Pharmaceutical composition for reducing local fat and its uses

Abstract

The present invention provides a pharmaceutical composition for reducing local fat, comprising a plurality of drug-containing micelles formed by a surfactant and curcumin encapsulated in the drug-containing micelles. The pharmaceutical composition for reducing local fat can reduce the fat at the administration site and has the advantages of high stability, high bioavailability in adipose tissue, low side effects, and sustained release.

Description

[0001] This application is a divisional application of an application with an application date of August 26, 2016, an application number of 201680046418.3, and an invention title of "Pharmaceutical Composition for Reducing Local Fat and Its Use". Technical Field

[0002] The present invention relates to a pharmaceutical composition for reducing local fat, particularly to a pharmaceutical composition comprising a plurality of drug-containing micelles and curcumin encapsulated in the drug-containing micelles, and the pharmaceutical composition is for reducing local fat. Background Art

[0003] In recent years, due to the changing concept of beauty among more and more people and the improvement of the standards for self-health and body shape, the issues that people are concerned about are no longer simply weight loss, but rather pay more attention to reducing local fat or sculpting curves to achieve a healthier and more beautiful body shape. Moreover, general weight loss methods, whether diet or exercise, cannot reduce the fat in a specific single part. If one wants to reduce the fat in a specific part (such as the waist, abdomen, legs, arms, chin, and face, etc.), the current technology can only be achieved by methods such as liposuction surgery.

[0004] Currently, the main method for reducing local fat is liposuction surgery. However, the liposuction process can cause serious damage to nerves, blood vessels, and other body tissues, and has risks of infection, large bleeding volume, overly long anesthesia time, and unpreventable fat embolism and anesthesia allergy leading to death. In addition, after liposuction surgery, there are also easily problems such as severe bruising and swelling, intense pain, a recovery period of up to 3 months to more than 6 months, and unevenness in the liposuction area. Therefore, statistics show that although most people want to use liposuction to reduce the subcutaneous fat accumulated in local parts or improve the body curve, the actual number of people undergoing liposuction surgery is less than 40%, indicating that most consumers who want to improve the body curve or reduce local fat will give up due to problems such as the side effects of liposuction surgery, postoperative pain, or risks.

[0005] Although there are some non-surgical local fat reduction pharmaceutical compositions or instruments that can reduce some side effects, most of them have poor efficacy and will cause other side effects, such as necrosis of surrounding normal cells, inflammation of surrounding tissues, and intense pain, etc., and there are also certain limitations in the implementation site. Therefore, there is still a great lack in the market for a local fat reduction pharmaceutical composition that can effectively reduce local fat and has lower side effects, better stability, and a shorter recovery period.

[0006] In the situation where there is a high demand from both consumers and physicians, developing a local fat reduction pharmaceutical composition that can break through the current technical limitations will be an urgent topic to be explored and solved. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a pharmaceutical composition for reducing local fat, comprising a plurality of drug-loaded micelles formed by a surfactant, and curcumin encapsulated in the drug-loaded micelles. The pharmaceutical composition for reducing local fat can reduce the fat at the administration site, and has the advantages of high stability, high bioavailability in adipose tissue, low side effects, and sustained release.

[0008] The present invention can induce apoptosis in adipocytes at the administration site, so as to achieve the purpose of reducing local fat at the administration site. The present invention can significantly improve the adverse reactions and side effects of necrosis and inflammation of surrounding cells caused by the prior art, and the effect of local fat reduction is significantly better than that of other pharmaceutical compositions for non-surgical reduction of local fat. The present invention is applicable to be administered to the site that needs to reduce subcutaneous fat by means of direct injection, subcutaneous implantation, intravenous injection, implantable infusion, ointment, patch, or other transdermal absorption systems, without any intervention or assistance of surgery or instruments. Preferably, it is administered by subcutaneous fat injection to the subcutaneous fat layer at the local site. Preferably, the injection dosage form of the pharmaceutical composition of the present invention includes, but is not limited to, injection solution or powder for injection (powder for injection, or powder for solution for injection). The local fat referred to in the present invention includes, but is not limited to, the fat in the waist, abdomen, legs, arms, chin, and face and other parts.

[0009] In the present invention, the turmeric extract refers to a mixture of turmeric components extracted by any solvent and any extraction method, a commercially available turmeric extract, any mixture containing at least 75% (weight percentage) of curcumin, any mixture containing at least 75% (weight percentage) of curcuminoids, or a commercially available curcumin.

[0010] Among them, curcuminoids is the general term for curcumin, demethoxycurcumin, and bisdemethoxycurcumin.

[0011] In the present invention, resveratrol refers to resveratrol obtained by extraction from natural plants or commercially available. Preferably, the purity of resveratrol is 90% to 100% (weight percentage).

[0012] In the present invention, the green tea extract refers to a mixture of green tea components extracted by any solvent and any extraction method, a commercially available green tea extract, any mixture containing at least 45% (weight percentage) epigallocatechin gallate (EGCG), or a commercially available epigallocatechin gallate.

[0013] In the present invention, a micelle refers to a micro-structure formed by a surfactant, the surfactant having a hydrophilic end and a lipophilic end (oleophilic end), and the surfactant forming the micro-structure with the hydrophilic end facing outwards and the lipophilic end (oleophilic end) facing inwards. Preferably, the micro-structure is spherical, quasi-spherical, or other micro-structures.

[0014] In the present invention, the drug-loaded micelle refers to a micelle containing curcuminoid substances, preferably, the drug-loaded micelle refers to a micelle containing curcumin; that is, the drug-loaded micelle refers to a micelle that encapsulates or contains curcuminoid substances, preferably, the drug-loaded micelle refers to a micelle that encapsulates or contains curcumin.

[0015] In the present invention, the second lipophilic drug micelle refers to a micelle containing other lipophilic drugs other than curcuminoid substances. That is, the second lipophilic drug micelle refers to a micelle that encapsulates or contains a second lipophilic drug.

[0016] Among them, other lipophilic drugs (or called the second lipophilic drug) refer to at least one or a combination of quercetin, synephrine, puerarin, resveratrol, and other lipophilic drugs other than curcuminoid substances; or, other lipophilic drugs refer to lipophilic drugs other than curcumin.

[0017] In the present invention, the water-soluble drug refers to at least one or a combination of green tea extract, epigallocatechin gallate, epicatechin, epicatechin gallate, epigallocatechin, gallocatechin gallate, gallocatechin, catechin gallate, catechin, epigallocatechin gallate (EGCG), caffeine, carnitine (also known as carnitine or carnitine), L-carnitine, synephrine, chlorogenic acid, and other water-soluble drugs.

[0018] In the present invention, the term "state without precipitate formation" means that no precipitate visible to the naked eye of a human is contained, that is, no artificial device is required.

[0019] In the present invention, the term "local subcutaneous fat" refers to the subcutaneous fat at the site where the pharmaceutical composition, subcutaneous injection, or subcutaneous fat layer injection of the present invention is administered.

[0020] In the present invention, the pharmaceutically acceptable aqueous solution is at least one or a combination of water for injection, aqueous injection solution, and physiological saline.

[0021] In the present invention, the local anesthetic is at least one or a combination of amides, para-aminobenzoate esters, amino ethers, and other local anesthetics. Preferably, the amides are at least one or a combination of dibucaine, lidocaine, mepivacaine HCl, bupivacaine HCl, pyrrocaine HCl, prilocaine HCl, digammacaine, and oxethazaine. Preferably, the para-aminobenzoate esters are at least one or a combination of butacaine, dimethocaine, and tutocaine. Preferably, the amino ethers are at least one or a combination of quinisocaine and pramocaine.

[0022] In the present invention, the antioxidant is at least one or a combination thereof selected from beta-carotene, lutein, lycopene, bilirubin, vitamin A, vitamin C (also known as ascorbic acid), vitamin E, uric acid, nitric oxide, nitroxide, pyruvate, catalase, superoxide dismutase, glutathione peroxidases, N-acetyl cysteine, naringenin, and other antioxidants.

[0023] In the present invention, when the pharmaceutical composition is subjected to an accelerated stability test under the conditions of a temperature of 25°C ± 2°C, a relative humidity of RH60% ± 5%, and avoiding direct sunlight, the pharmaceutical composition remains in a state without precipitation for at least 24 hours.

[0024] Alternatively, when the pharmaceutical composition is subjected to an accelerated stability test under the conditions of a temperature of 25°C ± 2°C, a relative humidity of RH60% ± 5%, and avoiding direct sunlight, the pharmaceutical composition remains in a state without precipitation for at least 6 months.

[0025] The present invention provides a pharmaceutical composition for topical administration to an individual, comprising:

[0026] a plurality of drug-loaded micelles; and

[0027] curcuminoid encapsulated in the drug-loaded micelles;

[0028] wherein the drug-loaded micelle is a micro-structure formed by a pharmaceutically acceptable polyoxyethylene castor oil derivative, and the hydrophilic-lipophilic balance value (HLB value) of the polyoxyethylene castor oil derivative is greater than 10.

[0029] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solution, and the plurality of drug-loaded micelles are uniformly distributed in the pharmaceutically acceptable aqueous solution.

[0030] Preferably, the polyoxyethylene castor oil derivative is at least one or a combination of polyoxyethylene 35 castor oil (Cremophor ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives.

[0031] Preferably, the weight ratio of the curcuminoid substance to the polyoxyethylene castor oil derivative is 1:8 to 1:500.

[0032] Preferably, the weight ratio of the curcuminoid substance to the polyoxyethylene castor oil derivative is 1:20 to 1:150.

[0033] Preferably, the concentration of the curcuminoid substance in the pharmaceutical composition is 0.3 to 120 mg / g.

[0034] Preferably, the concentration of the curcuminoid substance in the pharmaceutical composition is 2 to 91 mg / g.

[0035] Preferably, the particle size of the drug-loaded micelles is 3 to 50 nm.

[0036] Preferably, the particle size of the drug-loaded micelles is 5 to 20 nm.

[0037] Preferably, the curcuminoid substance is curcumin.

[0038] Preferably, the pharmaceutical composition further comprises a second lipid-soluble drug micelle, which is uniformly distributed in the pharmaceutically acceptable aqueous solution; the second lipid-soluble drug micelle is a second micro-structure formed by a second polyoxyethylene castor oil derivative, and a second lipid-soluble drug is encapsulated in the second lipid-soluble drug micelle.

[0039] Preferably, the hydrophilic-lipophilic balance value (HLB value) of the second polyoxyethylene castor oil derivative is greater than 10.

[0040] Preferably, the second polyoxyethylene castor oil derivative is at least one or a combination of polyoxyethylene 35 castor oil (Cremophor ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives.

[0041] Preferably, the second lipid-soluble drug is at least one or a combination of quercetin, synephrine, puerarin, resveratrol, and other lipid-soluble drugs other than curcuminoid substances.

[0042] Preferably, the weight ratio of the curcuminoid substance to the second fat-soluble drug is 30:1 to 1:10.

[0043] Preferably, the weight ratio of the curcuminoid substance to the second fat-soluble drug is 20:1 to 1:8.

[0044] Preferably, the pharmaceutically acceptable aqueous solution further contains a water-soluble drug.

[0045] Preferably, the water-soluble drug is at least one of or a combination of green tea extract, epigallocatechin gallate, epicatechin, epicatechin gallate, epigallocatechin, gallocatechin gallate, gallocatechin, catechin gallate, catechin, epigallocatechin gallate (EGCG), caffeine, carnitine (also known as carnitine or carnitine), L-carnitine, synephrine, chlorogenic acid, and other water-soluble drugs.

[0046] Preferably, the weight ratio of the curcuminoid substance to the water-soluble drug is 30:1 to 1:10.

[0047] Preferably, the concentration of epigallocatechin gallate in the pharmaceutical composition is 0.1 to 15 mg / mL.

[0048] Preferably, the pharmaceutical composition further contains a cosolvent to increase the drug solubility.

[0049] Preferably, the cosolvent is at least one of or a combination of polyethylene glycol, propylene glycol, ethanol, and other cosolvents.

[0050] Preferably, the polyethylene glycol is at least one of or a combination of polyethylene glycol 200 (PEG 200), polyethylene glycol 400 (PEG 400), polyethylene glycol 600 (PEG 600), and other polyethylene glycols.

[0051] Preferably, the pharmaceutical composition further comprises a suspending agent (also known as a suspension agent; suspending agent) for reducing the sedimentation rate of the drug or micelles.

[0052] Preferably, the suspending agent is at least one or a combination of sodium alginate, glycerol, carboxymethylcellulose sodium, mannitol, and other suspending agents.

[0053] Preferably, the pharmaceutical composition further comprises oil phase excipients for increasing the stability of the pharmaceutical composition and the solubility of the drug.

[0054] Preferably, the oil phase excipients are at least one or a combination of unsaturated fatty acids, glycerol, triglyceride, and other oil phase excipients.

