg / mL), then place the cell culture plate in an incubator at 37°C and 5% CO2 for 2 to 4 hours to form purple needle-shaped crystals of formazan. Thereafter, aspirate the solution, and add 500 μL of dimethyl sulfoxide (DMSO) to each well. Shake with a shaker for 5 to 10 minutes to completely dissolve the purple needle-shaped crystals of formazan, then use an ELISA reader to measure the absorbance at a wavelength of 570 or 595 nm, calculate the survival rate of the cells accordingly, and calculate the relative survival rate of each of the remaining groups with the blank group as the reference (i.e., set the survival rate of the blank group to 100%). The results are shown in Figures 7 and 8. Among them, the data of the experimental group serum taken at the 2nd hour after oral administration of the soft capsule are shown in Figure 7, and the data of the experimental group serum taken at the 1st, 4th, 8th, or 24th hour after oral administration of the soft capsule are shown in Figure 8.

[0088] As can be seen from Figures 7 and 8, compared with the blank group, the cancer cell survival rates of the "experimental serum (low)", "experimental serum (medium)", and "experimental serum (high)" groups are all lower, and are comparable to those of the "BP(50)" group or the "BP(100)" group; on the other hand, the degree of reduction in cancer cell survival rate is "experimental serum (low)" group < "experimental serum (medium)" group < "experimental serum (high)" group.

[0089] The above results show that the serum obtained after oral administration of the soft capsule containing the composition of the present invention has the effect of killing brain cancer cells and pancreatic cancer cells, and the aforementioned effect will increase with the increase in the concentration of the active ingredient in the serum, and is comparable to that of directly treating cancer cells with the active ingredient. This also shows that the active ingredient contained in the composition of the present invention has excellent bioavailability.

[0090] [Example] [6] [: Cancer cell cytotoxicity test] [II]

[0091] All cell cultures in this example were carried out in an environment of 95% relative humidity, 5% CO2, and 37°C.

[0092] [6-1.] [Cell culture]

[0093] The pancreatic cancer cell line Mia-PaCa2 was grouped and cultured in a 96-well cell culture plate with the following culture media for 24 hours: 1. "Z-BP" group: Mia-PaCa2 cell culture medium supplemented with Z-BP. 2. "Z-BP + sesame oil" group: "Z-BP + canola oil" group, "Z-BP + soybean oil" group: Mia-PaCa2 cell culture medium supplemented with a mixture of Z-BP and sesame oil, canola oil, or soybean oil in equal proportions. 3. "LG + sesame oil" group: "LG + canola oil" group, "LG + soybean oil" group: Mia-PaCa2 cell culture medium supplemented with a mixture of LG and sesame oil, canola oil, or soybean oil in equal proportions.

[0094] [6-2. MTT] [Analysis]

[0095] MTT analysis was performed in the same manner as in Example 5-2, and the concentration value (IC50) for the above different treatment methods to achieve 50% lethality of pancreatic cancer cells was calculated based on the measured survival rate. The results are shown in Table 6.

[0096] Table 6 Group IC50 (μg / mL) Z-BP 63.84 Z-BP + sesame oil 17.35 Z-BP + canola oil 23.25 Z-BP + soybean oil 40.66 LG 44.09 LG + sesame oil 20.69 LG + canola oil 25.98 LG + soybean oil 43.69

[0097] The results in Table 6 show that incorporating the compound of formula (I) of the present invention into the oily medium system of the present invention improves the efficacy of the compound of formula (I) in killing cancer cells.

[0098] As can be seen from the results of the above embodiments, the active ingredient of the present invention (i.e., the compound of formula (I) and / or its pharmaceutically acceptable salt) has good solubility in the oily medium system of the present invention and can stably exist in the oily medium system for a long time. In addition, when the active ingredient of the present invention is administered in the form incorporated into the oily medium system, the bioavailability of the active ingredient can be improved, thereby avoiding the problem of having to use an increased amount to provide the desired therapeutic benefit and reducing or eliminating unnecessary side effects.

[0099] None

Claims

1. A composition comprising: (1) an oily medium system containing vegetable oil but free from: phosphate buffer, polyethylene glycol, dimethyl methacrylate, ethanol, polypropylene glycol, polysorbate, ethyl acetate, hydroxyethyl 12-hydroxystearate, and tocopheryl polyethylene glycol succinate; and (2) an active ingredient selected from the group consisting of: butylenephthalide, pharmaceutically acceptable salts thereof, and combinations thereof, wherein, The vegetable oils are selected from the following groups: sesame oil, perilla oil, sacha inchi oil, brown rice oil, soybean oil, corn oil, peanut oil, grapeseed oil, safflower seed oil, pumpkin seed oil, walnut kernel oil, black soybean oil, pine nut oil, black cumin oil, evening primrose oil, rapeseed oil, cottonseed oil, sunflower oil, and combinations thereof.

2. As claimed in claim 1, wherein the vegetable oil is selected from the group consisting of: sesame oil, soybean oil, rapeseed oil, and combinations thereof.

3. The composition of claim 1 or 2, wherein the concentration of the active ingredient is from 0.001 mg / mL to 1,500 mg / mL per mL of the oily medium system.

4. The composition of claim 1 or 2, wherein the volume ratio of the oily medium system to the active ingredient is 1:15 to 30:

1.

5. A method for improving the bioavailability of an active ingredient, comprising incorporating the active ingredient into an oily medium system, wherein: (1) The oily medium system contains vegetable oil but does not contain the following substances: phosphate buffer, polyethylene glycol, dimethyl sulfoxide, ethanol, polypropylene glycol, polysorbate, ethyl acetate, hydroxyethyl 12-hydroxystearate, and tocopheryl polyethylene glycol succinate; and (2) the active ingredient is butylenephthalide, its pharmaceutically acceptable salt, and a combination thereof, wherein the vegetable oil is selected from the group consisting of: sesame oil, perilla oil, sacha inchi oil, brown rice oil, soybean oil, corn oil, peanut oil, grape seed oil, safflower seed oil, pumpkin seed oil, walnut oil, black soybean oil, pine nut oil, black cumin oil, evening primrose oil, rapeseed oil, cottonseed oil, sunflower oil, and a combination thereof.

6. The method of claim 5, wherein the vegetable oil is selected from the group consisting of: sesame oil, soybean oil, rapeseed oil, and combinations thereof.

7. The method of claim 5 or 6, wherein the concentration of the active ingredient is from 0.001 mg / mL to 1,500 mg / mL per mL of the oily medium system.

8. The method of claim 5 or 6, wherein the volume ratio of the oily medium system to the active ingredient is 1:15 to 30:

1.

9. The method of claim 5 or 6, wherein the oily medium system and the active ingredient are in the form of a pharmaceutical composition, a food composition, or a food additive composition.

10. The method of claim 9, wherein the combined oily media system and the active ingredient are in the form of a pharmaceutical composition, is for oral or sublingual administration.

11. The method of claim 9, wherein the combined oily medium system and the active ingredient are in the form of a food composition, is a health food, dietary supplement, functional food, nutritional supplement, or special nutritional food.

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