[0055] Preferably, the unsaturated fatty acid is at least one or a combination of oleic acid, castor oil, sesame oil, cottonseed oil, soybean oil, safflower oil, corn oil, and other unsaturated fatty acids.

[0056] Preferably, the triglyceride is at least one or a combination of medium chain triglycerides and other triglycerides.

[0057] Preferably, the pharmaceutically acceptable aqueous solution contains a local anesthetic.

[0058] Preferably, the pharmaceutically acceptable aqueous solution contains an antioxidant.

[0059] The present invention further provides a use of a pharmaceutical composition for preparing a subcutaneous injection, a subcutaneous fat layer injection, a subcutaneous implant device, a subcutaneous implant, an intravenous injection, an implantable infusion, an ointment, a patch, or other transdermal absorption systems.

[0060] Preferably, the pharmaceutical composition further comprises a second lipid-soluble drug micelle, which is uniformly distributed in the pharmaceutically acceptable aqueous solution; wherein the second lipid-soluble drug micelle is a second micro-structure formed by a second non-ionic surfactant, and a second lipid-soluble drug is encapsulated in the second lipid-soluble drug micelle.

[0061] Preferably, the second non-ionic surfactant is at least one or a combination of polyoxyethylene sorbitan monooleate (Tween 80), polyethylene glycol 15-hydroxystearate (Kolliphor HS 15), polyoxyethylene castor oil derivatives, and other non-ionic surfactants.

[0062] Preferably, the polyoxyethylene castor oil derivatives are at least one or a combination of polyoxyethylene 35 castor oil (Cremophor ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives.

[0063] Preferably, the pharmaceutically acceptable aqueous solution further comprises a water-soluble drug.

[0064] The present invention also provides a use of a pharmaceutical composition in the preparation of a drug or a subcutaneous injection formulation for reducing local subcutaneous fat; the pharmaceutical composition comprises:

[0065] A plurality of drug-loaded micelles; and

[0066] Curcuminoids encapsulated in the drug-loaded micelles;

[0067] Wherein, the drug-loaded micelle is a micro-structure formed by a pharmaceutically acceptable non-ionic surfactant, and the hydrophilic-lipophilic balance value (HLB value) of the non-ionic surfactant is greater than 10.

[0068] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solution, and the plurality of drug-loaded micelles are uniformly distributed in the pharmaceutically acceptable aqueous solution.

[0069] Preferably, the nonionic surfactant is at least one or a combination thereof selected from polysorbate 80 (Tween 80), polyethylene glycol 15-hydroxystearate (Kolliphor HS 15), polyoxyethylene castor oil derivatives, and other nonionic surfactants.

[0070] Preferably, the polyoxyethylene castor oil derivative is at least one or a combination thereof selected from polyoxyethylene 35 castor oil (Cremophor ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives.

[0071] Preferably, the weight ratio of the curcuminoid substance to the nonionic surfactant is 1:8 to 1:500.

[0072] Preferably, the concentration of the curcuminoid substance in the pharmaceutical composition is 0.3 to 120 mg / g.

[0073] Preferably, the particle size of the drug-loaded micelles is 3 to 50 nm.

[0074] Preferably, the pharmaceutical composition further comprises a second lipid-soluble drug micelle, and the second lipid-soluble drug micelle is uniformly distributed in the pharmaceutically acceptable aqueous solution; wherein, the second lipid-soluble drug micelle is a second microstructural formed by a second nonionic surfactant, and a second lipid-soluble drug is encapsulated in the second lipid-soluble drug micelle.

[0075] Preferably, the hydrophilic-lipophilic balance value (HLB value) of the second nonionic surfactant is greater than 10.

[0076] Preferably, the second nonionic surfactant is at least one or a combination thereof selected from polysorbate 80 (Tween 80), polyethylene glycol 15-hydroxystearate (Kolliphor HS 15), polyoxyethylene castor oil derivatives, and other nonionic surfactants.

[0077] Preferably, the second nonionic surfactant is at least one or a combination thereof selected from polyoxyethylene 35 castor oil (Cremophor ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives.

[0078] Preferably, the second fat-soluble drug is at least one or a combination of other fat-soluble drugs other than quercetin, synephrine, puerarin, resveratrol, and curcuminoids.

[0079] Preferably, the weight ratio of the curcuminoids to the second fat-soluble drug is 30:1 to 1:10.

[0080] Preferably, the weight ratio of the curcuminoids to the second fat-soluble drug is 20:1 to 1:8.

[0081] Preferably, the pharmaceutically acceptable aqueous solution further contains a water-soluble drug.

[0082] Preferably, the water-soluble drug is at least one or a combination of green tea extract, epigallocatechin gallate, epicatechin, epicatechin gallate, epigallocatechin, gallocatechin gallate, gallocatechin, catechin gallate, catechin, epigallocatechin gallate (EGCG), caffeine, carnitine (also known as carnitine or carnitine), L-carnitine, synephrine, chlorogenic acid, and other water-soluble drugs.

[0083] Preferably, the weight ratio of the curcuminoids to the water-soluble drug is 30:1 to 1:10.

[0084] Preferably, the weight ratio of the curcuminoids to the water-soluble drug is 20:1 to 1:8.

[0085] Preferably, the dosage form of the drug is a subcutaneous injection, a subcutaneous fat layer injection, an implantable infusion, an ointment dosage form, a patch dosage form, or other transdermal absorption system dosage forms.

[0086] Preferably, the drug is administered to an administration site to reduce the subcutaneous fat at the administration site.

[0087] Preferably, the dosage form of the drug is a subcutaneous injection or a subcutaneous adipose tissue injection, and the dosage of the drug is 0.02 to 20 mg per square centimeter of injection.

[0088] The dosage form of the drug is a subcutaneous injection or a subcutaneous adipose tissue injection, and the dosage of the drug is 0.04 to 16 mg per square centimeter of injection.

[0089] Preferably, the dosage form of the drug is a subcutaneous injection or a subcutaneous adipose tissue injection, and the dosage of the drug is 0.01 to 40 mg per kilogram.

[0090] Preferably, the dosage form of the drug is a subcutaneous injection or a subcutaneous adipose tissue injection, and the dosage of the drug is 0.1 to 20 mg per kilogram.

[0091] Preferably, the administration frequency of the drug is 1 to 12 times at the administration site every 1 to 30 days.

[0092] Preferably, the administration frequency of the drug is 1 to 6 times at the administration site every 1 to 30 days.

[0093] Preferably, the curcuminoid substance is curcumin.

[0094] Preferably, the pharmaceutical composition further comprises at least one or a combination of a cosolvent, a suspending agent, and an oil phase excipient.

[0095] Preferably, at least one of the oil phase excipient and the cosolvent forms the micro-structure together with the nonionic surfactant.

[0096] The present invention further provides a use of a pharmaceutical composition in the preparation of a drug or a subcutaneous injection for reducing body weight; the pharmaceutical composition comprises:

[0097] A plurality of drug-loaded micelles; and

[0098] A curcuminoid substance encapsulated in the drug-loaded micelles;

[0099] Wherein, the drug-loaded micelle is a micro-structure formed by a pharmaceutically acceptable nonionic surfactant, and the hydrophilic-lipophilic balance value (HLB value) of the nonionic surfactant is greater than 10.

[0100] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solution, and the plurality of drug-loaded micelles are uniformly distributed in the pharmaceutically acceptable aqueous solution.

[0101] Preferably, the curcuminoid substance is curcumin.

[0102] Preferably, the pharmaceutical composition further comprises a second lipid-soluble drug micelle, and the second lipid-soluble drug micelle is uniformly distributed in the pharmaceutically acceptable aqueous solution; wherein, the second lipid-soluble drug micelle is a second micro-structure formed by a second non-ionic surfactant, a second lipid-soluble drug is encapsulated in the second lipid-soluble drug micelle, and the second lipid-soluble drug is at least one or a combination of other lipid-soluble drugs other than quercetin, synephrine, puerarin, resveratrol, and curcuminoid.

[0103] Preferably, the pharmaceutically acceptable aqueous solution further comprises a water-soluble drug, and the water-soluble drug is at least one or a combination of green tea extract, epigallocatechin gallate, epicatechin, epicatechin gallate, epigallocatechin, gallocatechin gallate, gallocatechin, catechin gallate, catechin, epigallocatechin gallate (EGCG), caffeine, carnitine (also known as carnitine or carnitine), L-carnitine, synephrine, chlorogenic acid, and other water-soluble drugs.

[0104] Preferably, the dosage form of the drug is a subcutaneous injection, an intravenous injection, or a subcutaneous adipose tissue injection, and the administration dose of the drug is 0.2 to 16 mg per square centimeter of injection.

[0105] Preferably, the dosage form of the drug is a subcutaneous injection, an intravenous injection, or a subcutaneous adipose tissue injection, and the administration dose of the drug is 0.4 to 8 mg per square centimeter of injection.

[0106] Preferably, the dosage form of the drug is a subcutaneous injection, an intravenous injection, or a subcutaneous adipose tissue injection, and the dosage of the drug is 0.4 to 40 mg per kg of injection.

[0107] Preferably, the dosage form of the drug is a subcutaneous injection, an intravenous injection, or a subcutaneous adipose tissue injection, and the dosage of the drug is 0.8 to 20 mg per kg of injection.

[0108] Preferably, the administration frequency of the drug is 3 to 60 times at the administration site every 1 to 20 days.

[0109] Preferably, the administration frequency of the drug is 6 to 42 times at the administration site every 1 to 14 days.

[0110] Preferably, the pharmaceutical composition further comprises at least one or a combination of a cosolvent, a suspending agent, and an oil phase excipient.

[0111] Preferably, at least one of the oil phase excipient and the cosolvent forms the micro-structure together with the nonionic surfactant.

[0112] The present invention provides a method for reducing the subcutaneous fat amount at a local site of an individual, comprising administering a pharmaceutical composition to the local site of the individual, wherein the pharmaceutical composition comprises:

[0113] A plurality of drug-loaded micelles; and

[0114] A curcuminoid substance encapsulated in the drug-loaded micelles;

[0115] Wherein, the drug-loaded micelle is a micro-structure formed by a pharmaceutically acceptable nonionic surfactant, and the hydrophilic-lipophilic balance value (HLB value) of the nonionic surfactant is greater than 10.

[0116] Preferably, the pharmaceutical composition is administered to the local site of the individual by using a subcutaneous injection, a subcutaneous adipose tissue injection, an implantable infusion solution, an ointment dosage form, a patch dosage form, or other transdermal absorption systems.

[0117] Preferably, the dosage of the pharmaceutical composition administered to the local site of the individual is 0.02 to 20 mg per square centimeter of injection.

[0118] Preferably, the dosage of the pharmaceutical composition administered to the local part of the individual is 0.01 to 40 mg per kg of injection.

[0119] Preferably, the frequency of administering the pharmaceutical composition to the local part of the individual is 1 to 12 times every 1 to 30 days.

[0120] The present invention provides a method for reducing the body weight of an individual, comprising administering a pharmaceutical composition to the individual, wherein the pharmaceutical composition comprises:

[0121] A plurality of drug-containing micelles; and

[0122] A curcuminoid substance encapsulated in the drug-containing micelles;

[0123] Wherein, the drug-containing micelle is a micro-structure formed by a pharmaceutically acceptable nonionic surfactant, and the hydrophilic-lipophilic balance value (HLB value) of the nonionic surfactant is greater than 10.

[0124] Preferably, the pharmaceutical composition is administered to a local part of the individual by using a subcutaneous injection, an intravenous injection, or a subcutaneous adipose layer injection.

[0125] Preferably, the dosage of the pharmaceutical composition administered to the local part of the individual is 0.2 to 16 mg per square centimeter of injection.

[0126] Preferably, the dosage of the pharmaceutical composition administered to the local part of the individual is 0.4 to 40 mg per kg of injection.

[0127] Preferably, the frequency of administering the pharmaceutical composition to the local part of the individual is 3 to 60 times every 1 to 20 days.

[0128] Preferably, the pharmaceutical composition is administered to a local part of the individual by using a subcutaneous injection, a subcutaneous adipose layer injection, an intravenous injection, an implanted infusion solution, an ointment, a patch, or other transdermal absorption systems. Description of the Drawings

[0129] Figure 1A : Bar graph showing the effect of orally administered turmeric extract on the subcutaneous fat mass of rats.

[0130] Figure 1B : Bar graph showing the effect of orally administered turmeric extract on the total weight gain of rats.

[0131] Figure 2A:Bar graph showing the effect of curcumin subcutaneous injection preparations made with different excipients on the local subcutaneous fat mass in rats.

[0132] Figure 2B :Bar graph showing the effect of curcumin subcutaneous injection preparations made with different excipients on the total body weight gain in rats.

[0133] Figure 3 :Bar graph showing the effect of micelles on the local subcutaneous fat mass in rats.

[0134] Figure 4 :Bar graph showing the effect of micelles on the total body weight gain in rats.

[0135] Figure 5 :Bar graph showing the effect of resveratrol subcutaneous injection preparation without excipient on the local subcutaneous fat mass in rats.

[0136] Figure 6 :Bar graph showing the effect of curcumin-resveratrol combination subcutaneous injection preparations made with different excipients on the local subcutaneous fat mass in rats.

[0137] Figure 7 :Bar graph showing the effect of curcumin single-agent, resveratrol single-agent, and curcumin-resveratrol combination subcutaneous injection preparations containing micelles on the local subcutaneous fat mass in rats.

[0138] Figure 8A :Bar graph showing the effect of the dosing frequency of curcumin-resveratrol combination pharmaceutical composition on the local subcutaneous fat mass in rats.

[0139] Figure 8B :Bar graph showing the effect of the dosing frequency of curcumin-resveratrol combination pharmaceutical composition on the total body weight gain in rats.

[0140] Figure 9 :Bar graph showing the effect of the dosing dose of curcumin-resveratrol combination pharmaceutical composition on the local subcutaneous fat mass in rats.

[0141] Figure 10 :Effect of curcumin - other lipophilic drug combination pharmaceutical composition on apoptosis of mature adipocytes.

[0142] Figure 11 :Bar graph showing the effect of green tea extract subcutaneous injection preparation without excipient on the local subcutaneous fat mass in rats.

[0143] Figure 12A :Bar graph showing the effect of the dosing frequency of curcumin - green tea extract combination pharmaceutical composition on the local subcutaneous fat mass in rats.

[0144] Figure 12B:Bar graph showing the effect of the dosing frequency of the curcumin - green tea extract compound pharmaceutical composition on the total weight gain of rats.

[0145] Figure 13 :Effect of the curcumin - other water - soluble drug compound pharmaceutical composition on the apoptosis of mature adipocytes. Detailed implementation method

[0146] Experiment 1: Effect of oral administration of turmeric extract on subcutaneous fat mass and body weight of rats

[0147] Prepare the turmeric extract oral solution in the following way: Add an appropriate amount of turmeric extract to an appropriate amount of sterile injection water, mix well, and it is the turmeric extract oral solution.

[0148] Use 7 - week - old male Sprague - Dawley rats for the experiment. First, feed 12 rats with a high - fat diet (brand: Research Diets, Inc.; model: #D12492) to induce an increase in subcutaneous fat. After continuously feeding until the body weight of the rats reaches 330 ± 10 g, randomly divide the rats into 2 groups, namely the high - fat control group and the turmeric extract oral group, with 6 rats in each group, so that there is no statistical difference in the body weight of each group of rats. Record the body weight of each rat, which is defined as the "pre - test body weight" of each rat. Then, administer the drugs in the following way.

[0149] Turmeric extract oral group: Feed the rats a high - fat diet every day, and administer the turmeric extract oral solution to the rats by gavage, so that the dose of the administered turmeric extract is 100 mg / kg / day, and continuously feed for 20 days. In the administered turmeric extract, the weight percentage of curcumin is 95%.

[0150] High - fat control group: Feed the rats a high - fat diet every day, but do not feed turmeric extract.

[0151] Record the body weight change every day during the experiment, record the water intake and food intake once a week, fast on the 20th day of the experiment, and sacrifice on the 21st day.

[0152] Record the body weight of each rat, which is defined as the "post - test body weight" of each rat. Subtract the "pre - test body weight" of each rat from the "post - test body weight" to obtain the "total weight gain". Finally, weigh the subcutaneous fat on the left and right lower inguinal regions of the rats, and calculate the subcutaneous fat mass in the lower inguinal region of each group of rats. Present the data in the form of mean ± SD and conduct statistics. The statistical results are represented by symbols or English letters. Different symbols or letters indicate statistical differences between groups (p < 0.05), and the same symbols or letters indicate no statistical differences between groups (p > 0.05).

[0153] Please refer toFigure 1A and Figure 1B 。 Figure 1A It is a bar graph showing the effect of orally administered turmeric extract on the subcutaneous fat mass of rats. Among them, the subcutaneous fat mass in the lower inguinal region is the sum of the subcutaneous fat masses on the left and right lower inguinal regions. Figure 1B It is a bar graph showing the effect of orally administered turmeric extract on the total weight gain of rats.

[0154] Figure 1A The results showed that the subcutaneous fat mass in the lower inguinal region of the high-fat control group rats was 6.4 ± 1.5 g, and that of the rats in the oral turmeric extract group was 6.1 ± 0.8 g. There was no significant difference between the two (p > 0.05), indicating that orally administered turmeric extract could not reduce local fat.

[0155] Figure 1B The results showed that the total weight gain of the high-fat control group rats was 135 ± 12 g, and that of the rats in the oral turmeric extract group was 142 ± 10 g. There was no significant difference between the two (p > 0.05), indicating that orally administered turmeric extract could not reduce body weight.

[0156] From the above experiments, it can be seen that orally administered turmeric extract could not reduce local fat or body weight. In order to overcome this problem, the inventor further studied and developed the pharmaceutical composition containing curcumin and its subcutaneous injection of the present invention.

[0157] Experiment 2: Effects of curcumin subcutaneous injection on subcutaneous fat mass and body weight of rats

[0158] Prepare curcumin physiological saline solution, curcumin PEG solution, and curcumin ELP solution in the following manner.

[0159] Preparation method of curcumin physiological saline solution:

[0160] Mix 450 mg of curcumin with an appropriate amount of injectable physiological saline to make the final volume reach 90 mL. Stir evenly to completely dissolve curcumin, and curcumin physiological saline solution can be obtained, and the curcumin concentration in the curcumin physiological saline solution is 5 mg / mL.

[0161] Preparation method of curcumin PEG solution:

[0162] Mix 15 g of polyethylene glycol 400 (abbreviated as PEG400), 15 g of glycerol, and an appropriate amount of physiological saline for injection to make the final volume reach 100 mL. Stir evenly to completely dissolve the polyethylene glycol 400 and glycerol to obtain a polyethylene glycol and glycerol mixture. Mix 450 mg of curcumin with an appropriate amount of the polyethylene glycol and glycerol mixture to make the final volume reach 90 mL. Stir evenly to completely dissolve the curcumin to obtain a curcumin PEG solution. The concentration of curcumin in the curcumin PEG solution is 5 mg / mL.

[0163] Preparation method of curcumin ELP solution:

[0164] Mix 450 mg of curcumin with 80 - 140 mL of dichloromethane and stir at 150 - 500 rpm at room temperature until the curcumin is completely dissolved. Add 18 g of polyoxyl 35 castor oil (Kolliphor ELP, abbreviated as ELP), stir evenly at a rotation speed of 100 - 300 rpm to volatilize the dichloromethane. After the dichloromethane is completely volatilized, slowly add physiological saline for injection to make the final volume reach 90 mL, stir evenly to obtain a curcumin ELP solution. The concentration of curcumin in the curcumin ELP solution is 5 mg / mL, the concentration of polyoxyl 35 castor oil (ELP) is about 20% (weight percentage), and the weight ratio of curcumin to polyoxyl 35 castor oil is 1:40.

[0165] Use 6-week-old male Sprague-Dawley rats for the experiment. First, feed 20 rats with a high-fat diet (brand: Research Diets, Inc.; model: #D12492) to induce subcutaneous fat increase. After continuously feeding until the body weight of the rats reaches 330 ± 10 g, randomly divide the rats into 4 groups, namely the control group, the physiological saline group, the PEG group, and the ELP group, with 5 rats in each group, so that there is no statistical difference in the body weight of each group of rats. Record the body weight of each rat, defined as the "pre-test body weight" of each rat. Then, administer the drugs in the following manner.

[0166] Curcumin physiological saline, curcumin PEG solution, and curcumin ELP solution were respectively injected into the subcutaneous fat layer of the lower groin of rats in the physiological saline group, PEG group, and ELP group. The injection volume each time was 4 mL per kilogram of body weight (4 mL / kg), so that the injection dose each time was 20 mg of curcumin per kilogram of body weight (20 mg / kg; calculation method: 4 mL / kg × 5 mg / mL = 20 mg / kg). The control group was given the same volume of injectable physiological saline by the same injection method as above.

[0167] The above injection site was the lower groin fat of rats, and the injection was evenly distributed on the left and right sides. Each injection was given once on the 1st, 2nd, 3rd, and 4th days of the experiment. During the experiment, a high-fat diet was continuously given, and the body weight change was recorded daily, and the water intake and food intake were recorded once a week. The experiment was carried out for 14 days, and the rats were sacrificed with carbon dioxide on the 15th day.

[0168] The body weight of each rat was recorded, which was defined as the "post-experiment body weight" of each rat. The "post-experiment body weight" of each rat was subtracted from the "pre-experiment body weight" to obtain the "total weight gain". The total weight gain of the rats in each group was divided by the total weight gain of the rats in the control group to obtain the "relative total weight gain".

[0169] The subcutaneous fat of the lower groin on the left and right sides of the rats was weighed, and the subcutaneous fat amounts on the left and right sides of the lower groin were added together to calculate the subcutaneous fat amount of the lower groin. The subcutaneous fat amount of the lower groin of the rats in each group was divided by the subcutaneous fat amount of the lower groin of the rats in the control group to obtain the "relative weight of the subcutaneous fat of the lower groin".

[0170] The data were presented in the form of mean ± SD, and statistical analysis was performed by one-way ANOVA. The statistical results were represented by symbols or English letters. Different symbols or letters indicated statistical differences between groups (p < 0.05), and the same symbols or letters indicated no statistical differences between groups (p > 0.05).

[0171] Please refer to Figure 2A and Figure 2B . Figure 2A It is a bar graph showing the effect of curcumin subcutaneous injection preparations prepared with different excipients on the local subcutaneous fat amount of rats. Figure 2B It is a bar graph showing the effect of curcumin subcutaneous injection preparations prepared with different excipients on the total weight gain of rats.

[0172] Figure 2AThe results showed that the relative weight of subcutaneous fat in the lower inguinal region of rats in the control group was 100±27.6%, that in the physiological saline group was 99.8±8.0%, that in the PEG group was 93.6±5.8%, and that in the ELP group was 62.8±20.5%. There was no significant difference in the relative weight of subcutaneous fat in the lower inguinal region between the rats in the physiological saline group and those in the control group, indicating that directly injecting curcumin into the subcutaneous fat layer at the administration site could not reduce the fat (local fat) at the administration site. There was no significant difference in the relative weight of subcutaneous fat in the lower inguinal region between the rats in the PEG group and those in the control group; there was a significant difference between the rats in the ELP group and those in the control group (p<0.05), and the relative weight of subcutaneous fat in the lower inguinal region of the rats in the ELP group decreased by 37.2%.

[0173] Figure 2B The results showed that the relative total weight gain of rats in the control group was 100.0±30.8%, that in the physiological saline group was 110.0±18.7%, that in the PEG group was 112.5±20.7%, and that in the ELP group was 87.1±33.1%. There was no significant difference among the four groups (p>0.05), but the body weight of the rats in the ELP group was 12.9% less than that of the rats in the control group, indicating a tendency for the body weight of the rats in the ELP group to decrease.

[0174] From the above experiments, it can be seen that directly injecting curcumin into the subcutaneous fat layer at the administration site could not reduce the fat (local fat) at the administration site nor the body weight. Injecting the curcumin composition added with the excipient PEG (a commonly used co-solvent) into the subcutaneous fat layer at the administration site could not reduce the fat (local fat) at the administration site nor the body weight; however, injecting the curcumin composition added with the non-ionic surfactant ELP into the subcutaneous fat layer at the administration site could not only significantly reduce the fat (local fat) at the administration site but also showed a tendency to reduce the body weight. Therefore, it is necessary to further explore whether the curcumin composition must contain a non-ionic surfactant to reduce the subcutaneous fat (local fat) at the administration site and reduce the body weight.

[0175] Further analysis found that there were no micelles in the above-mentioned curcumin PEG solution administered, while there were micelles in the curcumin ELP solution, and curcumin was encapsulated in the micelles formed by ELP. Therefore, it is also necessary to further explore the effect of micelles on reducing local fat and reducing body weight.

[0176] Experiment 3: Effects of a subcutaneous injection preparation of a curcumin single composition containing a non-ionic surfactant on the subcutaneous fat amount and body weight of rats

[0177] Prepare curcumin ELP partial micelle formulations, curcumin HS-15 partial micelle formulations, curcumin ELP micelle formulations, and curcumin HS-15 micelle formulations in the following manner.

[0178] Method for preparing curcumin ELP partial micelle formulation: Mix 20 g of polyoxyl 35 castor oil (i.e., ELP) with an appropriate amount of physiological saline for injection to make the final weight reach 100 g. Stir evenly to completely dissolve polyoxyl 35 castor oil (i.e., ELP) to obtain a 20% ELP solution. Mix 400 mg of curcumin with an appropriate amount of the 20% ELP solution to make the final weight reach 80 g. Stir evenly to completely dissolve curcumin, and the curcumin ELP partial micelle formulation can be obtained. The curcumin concentration in the curcumin ELP partial micelle formulation is approximately 5 mg / mL, the concentration of polyoxyl 35 castor oil (ELP) is approximately 20% (weight percentage), and the weight ratio of curcumin to polyoxyl 35 castor oil is approximately 1:40.

[0179] Method for preparing curcumin HS-15 partial micelle formulation: Mix 20 g of polyethylene glycol 15-hydroxystearate (Kolliphor HS 15, abbreviated as HS-15) with an appropriate amount of physiological saline for injection to make the final weight reach 100 g. Stir evenly to completely dissolve polyethylene glycol 15-hydroxystearate (i.e., HS-15) to obtain a 20% HS-15 solution. Mix 400 mg of curcumin with an appropriate amount of the 20% HS-15 solution to make the final weight reach 80 g. Stir evenly to completely dissolve curcumin, and the curcumin HS-15 partial micelle formulation can be obtained. The curcumin concentration in the curcumin HS-15 partial micelle formulation is approximately 5 mg / mL, the concentration of polyethylene glycol 15-hydroxystearate (i.e., HS-15) is approximately 20% (weight percentage), and the weight ratio of curcumin to polyethylene glycol 15-hydroxystearate (i.e., HS-15) is approximately 1:40.

[0180] Method for preparing curcumin ELP micelle formulation: The same as the method for preparing the curcumin ELP solution in Experiment 2.

[0181] Preparation method of curcumin HS-15 micelle formulation: Mix 500 mg of curcumin with 80 - 140 mL of dichloromethane, and stir at 150 - 500 rpm at room temperature until the curcumin is completely dissolved. Add 20 g of polyethylene glycol 15-hydroxystearate (Kolliphor HS 15, abbreviated as HS-15), stir evenly at a rotation speed of 100 - 300 rpm to volatilize the dichloromethane. After the dichloromethane is completely volatilized, slowly add physiological saline for injection to make the final volume reach 100 g, and stir evenly to form multiple drug-loaded micelles, thus obtaining the curcumin HS-15 micelle formulation. The concentration of curcumin in the curcumin HS-15 micelle formulation is about 5 mg / g, the concentration of polyethylene glycol 15-hydroxystearate (HS-15) is 20% (weight percentage), and the weight ratio of curcumin to polyethylene glycol 15-hydroxystearate (HS-15) is 1:40.

[0182] Use a particle size analyzer to determine whether micelles are contained in the curcumin ELP partial micelle formulation, curcumin HS-15 partial micelle formulation, curcumin ELP micelle formulation, and curcumin HS-15 micelle formulation, and measure the particle size of the micelles.

[0183] The results show that both the curcumin ELP partial micelle formulation and the curcumin HS-15 partial micelle formulation contain drug precipitates (curcumin precipitates), and the number of drug-loaded micelles is small. The curcumin ELP micelle formulation and the curcumin HS-15 micelle formulation are clear and without stratification, and the number of drug-loaded micelles is large.

[0184] In addition, the particle sizes of the curcumin ELP partial micelle formulation, curcumin HS-15 partial micelle formulation, curcumin ELP micelle formulation, and curcumin HS-15 micelle formulation are 13.16 ± 0.18 nm, 13.18 ± 1.45 nm, 12.43 ± 0.40 nm, and 11.46 ± 0.41 nm in sequence, and the PDI values are 0.22 ± 0.03, 0.18 ± 0.05, 0.28 ± 0.05, and 0.18 ± 0.04 in sequence.

[0185] It can be seen from this that although both the curcumin ELP partial micelle formulation and the curcumin HS-15 partial micelle formulation contain drug precipitates (curcumin precipitates), the supernatant still contains micelles (particle size less than 250 nm and PDI value less than 0.4). Therefore, the curcumin ELP partial micelle formulation, curcumin HS-15 partial micelle formulation, curcumin ELP micelle formulation, and curcumin HS-15 micelle formulation are all pharmaceutical compositions of the present invention.

[0186] The experiment was conducted using 6-week-old male Sprague-Dawley rats. First, 20 rats were fed a high-fat diet (Research Diets, Inc.; model #D12492) to induce subcutaneous fat increase. After continuous feeding until the rats reached a body weight of 330 ± 10 g, the rats were randomly divided into 5 groups: a control group, an ELP partial micelle group, an HS-15 partial micelle group, an ELP micelle group, and an HS-15 micelle group, with 4 rats in each group, so that there was no statistical difference in the body weights of the rats in each group. The body weight of each rat was recorded and defined as the "pre-experiment body weight" of each rat. Then, the drugs were administered in the following manner.

[0187] After the curcumin ELP partial micelle formulation, curcumin HS-15 partial micelle formulation, curcumin ELP micelle formulation, and curcumin HS-15 micelle formulation were respectively prepared and mixed evenly (to evenly suspend the precipitate in the partial micelle formulation), they were respectively injected into the subcutaneous fat layer of the lower inguinal region of the rats in the ELP partial micelle group, HS-15 partial micelle group, ELP micelle group, and HS-15 micelle group. The injection volume each time was 4 mL per kilogram of body weight (4 mL / kg), so that the injection dose each time was 20 mg of curcumin per kilogram of body weight (20 mg / kg; calculation method: 4 mL / kg × 5 mg / mL = 20 mg / kg). The control group was given the same volume of injectable physiological saline in the same injection manner as above.

[0188] The above injection site was the lower inguinal fat of the rats, and the injection was evenly distributed on the left and right sides. Each injection was given once on days 1, 2, 3, 4, 5, and 6 of the experiment. During the experiment, the high-fat diet was continuously given, and the body weight changes were recorded daily, and the water intake and food intake were recorded once a week. The experiment was carried out for 14 days, and the rats were sacrificed with carbon dioxide on the 15th day.

[0189] The body weight of each rat was recorded and defined as the "post-experiment body weight" of each rat. The "post-experiment body weight" of each rat was subtracted from the "pre-experiment body weight" to obtain the "total weight gain". The total weight gain of the rats in each group was divided by the total weight gain of the rats in the control group to obtain the "relative total weight gain".

[0190] The subcutaneous fat of the lower inguinal region on the left and right sides of the rats was weighed, and the subcutaneous fat amounts on the left and right sides of the lower inguinal region were added together to calculate the subcutaneous fat amount of the lower inguinal region. The subcutaneous fat amount of the lower inguinal region of the rats in each group was divided by the subcutaneous fat amount of the lower inguinal region of the rats in the control group to obtain the "relative weight of the subcutaneous fat of the lower inguinal region".

[0191] Data are presented as mean ± SD and analyzed statistically by one-way ANOVA. Statistical results are represented by symbols or English letters. Different symbols or letters indicate statistical differences between groups (p < 0.05), while the same symbols or letters indicate no statistical differences between groups (p > 0.05).

[0192] As can be seen from the above dosage form preparation method and particle size analysis results, the ELP concentration and curcumin concentration in the curcumin ELP partial micelle dosage form and the curcumin ELP micelle dosage form are the same, and only the number of drug-loaded micelles is different. Therefore, compared with the control group, if the curcumin ELP partial micelle dosage form cannot significantly reduce the local fat at the administration site, but the curcumin ELP micelle dosage form can significantly reduce the local fat at the administration site, it indicates that the formation of drug-loaded micelles is the key factor for the curcumin composition to significantly reduce the local fat at the administration site.

[0193] Similarly, the HS-15 concentration and curcumin concentration in the curcumin HS-15 partial micelle dosage form and the curcumin HS-15 micelle dosage form are the same, and only the number of drug-loaded micelles is different. Therefore, compared with the control group, if the curcumin HS-15 partial micelle dosage form cannot significantly reduce the local fat at the administration site, but the curcumin HS-15 micelle dosage form can significantly reduce the local fat at the administration site, it indicates that the formation of drug-loaded micelles is the key factor for the curcumin composition to significantly reduce the local fat at the administration site.

[0194] On the other hand, since the ELP concentration and curcumin concentration in the curcumin ELP partial micelle dosage form and the curcumin ELP micelle dosage form are the same, and only the number of drug-loaded micelles is different. Therefore, compared with the control group, if the curcumin ELP partial micelle dosage form cannot significantly reduce the body weight, but the curcumin ELP micelle dosage form can significantly reduce the body weight, it indicates that the formation of drug-loaded micelles is the key factor for the curcumin composition to significantly reduce the body weight.

[0195] Please refer to Figure 3 , Figure 3 is a bar graph showing the effect of micelles on the local subcutaneous fat mass of rats. Figure 3 In, the vertical axis is the relative weight (%) of the subcutaneous fat in the lower groin, and the horizontal axis from left to right is the control group, the HS-15 partial micelle group, the HS-15 micelle group, the ELP partial micelle group, and the ELP micelle group in sequence.

[0196] Figure 3The relative weight of subcutaneous fat in the lower inguinal region of rats in the control group was 100.0 ± 26.4%, that of rats in the HS-15 partial micelle group was 74.7 ± 10.1, that of rats in the HS-15 micelle group was 67.6 ± 8.6%, that of rats in the ELP partial micelle group was 71.8 ± 22.9%, and that of rats in the ELP micelle group was 65.0 ± 7.2%.

[0197] Compared with the rats in the control group, the relative weight of subcutaneous fat in the lower inguinal region of rats in the HS-15 partial micelle group and the ELP partial micelle group showed a decreasing trend, but the statistical difference was not reached (p > 0.05). The relative weight of subcutaneous fat in the lower inguinal region of rats in the HS-15 micelle group and the ELP micelle group decreased significantly (p < 0.05), by 32.4% and 35% respectively.

[0198] The above data showed that although the concentration of non-ionic surfactant and curcumin in the partial micelle dosage form were the same as those in the micelle dosage form, and it contained a part of micelles, the partial micelle dosage form could only make the local fat show a decreasing trend and could not significantly reduce the local fat. On the contrary, the micelle dosage form containing a large number of drug-loaded micelles could significantly reduce the local fat.

[0199] It can be seen from this that the formation of drug-loaded micelles is the key factor for the curcumin composition to significantly reduce the local fat at the application site. That is, the curcumin composition containing a small amount of drug-loaded micelles can make the local fat show a decreasing trend, while the curcumin composition containing a large number of drug-loaded micelles can significantly reduce the local fat.

[0200] Please refer to Figure 4 , Figure 4 which is a bar chart showing the effect of micelles on the total weight gain of rats. Figure 4 In, the vertical axis is the relative total weight gain (%), and the horizontal axis is, from left to right in sequence, the control group, the HS-15 partial micelle group, the HS-15 micelle group, the ELP partial micelle group, and the ELP micelle group.

[0201] Figure 4 The results showed that the relative total weight gain of rats in the control group was 100.0 ± 20.6%, that of rats in the HS-15 partial micelle group was 100.0 ± 17.3%, that of rats in the HS-15 micelle group was 96.4 ± 18.5%, that of rats in the ELP partial micelle group was 73.8 ± 11.2%, and that of rats in the ELP micelle group was 54.8 ± 14.3%.

[0202] Compared with the control group of rats, the relative total weight gain of rats in the ELP partial micelle group showed a decreasing trend, but the statistical difference was not reached (p > 0.05). The relative total weight gain of rats in the ELP micelle group decreased by 45.2%, and there was a significant difference compared with the control group (p < 0.05).

[0203] The above data show that although the concentrations of the non-ionic surfactant and curcumin in the ELP partial micelle dosage form are the same as those in the ELP micelle dosage form, and it contains a part of micelles, the ELP partial micelle dosage form can only make the body weight show a decreasing trend and cannot significantly reduce the body weight; on the contrary, the ELP micelle dosage form containing a large number of drug-loaded micelles can significantly reduce the body weight.

[0204] It can be seen from this that the formation of drug-loaded micelles is the key factor for the curcumin composition to significantly reduce body weight. That is, the curcumin composition containing a small amount of drug-loaded micelles can make the body weight show a decreasing trend, and the curcumin composition containing a large number of drug-loaded micelles can make the body weight significantly decrease.

[0205] Although the HS-15 micelle dosage form containing a large number of micelles in this experiment did not significantly reduce the body weight, according to the inventor's experience, if the application frequency or dosage is increased, the HS-15 micelle dosage form containing a large number of micelles can also significantly reduce the body weight. Therefore, this non-ionic surfactant HS-15 should also be included in the scope of the patent application of the present invention.

[0206] Experiment 4: Preparation of the pharmaceutical composition of the present invention

[0207] It can be seen from the above experiments that the formation of micelles using a non-ionic surfactant is the key factor for the curcumin composition to significantly reduce local fat. Accordingly, the present invention provides a curcumin single-component pharmaceutical composition capable of reducing local fat, which is characterized in that the curcumin single-component pharmaceutical composition contains a plurality of drug-loaded micelles.

[0208] The steps for preparing the curcumin single-component pharmaceutical composition are as follows:

[0209] (a) Mix a first weight of curcumin with a solvent and stir at 150 - 500 rpm at room temperature until the curcumin is completely dissolved;

[0210] (b) Add a second weight of a pharmaceutically acceptable surfactant and stir evenly at a rotation speed of 100 - 300 rpm to volatilize the solvent, wherein the hydrophilic-lipophilic balance value (HLB value) of the surfactant is greater than 10; and

[0211] (c) After the solvent is completely volatilized, slowly add a third weight of a pharmaceutically acceptable aqueous solution to obtain a plurality of drug-loaded micelles; and

[0212] (d) After filtering through a 0.2 μm filter membrane, the filtrate containing the drug-loaded micelles is stored in the dark and refrigerated.

[0213] Among them, in step (c), the drug-loaded micelle is a micro-structure formed by a surfactant, and curcumin is encapsulated in the drug-loaded micelle; the third weight is greater than or equal to 0 g.

[0214] Preferably, the operation steps of step (c) are: after the solvent is completely volatilized, a third weight of a pharmaceutically acceptable aqueous solution is slowly added and stirred evenly to form a plurality of drug-loaded micelles.

[0215] Preferably, in step (a), the boiling point of the solvent is less than that of pure water.

[0216] Preferably, in step (a), the solvent is a hydrophilic solvent.

[0217] Preferably, the hydrophilic solvent is at least one or a combination of methanol, ethanol, acetone, and other hydrophilic solvents.

[0218] Preferably, the solvent in step (a) is a lipophilic solvent.

[0219] Preferably, the lipophilic solvent is at least one or a combination of ether, benzene, chloroform, ethyl acetate, dichloromethane, hexane, and other lipophilic solvents.

[0220] Preferably, in step (b), the surfactant is a non-ionic surfactant.

[0221] Preferably, the non-ionic surfactant is at least one or a combination of polysorbate 80 (Tween 80), polyethylene glycol 15-hydroxystearate (Kolliphor HS 15), polyoxyethylene castor oil derivatives, and other non-ionic surfactants.

[0222] Preferably, the polyoxyethylene castor oil derivative is at least one or a combination of polyoxyethylene 35 castor oil (Cremophor ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives.

[0223] Preferably, in steps (a) and (b), the weight ratio of the first weight of curcumin to the second weight of the surfactant is 1:5 to 1:500.

[0224] Preferably, in steps (a) and (b), the weight ratio of the curcumin of the first weight to the surfactant of the second weight is 1:20 to 1:150.

[0225] Preferably, in steps (a) and (c), the weight ratio of the curcumin of the first weight to the pharmaceutically acceptable aqueous solution of the third weight is 1:400 to 3:50.

[0226] Preferably, in step (c), the pharmaceutically acceptable aqueous solution is water for injection, aqueous injection solution, or physiological saline.

[0227] Preferably, in step (c), the pharmaceutically acceptable aqueous solution contains a local anesthetic.

[0228] Preferably, in step (c), the pharmaceutically acceptable aqueous solution contains an antioxidant.

[0229] Experiment Five: Determination of the Quality of the Pharmaceutical Composition

[0230] Experiment 5-1 Component Analysis

[0231] Let the pharmaceutical composition stand for at least 20 minutes. If no layering occurs, further test it using a particle size analyzer.

[0232] Use a particle size analyzer to determine whether micelles are contained in the pharmaceutical composition. If, after the pharmaceutical composition is analyzed by the particle size analyzer, the measured particle size is less than 250 nm, the PDI value is less than 0.4, the solution in the pharmaceutical composition is found to be clear and transparent to the naked eye, and a light path can be observed after irradiating the solution in the pharmaceutical composition with a laser, it means that micelles are present in the pharmaceutical composition.

[0233] If the pharmaceutical composition has micelles, the prepared pharmaceutical composition is the pharmaceutical composition of the present invention that can be used to reduce local fat.

[0234] Preferably, if the pharmaceutical composition does not layer and does not contain precipitates after standing, the prepared pharmaceutical composition is the better pharmaceutical composition of the present invention.

[0235] Experiment 5-2 Analysis of the Stability of the Pharmaceutical Composition Using the Particle Size Distribution

[0236] Use a particle size analyzer (purchased from Malvern) to measure the particle size distribution and polydispersity index (PDI). If the polydispersity index is less than 0.4, it means that the stability of the pharmaceutical composition is good, that is, the micelles in the pharmaceutical composition can exist stably.

[0237] Experiment 5-3 Determination of the Stability of Pharmaceutical Compositions by Accelerated Stability Testing

[0238] The storage condition of the pharmaceutical composition of the present invention is 2-8°C. To test the stability of the pharmaceutical composition, the inventor placed the pharmaceutical composition in an environment with relatively high temperature and high humidity (temperature 25°C ± 2°C, relative humidity RH 60% ± 5%) for accelerated stability testing, and observed how long the micelles in the pharmaceutical composition could stably exist under relatively high temperature conditions, so as to calculate how long the pharmaceutical composition could be stored at 2-8°C according to the accelerated stability formula, as described below.

[0239] If the pharmaceutical composition can be stored for n months at 25°C, the length of time it can be stored at 5°C is 2 ((25-5) / 10) times that of n months. That is, the length of time the pharmaceutical composition can be stored at 5°C is 2 2 times, which is 4 times.

[0240] For example, if the pharmaceutical composition can be stored for 6 months at 25°C, the length of time it can be stored at 5°C is 24 months (6 months × 4 times = 24 months).

[0241] Preferably, when the accelerated stability test is carried out under the conditions of temperature 25°C ± 2°C, relative humidity RH 60% ± 5%, and avoiding direct light, the pharmaceutical composition still maintains a state without precipitation for at least 24 hours.

[0242] Preferably, when the accelerated stability test is carried out under the conditions of temperature 25°C ± 2°C, relative humidity RH 60% ± 5%, and avoiding direct light, the pharmaceutical composition still maintains a state without precipitation for at least 6 months.

[0243] Preferably, under the condition of temperature 2-8°C, the pharmaceutical composition still maintains a state without precipitation for at least 24 months.

[0244] Experiment Six: Maximum Drug Loading of Drug-loaded Micelles Formed by Various Non-ionic Surfactants

[0245] Since the maximum drug loading of drug-loaded micelles directly affects the injection volume, it has a great impact on the volume of the drug that must be accommodated in the local subcutaneous fat layer (such as the subcutaneous fat layer of the face) at one time, side effects, and burden. Therefore, this experiment will explore the maximum drug loading of drug-loaded micelles formed by various non-ionic surfactants to evaluate which non-ionic surfactant is the best excipient for preparing the pharmaceutical composition of the present invention.

[0246] Four non-ionic surfactants were selected for this experiment. The four non-ionic surfactants are polyoxyethylene 35 castor oil (i.e., ELP), polyethylene glycol 15-hydroxystearate (i.e., HS-15), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40, abbreviated as RH 40), and polysorbate 80 (i.e., Tween 80).

[0247] The experiment was divided into four groups, namely the ELP group, the HS-15 group, the RH40 group, and the Tween 80 group.

[0248] Experimental procedure:

[0249] (a’) Mix 2.0 g (an example of the first weight) of curcumin with 300 - 500 mL of dichloromethane, and stir at 150 - 500 rpm at room temperature until the curcumin is completely dissolved;

[0250] (b’) Add 18.0 g (an example of the second weight) of a single one of the above non-ionic surfactants, stir evenly at a rotation speed of 100 - 300 rpm, and let the dichloromethane evaporate; and

[0251] (c’) After the solvent has completely evaporated, obtain a composition, a total of 20 g; take 2 g of this composition, slowly add 8 g (an example of the third weight) of physiological saline for injection, and stir evenly to obtain the composition to be tested. The concentration of curcumin in the composition to be tested is 20 mg / g, and the concentration of the non-ionic surfactant is 18%.

[0252] Let the compositions to be tested of the ELP group, the HS-15 group, the RH40 group, and the Tween 80 group stand for at least 20 minutes, and observe whether stratification occurs. If stratification occurs, it means that the concentration of curcumin is too high and the micelles in the composition to be tested are ruptured, that is, the pharmaceutical composition of the present invention with a curcumin concentration of 20 mg / g cannot be prepared using this non-ionic surfactant.

[0253] The experimental results show that stratification occurred in the compositions to be tested of the HS-15 group and the RH40 group, and only the compositions to be tested of the ELP group and the Tween 80 group did not show stratification. It can be seen from this that the maximum drug loading of the drug-containing micelles formed by HS-15 and RH40 is less than 20 mg / g. The drug-containing micelles formed by ELP and Tween 80 can both prepare pharmaceutical compositions with a curcumin concentration of 20 mg / g.

[0254] However, due to the toxicity of Tween 80, pharmacopoeias in various countries limit the injection concentration of Tween 80 to be less than 0.4% to avoid adverse reactions or toxicity. Therefore, the upper limit of the drug loading of the drug-loaded micelles formed by Tween 80 should be 0.44 mg / g (calculation method: 20 mg / g × (0.4% / 18%) = 0.44 mg / g).

[0255] To determine the maximum drug loading limit of ELP, the inventors conducted subsequent Experiment 6 and found that the maximum drug loading of ELP is greater than or equal to 111 mg / g (when the ratio of curcumin to ELP is 1:8, the prepared pharmaceutical composition contains 111 mg / g of curcumin).

[0256] From the above results, it can be seen that ELP is the best excipient for preparing the pharmaceutical composition of the present invention. In the pharmaceutical composition prepared using ELP, the curcumin concentration can reach 111 mg / g, while in the pharmaceutical compositions prepared using other non-ionic surfactants, the curcumin concentration is less than 20 mg / g (please refer to Table 1).

[0257] To determine which non-ionic surfactant has the lowest drug loading upper limit among HS-15 and RH40, the inventors further prepared the pharmaceutical composition of the present invention with a curcumin concentration of 10 mg / g using these non-ionic surfactants. The results showed that ELP, HS-15, RH40, and Tween 80 can all prepare the pharmaceutical composition of the present invention with a curcumin concentration of 10 mg / g, and the pharmaceutical compositions of the present invention with a curcumin concentration of 10 mg / g are all clear and without stratification. The particle sizes are 15.95 ± 0.24 nm, 88.23 ± 116.06 nm, 21.63 ± 9.34 nm, and 11.37 ± 0.13 nm in sequence, and the PDI values are 0.32 ± 0.02, 0.48 ± 0.27, 0.26 ± 0.09, and 0.33 ± 0.04 in sequence.

[0258] Among them, when preparing the pharmaceutical composition of the present invention with a curcumin concentration of 10 mg / g using HS-15, the PDI of the prepared pharmaceutical composition is greater than 0.4, which does not meet the definition of the pharmaceutical composition having micelles in the present invention (the measured particle size is less than 250 nm, the PDI value is less than 0.4, the solution in the pharmaceutical composition is observed to be clear and transparent with the naked eye, and the light path can be observed after irradiating the solution in the pharmaceutical composition with a laser). It can be seen that among the non-ionic surfactants selected in this experiment, HS-15 has the lowest drug loading upper limit.

[0259] Table 1 Maximum drug loadings of drug-loaded micelles formed by various non-ionic surfactants

[0260]

[0261] Experiment 6: Preparation of Pharmaceutical Composition Using Polyoxyethylene 35 Castor Oil (ELP)

[0262] In this experiment, a series of pharmaceutical compositions of the present invention were prepared by varying the ratio of curcumin and polyoxyethylene 35 castor oil (ELP), and stability analysis was carried out to determine the appropriate ratio of curcumin and polyoxyethylene 35 castor oil (ELP) and the maximum drug loading capacity when using ELP to prepare the pharmaceutical composition of the present invention.

[0263] The experiment was divided into 9 groups, namely groups 1 - 9. The preparation methods of the pharmaceutical compositions in each group were roughly the same as the experimental steps in Experiment 5, only the weight of curcumin (the first weight in step (a')), the weight of ELP (the second weight in step (b')), and the weight of physiological saline for injection (the third weight in step (c')) were different. In this experiment, the addition principles of the weight of curcumin (the first weight), the weight of ELP (the second weight), and the weight of physiological saline for injection (the third weight) are shown in Table 2.

[0264] In this experiment, the ratios of curcumin to ELP (weight ratio) in groups 1 - 9 were 1:4, 1:5, 1:8, 1:10, 1:20, 1:40, 1:100, 1:150, and 1:500 in sequence. And in the pharmaceutical compositions prepared in groups 1 - 9, the final concentrations of curcumin were 200 mg / g, 167 mg / g, 111 mg / g, 91 mg / g, 47.62 mg / g, 7.5 mg / g, 3 mg / g, 2 mg / g, and 0.5 mg / g in sequence. That is to say, in the preparation methods of the pharmaceutical compositions in groups 1 - 9, the weight ratio of curcumin in step (a') to ELP in step (b') (the ratio of the first weight to the second weight) was 1:4, 1:5, 1:8, 1:10, 1:20, 1:40, 1:100, 1:150, and 1:500 in sequence. And after adding the third weight of physiological saline for injection in step (c'), pharmaceutical compositions with final curcumin concentrations of 200 mg / g, 167 mg / g, 111 mg / g, 91 mg / g, 47.62 mg / g, 7.5 mg / g, 3 mg / g, 2 mg / g, and 0.5 mg / g were prepared in sequence. Among them, when the final drug concentration is expressed in mg / g, it represents the number of milligrams of curcumin contained in each gram of the pharmaceutical composition.

[0265] Use a particle size analyzer to determine whether micelles are contained in the pharmaceutical composition and measure the particle size of the micelles.

[0266] The particle size distribution and polydispersity index (PDI) were determined using a particle size analyzer to evaluate the stability of the pharmaceutical composition. The curcumin content in the micelles was analyzed using high performance liquid chromatography (HPLC; such as HPLC-UV), which was defined as the "initial drug content".

[0267] Table 2 Sample preparation table for preparing pharmaceutical compositions using ELP

[0268]

[0269]

[0270] The accelerated stability test was used to observe whether layering occurred in the pharmaceutical composition under high temperature storage conditions (25 ± 2 °C) for 3 months, and the drug content in the micelles was analyzed using high performance liquid chromatography (HPLC; such as HPLC-UV), which was defined as the "drug content after accelerated experiment". The "drug content after accelerated experiment" was divided by the "initial drug content" to obtain the "drug content percentage". If the drug content percentage was greater than or equal to 95%, it represented that the stability of the pharmaceutical composition was excellent.

[0271] Please refer to Table 3, which shows the stability analysis results of the pharmaceutical composition. Table 3 shows that micelles were present in the pharmaceutical compositions of Groups 2-9. Therefore, the pharmaceutical compositions prepared with a curcumin to ELP ratio of 1:5 to 1:500 are all pharmaceutical compositions of the present invention that can be used to reduce local fat.

[0272] In terms of stability, when the ratio of curcumin to ELP was 1:4 and 1:5, the PDI was greater than 0.4; when the ratio of curcumin to ELP was 1:8 to 1:500, the PDI was less than 0.4. Therefore, to prepare a pharmaceutical composition of the present invention for reducing local fat with better stability, the ratio of curcumin to ELP should be less than one-fifth (1 / 5). That is, to prepare a pharmaceutical composition of the present invention for reducing local fat with better stability, based on 1 weight unit of curcumin, the weight of ELP should be greater than 5 weight units. Preferably, based on 1 weight unit of curcumin, the weight of ELP is 8-500 weight units. More preferably, based on 1 weight unit of curcumin, the weight of ELP is 20-150 weight units.

[0273] As can be seen from the data in Table 3, when the pharmaceutical compositions in Groups 5 - 8 were stored at 25°C for 3 months, the percentage of curcumin drug content in each sample was greater than 95%, and there was no obvious downward trend compared with the initial drug content. From this result, it can be known that the said pharmaceutical compositions have good stability, and according to the formula of the accelerated experiment, these pharmaceutical compositions can be stored for at least 24 months under the condition of refrigeration at 2 - 8°C.

[0274] Table 3 Stability Analysis of Pharmaceutical Compositions

[0275]

[0276] In the above table, the blank fields represent no analysis was conducted.

[0277] Experiment 7: Effect of Curcumin - Resveratrol Compound Subcutaneous Injection on Subcutaneous Fat in Rats

[0278] Experiment 7 - 1 Effect of Resveratrol Single - Component Subcutaneous Injection on Subcutaneous Fat in Rats

[0279] Preparation method of resveratrol subcutaneous injection:

[0280] Mix resveratrol with an appropriate amount of physiological saline for injection to obtain resveratrol subcutaneous injection.

[0281] The rats were divided into a high - fat control group and a resveratrol group, with 6 rats in each group. The way of feeding the rats was the same as in Experiment 2. Inject the resveratrol subcutaneous injection into the subcutaneous fat layer of the lower groin of the resveratrol group, and the injection amount each time is 8 mg of resveratrol per kilogram of body weight (8 mg / kg). The high - fat control group was given the same volume of injection water by the same injection method as above.

[0282] The above injection site is the lower groin fat of the rats, and the injection is evenly distributed on the left and right sides. Inject once on each of the 1st, 3rd, and 5th days of the experiment. During the experiment, continuously feed the high - fat diet, record the body weight change daily, record the water intake and food intake once a week, the experiment lasts for 20 days, and sacrifice the rats with carbon dioxide on the 21st day.

[0283] Please refer to Figure 5 。 Figure 5 is a bar graph showing the effect of resveratrol subcutaneous injection without excipients on the local subcutaneous fat in rats.

[0284] Figure 5The results showed that the relative weight of subcutaneous fat in the lower inguinal region of rats in the high-fat control group was 100.00 ± 21.51%, and that of rats in the resveratrol group was 111.59 ± 11.288%. There was no significant difference in the relative weight of subcutaneous fat in the lower inguinal region between the resveratrol group and the high-fat control group, indicating that the fat-soluble plant extract without excipient - resveratrol could not reduce the fat at the administration site (local fat). Therefore, the inventors believe that directly mixing resveratrol and curcumin and injecting them into subcutaneous fat should also not be able to reduce local fat, and will further explore whether the excipient is helpful for the local fat-dissolving effect of the compound drug (resveratrol + curcumin).

[0285] Experiment 7-2 Effect of Curcumin-Resveratrol Compound with Excipient on Subcutaneous Fat in Rats

[0286] Prepare a curcumin-resveratrol compound solution containing Formulation A and a curcumin-resveratrol compound solution containing ELP in the following manner.

[0287] Preparation method of the curcumin-resveratrol compound solution containing Formulation A:

[0288] Grind 0.05 g of resveratrol, 0.2 g of curcumin, and 2 g of mannitol and mix them evenly to obtain a powder. Mix 0.05 g of carboxymethylcellulose (CMC) with 40 mL of sterile water, heat it to 60°C - 70°C to dissolve it, add 0.055 g of polysorbate 80 (Tween 80), stir until completely dissolved, and finally make up the volume to 50 mL to obtain a liquid agent. Pour the liquid agent into the powder, mix evenly, and then obtain the curcumin-resveratrol compound solution containing Formulation A. The so-called Formulation A refers to Tween 80 plus mannitol. The curcumin-resveratrol compound solution containing Formulation A does not contain micelles, the curcumin concentration is 4 mg / mL, and the resveratrol concentration is 1 mg / mL.

[0289] Preparation method of the curcumin-resveratrol compound solution containing ELP:

[0290] Mix 0.2 g of resveratrol, 0.8 g of curcumin, and 150 - 200 mL of dichloromethane, and stir at 150 - 500 rpm at room temperature until the curcumin is completely dissolved. Add 40 g of polyoxyl 35 castor oil (Kolliphor ELP, abbreviated as ELP), stir evenly at a rotation speed of 100 - 300 rpm to volatilize the dichloromethane. After the dichloromethane is completely volatilized, slowly add physiological saline for injection to make the final volume reach 200 mL, and stir evenly to obtain a curcumin-resveratrol compound solution containing ELP. The curcumin-resveratrol compound solution containing ELP contains micelles, the concentration of curcumin is 4 mg / mL, the concentration of resveratrol is 1 mg / mL, the concentration of polyoxyl 35 castor oil (ELP) is about 20% (weight percentage), and the weight ratio of curcumin, resveratrol, and polyoxyl 35 castor oil is 4:1:200.

[0291] Randomly divide the rats into 4 groups, namely the high-fat control group, the compound low-dose group of Formulation A, the compound high-dose group of Formulation A, and the compound low-dose group of ELP. The method of feeding the rats is the same as in Experiment 2.

[0292] Inject the curcumin-resveratrol compound solution containing Formulation A into the subcutaneous fat layer of the lower inguinal region of the rats in the compound low-dose group of Formulation A. The injection volume per time is 0.2 mL of the curcumin-resveratrol compound solution containing Formulation A per kilogram of body weight (0.2 mL / kg), so that the injection volume per time is 1 mg of the curcumin-resveratrol compound drug per kilogram of body weight (1 mg / kg); inject the curcumin-resveratrol compound solution containing Formulation A into the subcutaneous fat layer of the lower inguinal region of the rats in the compound high-dose group of Formulation A. The injection volume per time is 1 mL of the curcumin-resveratrol compound solution containing Formulation A per kilogram of body weight (1 mL / kg), so that the injection volume per time is 5 mg of the curcumin-resveratrol compound drug per kilogram of body weight (5 mg / kg); inject the curcumin-resveratrol compound solution containing ELP into the subcutaneous fat layer of the lower inguinal region of the rats in the compound low-dose group of ELP. The injection volume per time is 0.2 mL of the curcumin-resveratrol compound solution containing ELP per kilogram of body weight (0.2 mL / kg), so that the injection volume per time is 1 mg of the curcumin-resveratrol compound drug per kilogram of body weight (1 mg / kg). The high-fat diet group is given the same volume of physiological saline for injection by the same injection method as above.

[0293] The above injection site is the lower inguinal fat of the rats, and the injection is evenly distributed on the left and right sides. Inject once on the 1st, 3rd, and 5th days of the experiment. During the experiment, continuously feed the high-fat diet. The experiment lasts for 20 days, and the rats are sacrificed by carbon dioxide on the 21st day.

[0294] Please refer to Figure 6 。Figure 6 It is a bar graph showing the effect of curcumin-resveratrol compound subcutaneous injection preparations prepared using different excipients on the local subcutaneous fat mass of rats.

[0295] Figure 6 The results showed that the relative weight of subcutaneous fat in the lower inguinal region of rats in the high-fat control group was 100.0 ± 13%, the relative weight of subcutaneous fat in the lower inguinal region of rats in the low-dose compound group of Formulation A was 134.9 ± 39%, the relative weight of subcutaneous fat in the lower inguinal region of rats in the high-dose compound group of Formulation A was 134.9 ± 14%, and the relative weight of subcutaneous fat in the lower inguinal region of rats in the low-dose compound group of ELP was 71.1 ± 14%. Compared with the high-fat control group, neither the low-dose compound group of Formulation A nor the high-dose compound group of Formulation A could significantly reduce the fat at the administration site (local fat) (p > 0.05).

[0296] There was a significant difference (p < 0.05) between the relative weight of subcutaneous fat in the lower inguinal region of rats in the low-dose compound group of ELP and that of rats in the control group, and the relative weight of subcutaneous fat in the lower inguinal region of rats in the low-dose compound group of ELP decreased by 28.9%. From the above preparation method, it can be seen that the curcumin-resveratrol compound solution containing ELP administered to this group contains drug micelles (containing curcumin) and second lipid-soluble drug micelles (containing resveratrol). Thus, similar to the curcumin single-drug composition, the formation of micelles is also a key factor for the curcumin-resveratrol compound to significantly reduce local fat.

[0297] Experiment 7-3 Comparison between the curcumin-resveratrol compound pharmaceutical composition and the single-drug composition

[0298] The curcumin single-drug composition, resveratrol single-drug composition, and curcumin-resveratrol compound pharmaceutical composition of the present invention were prepared in the following manner.

[0299] Preparation method of the curcumin single-drug composition: The same as the preparation method of the curcumin ELP solution in Experiment 2. Among them, the concentration of curcumin was 5 mg / mL.

[0300] Preparation method of the resveratrol single-drug composition: It was roughly the same as the preparation method of the curcumin ELP solution in Experiment 2, only replacing curcumin with resveratrol. In the prepared resveratrol single-drug composition, the concentration of resveratrol was 5 mg / mL.

[0301] Preparation method of the curcumin-resveratrol compound pharmaceutical composition: The same as the preparation method of the curcumin-resveratrol compound solution containing ELP in Experiment 7-2. Among them, the total concentration of curcumin and resveratrol was 5 mg / mL, and the ratio of curcumin to resveratrol was 4:1.

[0302] The rats were divided into a high-fat control group, a curcumin group, a resveratrol group, and a curcumin-resveratrol compound group, with 5 rats in each group. The way of feeding the rats was the same as in Experiment 2.

[0303] The curcumin single medicine composition, the resveratrol single medicine composition, and the curcumin-resveratrol compound medicine composition were respectively injected into the subcutaneous fat layer of the lower inguinal region of the rats in the curcumin group, the resveratrol group, and the curcumin-resveratrol compound group. The injection volume each time was 2 mL per kilogram of body weight (2 mL / kg), so that the injection dose each time was 10 mg of the drug per kilogram of body weight (10 mg / kg). That is, 10 mg of curcumin was administered to the rats in the curcumin group per kilogram of body weight; 10 mg of resveratrol was administered to the rats in the resveratrol group per kilogram of body weight; 8 mg of curcumin and 2 mg of resveratrol were administered to the rats in the curcumin-resveratrol compound group per kilogram of body weight. The high-fat control group was given the same volume of injectable physiological saline by the same injection method as above.

[0304] The above injection site was the lower inguinal fat of the rats, and the injection was evenly distributed on the left and right sides. Each injection was given once on the 1st, 2nd, 3rd, and 4th days of the experiment. During the experiment, the high-fat diet was continuously given, and the experiment was carried out for 14 days. The rats were sacrificed by carbon dioxide on the 15th day.

[0305] Since 10 mg / kg of the drug was administered to each group each time, the local fat-dissolving effect of the rats in the curcumin-resveratrol compound group should be between that of the curcumin group and the resveratrol group. If the local fat-dissolving effect of the rats in the curcumin-resveratrol compound group is better than that of the curcumin group and the resveratrol group, it means that the curcumin-resveratrol compound medicine composition has a synergistic effect in the local fat-dissolving efficacy.

[0306] Please refer to Figure 7 。 Figure 7 is a bar graph showing the effect of curcumin single, resveratrol single, and curcumin-resveratrol compound subcutaneous injection needles containing micelles on the local subcutaneous fat mass of rats.

[0307] Figure 7 The results showed that the relative weight of the subcutaneous fat in the lower inguinal region of the rats in the high-fat control group was 100.0 ± 14.6%, the relative weight of the subcutaneous fat in the lower inguinal region of the rats in the curcumin group was 93.5 ± 6.5%, the relative weight of the subcutaneous fat in the lower inguinal region of the rats in the resveratrol group was 91.6 ± 27.8%, and the relative weight of the subcutaneous fat in the lower inguinal region of the rats in the curcumin-resveratrol compound group was 80.0 ± 5.8%. Compared with the high-fat control group, the curcumin group or the resveratrol group could not significantly reduce the fat at the application site (local fat) (p > 0.05).

[0308] There was a significant difference in the relative weight of subcutaneous fat in the lower inguinal region between the rats in the curcumin-resveratrol compound group and those in the high-fat control group (p<0.05), and the relative weight of subcutaneous fat in the lower inguinal region of the rats in the curcumin-resveratrol compound group decreased by 20%.

[0309] Comparing the local fat-dissolving effects of the curcumin group, resveratrol group, and curcumin-resveratrol compound group of rats, it can be seen that the curcumin-resveratrol compound pharmaceutical composition has a synergistic effect in the efficacy of local fat dissolution (synergy).

[0310] Experiment 7-4 Effects of administration frequency on subcutaneous fat mass and body weight of rats

[0311] In this experiment, when the curcumin-resveratrol compound pharmaceutical composition was administered to each group of rats, the total dose administered was the same but the administration frequency was different, in order to evaluate the effects of administration frequency on subcutaneous fat mass and body weight of rats. In this experiment, the main component of the commercially available local fat-dissolving injection was also administered to the rats, in order to simultaneously compare the effects of the curcumin-resveratrol compound pharmaceutical composition of the present invention and the commercially available local fat-dissolving injection on subcutaneous fat mass and body weight of rats.

[0312] Prepare the sodium deoxycholate solution and the curcumin-resveratrol compound pharmaceutical composition in the following manner.

[0313] Method for preparing the sodium deoxycholate solution: Mix an appropriate amount of sodium deoxycholate with sterile injection water to make the concentration of sodium deoxycholate 2.575 mg / mL, and stir evenly to obtain the sodium deoxycholate solution. Among them, sodium deoxycholate (purchased from Sigma-Aldrich, product number D6750) is the main component of the commercially available local fat-dissolving injection ATX-101 (trade name Kybella).

[0314] Method for preparing the curcumin-resveratrol compound pharmaceutical composition: The same as the method for preparing the curcumin-resveratrol compound solution containing ELP in Experiment 7-2. Among them, the total concentration of curcumin and resveratrol is 5 mg / mL, and the ratio of curcumin to resveratrol is 4:1.

[0315] Randomly divide the rats into 4 groups, namely the high-fat control group, the sodium deoxycholate group, the curcumin-resveratrol high administration frequency group (abbreviated as the high administration frequency group in this experiment), and the curcumin-resveratrol low administration frequency group (abbreviated as the low administration frequency group in this experiment. The method of feeding the rats is the same as that in Experiment 2.

[0316] The following describes the method of administering the drug:

[0317] Sodium deoxycholate group: Inject the sodium deoxycholate solution into the subcutaneous fat layer of the lower groin of the rats in the sodium deoxycholate group. The injection volume each time is 4 mL per kilogram of body weight (4 mL / kg), so that the dosage each time is 10.3 mg (10.3 mg / kg; the calculation method is 2.575 mg / mL × 4 mL / kg = 10.3 mg / kg). Inject once on each of the 1st, 3rd, and 5th days of the experiment, for a total of 3 injections, so that the total dosage is 30.9 mg / kg (10.3 mg / kg × 3 times = 30.9 mg / kg).

[0318] High dosing frequency group: Inject the curcumin-resveratrol compound pharmaceutical composition into the subcutaneous fat layer of the lower groin of the rats in the high dosing frequency group. The injection volume each time is 4 mL per kilogram of body weight (4 mL / kg), so that the dosage each time is 20 mg (20 mg / kg; the calculation method is 5 mg / mL × 4 mL / kg = 20 mg / kg). Inject once on each of the 1st, 3rd, 5th, 7th, 9th, and 11th days of the experiment, for a total of 6 injections, so that the total dosage is 120 mg / kg (20 mg / kg × 6 times = 120 mg / kg).

[0319] Low dosing frequency group: Inject the curcumin-resveratrol compound pharmaceutical composition into the subcutaneous fat layer of the lower groin of the rats in the low dosing frequency group. The injection volume each time is 8 mL per kilogram of body weight (8 mL / kg), so that the dosage each time is 40 mg (40 mg / kg; the calculation method is 5 mg / mL × 8 mL / kg = 40 mg / kg). Inject once on each of the 1st, 3rd, and 5th days of the experiment, for a total of 3 injections, so that the total dosage is 120 mg / kg (40 mg / kg × 3 times = 120 mg / kg).

[0320] High-fat control group: Administer the same volume of injection water by the same injection method as above.

[0321] Continue to feed the high-fat diet during the experiment. The experiment lasts for 20 days, and sacrifice the rats with carbon dioxide on the 21st day.

[0322] Please refer to Figure 8A and 8B . Figure 8A is the bar graph of the effect of the dosing frequency of the curcumin-resveratrol compound pharmaceutical composition on the local subcutaneous fat mass of rats, Figure 8B is the bar graph of the effect of the dosing frequency of the curcumin-resveratrol compound pharmaceutical composition on the total weight gain of rats.

[0323] Figure 8AThe results showed that the relative weight of subcutaneous fat in the lower inguinal region of rats in the high-fat control group was 100.0 ± 22.6%, that in the sodium deoxycholate group was 88.8 ± 16.7%, that in the high dosing frequency group was 62.3 ± 5.1%, and that in the low dosing frequency group was 65.4 ± 11.3%.

[0324] Compared with the high-fat control group, both the high dosing frequency group and the low dosing frequency group could significantly reduce the fat at the administration site (local fat) (p < 0.05). It can be seen that if the concentration of the curcumin-resveratrol compound drug is sufficient, a low dosing frequency can achieve the local fat-dissolving effect.

[0325] Compared with the low dosing frequency group, the high dosing frequency group had a better local fat-dissolving effect. It can be seen that although there was no significant difference between the high dosing frequency and the low dosing frequency, the local fat-dissolving effect of the high dosing frequency had a better trend.

[0326] Figure 8B The results showed that the relative total weight gain of rats in the high-fat control group was 100.0 ± 11.6%, that in the sodium deoxycholate group was 100.2 ± 12.6%, that in the high dosing frequency group was 63.5 ± 5.5%, and that in the low dosing frequency group was 78.7 ± 11.5%. Compared with the relative total weight gain of rats in the high-fat control group, the relative total weight gain of rats in the low dosing frequency group and the high dosing frequency group both decreased significantly (p < 0.05), and the relative total weight gain decreased by 21.3% and 36.5% respectively, and the weight loss effect was quite significant.

[0327] It can be seen that the curcumin-resveratrol compound pharmaceutical composition of the present invention can significantly reduce body weight, and the weight loss effect of the high dosing frequency is significantly better than that of the low dosing frequency (p < 0.05).

[0328] According to the inventor's experience, when the dosing frequency applicable to rats is 3 to 6 times, the dosing frequency applicable to humans is 1 to 12 times. Preferably, the dosing frequency applied to humans is 1 to 6 times.

[0329] Preferably, the frequency applied to humans is 1 to 12 times every 1 to 30 days. Preferably, the frequency applied to humans is 1 to 6 times every 1 to 30 days. Or, preferably, the frequency applied to humans is 3 to 60 times every 1 to 20 days; preferably, the frequency applied to humans is 6 to 42 times every 1 to 14 days.

[0330] Experiment 7-5 Effect of dosing dose on subcutaneous fat amount in rats

[0331] When the curcumin-resveratrol compound pharmaceutical composition was administered to each group of rats in this experiment, different doses were administered to evaluate the effect of the administration dose on the subcutaneous fat mass of the rats. In this experiment, the main component of another local lipolytic injection that was already undergoing clinical trials was also administered to the rats to simultaneously compare the effects of the curcumin-resveratrol compound pharmaceutical composition of the present invention and another local lipolytic injection that was already undergoing clinical trials on the subcutaneous fat mass of the rats.

[0332] Prepare the ELP solution, LIPO-202 solution, and curcumin-resveratrol compound pharmaceutical composition in the following manner.

[0333] ELP solution: Mix 18 g of polyoxyl 35 castor oil (Kolliphor ELP, simply referred to as ELP) with an appropriate amount of physiological saline for injection to make the final volume reach 90 mL, and stir evenly to obtain the ELP solution. Among them, the concentration of ELP is approximately 20%.

[0334] Preparation method of LIPO-202 solution:

[0335] LIPO-202 is a local lipolytic injection that is already undergoing clinical trials, and its main component is salmeterol xinafonate.

[0336] (i) Mix 1 mg of salmeterol (purchased from Sigma-Aldrich) with an appropriate amount of methanol to make the total volume 1 mL to obtain a 1 mg / mL stock solution.

[0337] (ii) Continuously dilute the stock solution 10-fold with sterile water to prepare a salmeterol with a final concentration of 0.01 μg / mL, which is the LIPO-202 solution used in this experiment.

[0338] Preparation method of curcumin-resveratrol compound pharmaceutical composition: The same as the preparation method of the curcumin-resveratrol compound solution containing ELP in Experiment 7-2. Among them, the total concentration of curcumin and resveratrol is 5 mg / mL, the ratio of curcumin to resveratrol is 4:1, and the concentration of ELP is 20%.

[0339] Randomly divide the rats into 7 groups, namely the high-fat control group, the control group, the LIPO-202 group, the compound 1 mg / mL group, the compound 5 mg / mL group, the compound 10 mg / mL group, and the compound 20 mg / mL group. Feed the rats in the same manner as in Experiment 2.

[0340] The following describes the method of administering the drugs:

[0341] Control group: Inject the ELP solution into the subcutaneous fat layer of the lower groin of the rats in the control group, with an injection volume of 4 mL per kilogram of body weight (4 mL / kg) each time.

[0342] LIPO-202 group: Inject the LIPO-202 solution into the subcutaneous fat layer of the lower groin of the rats in the LIPO-202 group, with an injection volume of 4 mL per kilogram of body weight (4 mL / kg) each time, so that the dose per administration is 0.04 μg (0.04 μg / kg; calculation method: 0.01 μg / mL × 4 mL / kg = 0.04 μg / kg).

[0343] Compound 1 mg / mL group: Inject the curcumin-resveratrol compound pharmaceutical composition into the subcutaneous fat layer of the lower groin of the rats in the compound 1 mg / mL group, with an injection volume of 0.2 mL per kilogram of body weight (0.2 mL / kg) each time, so that the dose per administration is 1 mg (1 mg / kg; calculation method: 5 mg / mL × 0.2 mL / kg = 1 mg / kg).

[0344] Compound 5 mg / mL group: Inject the curcumin-resveratrol compound pharmaceutical composition into the subcutaneous fat layer of the lower groin of the rats in the compound 5 mg / mL group, with an injection volume of 1 mL per kilogram of body weight (1 mL / kg) each time, so that the dose per administration is 5 mg (5 mg / kg; calculation method: 5 mg / mL × 1 mL / kg = 5 mg / kg).

[0345] Compound 10 mg / mL group: Inject the curcumin-resveratrol compound pharmaceutical composition into the subcutaneous fat layer of the lower groin of the rats in the compound 10 mg / mL group, with an injection volume of 2 mL per kilogram of body weight (2 mL / kg) each time, so that the dose per administration is 10 mg (10 mg / kg; calculation method: 5 mg / mL × 2 mL / kg = 10 mg / kg).

[0346] Compound 20 mg / mL group: Inject the curcumin-resveratrol compound pharmaceutical composition into the subcutaneous fat layer of the lower groin of the rats in the compound 20 mg / mL group, with an injection volume of 4 mL per kilogram of body weight (4 mL / kg) each time, so that the dose per administration is 20 mg (20 mg / kg; calculation method: 5 mg / mL × 4 mL / kg = 20 mg / kg).

[0347] High-fat control group: Administer the same volume of injection water in the same injection method as above.

[0348] Inject once each on the 1st, 2nd, 3rd, and 4th days of the experiment. Continuously feed the high-fat diet during the experiment. The experiment lasts for 14 days, and sacrifice the rats with carbon dioxide on the 15th day.

[0349] Please refer to Figure 9 。Figure 9 It is a bar graph showing the effect of the dosage of the curcumin-resveratrol compound pharmaceutical composition on the local subcutaneous fat mass in rats.

[0350] Figure 9 The results showed that the relative weight of the lower inguinal subcutaneous fat in the high-fat control group of rats was 100 ± 15.2%, that in the control group of rats was 99.2 ± 22.0%, that in the LIPO-202 group of rats was 97.8 ± 12.8%, that in the compound 1 mg / mL group of rats was 90.1 ± 12.2%, that in the compound 5 mg / mL group of rats was 80.9 ± 13.9%, that in the compound 10 mg / mL group of rats was 73.9 ± 9.5%, and that in the compound 20 mg / mL group of rats was 64.1 ± 12.0%.

[0351] It can be seen from this that the curcumin-resveratrol compound pharmaceutical composition has a significant local fat-dissolving effect at a dose of 5 mg / kg, and the higher the dose, the more significant the effect. Although the curcumin-resveratrol compound pharmaceutical composition did not achieve a significant local fat-dissolving effect at a dose of 1 mg / kg, there was already a trend. According to the inventor's experience, as long as the administration frequency is increased, the curcumin-resveratrol compound pharmaceutical composition can also achieve a significant local fat-dissolving effect at a dose of 1 mg / kg.

[0352] According to the inventor's experience, when the dosage applicable to rats is 1 mg / kg to 20 mg / kg, the dosage applicable to humans is 0.01 to 40 mg / kg. Preferably, the dosage administered to humans is 0.1 to 20 mg / kg.

[0353] Preferably, the dosage administered to humans is 0.02 to 20 mg injected per square centimeter. Preferably, the dosage administered to humans is 0.04 to 16 mg injected per square centimeter. Preferably, the dosage administered to humans is 0.2 to 12 mg injected per square centimeter. Preferably, the dosage administered to humans is 0.4 to 8 mg injected per square centimeter.

[0354] Preferably, the dosage administered to humans is 0.01 to 40 mg injected per kilogram. Preferably, the dosage administered to humans is 0.4 to 40 mg injected per kilogram. Preferably, the dosage administered to humans is 0.8 to 20 mg injected per kilogram.

[0355] Experiment 8: Effect of the curcumin compound pharmaceutical composition on fat dissolution

[0356] In this experiment, a compound pharmaceutical composition was prepared using other lipophilic drugs besides curcumin and resveratrol to evaluate the lipolytic effect of various lipophilic compound pharmaceutical compositions on mature adipocytes.

[0357] In this experiment, puerarin, quercetin, and synephrine were selected to prepare various lipophilic compound pharmaceutical compositions.

[0358] Experiment 8-1 Cytotoxicity Test

[0359] The cell viability assay (MTT assay) was used to evaluate whether 50 ppm of curcumin, puerarin, quercetin, or synephrine was toxic to cells other than adipocytes. If it was not toxic, the lipolytic test was then carried out.

[0360] The experimental results showed that 50 ppm of curcumin, puerarin, quercetin, and synephrine were not cytotoxic to somatic cells other than rat adipocytes. Therefore, this dose would not affect somatic cells.

[0361] Experiment 8-2 Lipolytic Effect on Mature Adipocytes

[0362] The cell culture media of the DMSO control group, curcumin cell culture media, puerarin cell culture media, quercetin cell culture media, synephrine cell culture media, curcumin-puerarin compound cell culture media, curcumin-quercetin compound cell culture media, and curcumin-synephrine compound cell culture media were prepared in the following manner.

[0363] Cell culture media of the DMSO control group: DMSO was mixed with an appropriate amount of sterile water to prepare a 0.5% DMSO solution. The 0.5% DMSO solution was mixed with cell culture media (product name: Dulbecco's Modified Eagle Medium, purchased from Gibco) to prepare the cell culture media of the DMSO control group, where the volume ratio of the 0.5% DMSO solution to the cell culture media was 1:1000.

[0364] Curcumin cell culture medium: Mix curcumin with an appropriate amount of 0.5% DMSO solution to prepare a curcumin solution. Mix the curcumin solution with cell culture medium (product name: Dulbecco's Modified Eagle Medium, purchased from Gibco) to prepare a curcumin cell culture medium containing 50 ppm curcumin, where the volume ratio of the curcumin solution to the cell culture medium is 1:1000.

[0365] Puerarin cell culture medium: Mix puerarin (purchased from Sigma - Aldrich) with an appropriate amount of 0.5% DMSO solution to prepare a puerarin solution. Mix the puerarin solution with cell culture medium to prepare a puerarin cell culture medium containing 50 ppm puerarin, where the volume ratio of the puerarin solution to the cell culture medium is 1:1000.

[0366] Quercetin cell culture medium: Mix quercetin (purchased from Sigma - Aldrich) with an appropriate amount of 0.5% DMSO solution to prepare a quercetin solution. Mix the quercetin solution with cell culture medium to prepare a quercetin cell culture medium containing 50 ppm quercetin, where the volume ratio of the quercetin solution to the cell culture medium is 1:1000.

[0367] Synephrine cell culture medium: Mix synephrine (purchased from Sigma - Aldrich) with an appropriate amount of 0.5% DMSO solution to prepare a synephrine solution. Mix the synephrine solution with cell culture medium to prepare a synephrine cell culture medium containing 50 ppm synephrine, where the volume ratio of the synephrine solution to the cell culture medium is 1:1000.

[0368] Curcumin - puerarin compound cell culture medium: Mix curcumin, puerarin, and an appropriate amount of 0.5% DMSO solution to prepare a curcumin - puerarin compound solution. The weight ratio of curcumin to puerarin is 2:3. Mix the curcumin - puerarin compound solution with cell culture medium to prepare a curcumin - puerarin compound cell culture medium containing 50 ppm curcumin - puerarin compound drug, where the concentration of curcumin is 20 ppm, the concentration of puerarin is 30 ppm, and the volume ratio of the curcumin - puerarin compound solution to the cell culture medium is 1:1000.

[0369] Curcumin - Quercetin Compound Cell Culture Medium: Mix curcumin, quercetin, and an appropriate amount of 0.5% DMSO solution to prepare a curcumin - quercetin compound solution. Among them, the weight ratio of curcumin to quercetin is 2:3. Mix the curcumin - quercetin compound solution with cell culture medium to prepare a curcumin - quercetin compound cell culture medium containing 50 ppm of curcumin - quercetin compound drug, where the concentration of curcumin is 20 ppm, the concentration of quercetin is 30 ppm, and the volume ratio of the curcumin - quercetin compound solution to the cell culture medium is 1:1000.

[0370] Curcumin - Synephrine Compound Cell Culture Medium: Mix curcumin, synephrine, and an appropriate amount of 0.5% DMSO solution to prepare a curcumin - synephrine compound solution. Among them, the weight ratio of curcumin to synephrine is 2:3. Mix the curcumin - synephrine compound solution with cell culture medium to prepare a curcumin - synephrine compound cell culture medium containing 50 ppm of curcumin - synephrine compound drug, where the concentration of curcumin is 20 ppm, the concentration of synephrine is 30 ppm, and the volume ratio of the curcumin - synephrine compound solution to the cell culture medium is 1:1000.

[0371] Experimental steps for the lipolysis effect of mature adipocytes:

[0372] Inoculate 3T3 - L1 preadipocytes (purchased from the Food Industry Research and Development Institute, Taiwan, China, abbreviated as BCRC) in a 12 - well plate so that each well contains 1×10 5 cells. After culturing for 2 days, culture with cell induction differentiation medium (DMI medium; containing 0.5 μM IBMX (purchased from Sigma - Aldrich), 0.1 μM Dexamethasone (purchased from Sigma - Aldrich), and 5 μg / ml Insulin (purchased from Humunlin R.)) for 2 days. Then, culture with cell culture medium containing 5 μg / ml insulin for 6 days. When the cell morphology changes from spindle - shaped to spherical and many lipid droplets accumulate inside the cells, it indicates that they have differentiated into mature adipocytes.

[0373] Divide the mature adipocytes into 8 groups, namely the DMSO control group, curcumin group, puerarin group, quercetin group, synephrine group, curcumin - puerarin compound group, curcumin - quercetin compound group, and curcumin - synephrine compound group.

[0374] The mature adipocytes in the DMSO control group, curcumin group, puerarin group, quercetin group, synephrine group, curcumin-puerarin compound group, curcumin-quercetin compound group, and curcumin-synephrine compound group were cultured for 24 hours respectively with the cell culture media of the DMSO control group, curcumin, puerarin, quercetin, synephrine, curcumin-puerarin compound, curcumin-quercetin compound, and curcumin-synephrine compound.

[0375] After mixing Annexin V protein (purchased from eBioscience) and Propidium iodide stain (abbreviated as PI; purchased from eBioscience) with the cells in each group for a period of time, the proportions of the cells in each group labeled by Annexin V protein and PI stain were analyzed using flow cytometry, so as to evaluate the proportion of mature adipocytes undergoing apoptosis. Among them, the mature adipocytes labeled by both Annexin V protein and PI stain represent that they have entered the apoptosis program; the more mature adipocytes undergoing apoptosis, the better the lipid-dissolving effect of the administered drug, and it represents that lipid-dissolving is through the apoptosis program rather than causing cell necrosis.

[0376] The data were presented in the form of mean ± SD and statistically analyzed by one-way ANOVA. The statistical results were represented by symbols or English letters. Different symbols or letters indicate statistical differences between groups (p < 0.05), and the same symbols or letters indicate no statistical differences between groups (p > 0.05).

[0377] Since the total dose of the drug administered to each group is 50 ppm, the apoptosis effect of the curcumin-puerarin compound group should be between that of the curcumin group and the puerarin group. If the apoptosis effect of the curcumin-puerarin compound group is better than that of the curcumin group and the puerarin group, it represents that the curcumin-puerarin compound pharmaceutical composition has a synergistic effect in terms of lipid-dissolving efficacy. Similarly, the apoptosis effect of the curcumin-quercetin compound group should be between that of the curcumin group and the quercetin group. If the apoptosis effect of the curcumin-quercetin compound group is better than that of the curcumin group and the quercetin group, it represents that the curcumin-quercetin compound pharmaceutical composition has a synergistic effect in terms of lipid-dissolving efficacy. The apoptosis effect of the curcumin-synephrine compound group should be between that of the curcumin group and the synephrine group. If the apoptosis effect of the curcumin-synephrine compound group is better than that of the curcumin group and the synephrine group, it represents that the curcumin-synephrine compound pharmaceutical composition has a synergistic effect in terms of lipid-dissolving efficacy.

[0378] Please refer to Figure 10 . Figure 10 It is about the effect of the curcumin-other liposoluble drug compound pharmaceutical composition on apoptosis of mature adipocytes.

[0379] Figure 10 The results showed that the percentage of apoptotic cells in the DMSO control group was 0.8±0.2%, in the curcumin group was 78.4±5.4%, in the puerarin group was 2.0±1.6%, in the quercetin group was 1.8±0.6%, in the synephrine group was 0.9±0.2%, in the curcumin-puerarin compound group was 80.0±5.9%, in the curcumin-quercetin compound group was 80.4±7.0%, and in the curcumin-synephrine compound group was 80.8±4.8%.

[0380] By comparing the apoptosis effects of the curcumin group, the puerarin group, and the curcumin-puerarin compound group, it can be seen that the curcumin-puerarin compound pharmaceutical composition has a synergistic effect in the efficacy of dissolving fat (synergy).

[0381] By comparing the apoptosis effects of the curcumin group, the quercetin group, and the curcumin-quercetin compound group, it can be seen that the curcumin-quercetin compound pharmaceutical composition has a synergistic effect in the efficacy of dissolving fat (synergy).

[0382] By comparing the apoptosis effects of the curcumin group, the synephrine group, and the curcumin-synephrine compound group, it can be seen that the curcumin-synephrine compound pharmaceutical composition has a synergistic effect in the efficacy of dissolving fat (synergy).

[0383] From this, it can be known that the compound pharmaceutical compositions formed by curcumin and various liposoluble drugs can all achieve the effect of dissolving fat, and curcumin and various liposoluble drugs have a synergistic effect in the efficacy of dissolving fat (synergy). Therefore, the present invention uses curcumin and various liposoluble drugs to prepare drug-containing micelles and second liposoluble drug micelles, and further prepares a curcumin-other liposoluble drug compound pharmaceutical composition, which can be used as a pharmaceutical composition for local fat dissolution and weight loss.

[0384] The present invention provides a first method for preparing a curcumin-other liposoluble drug compound pharmaceutical composition, and the curcumin-other liposoluble drug compound pharmaceutical composition contains a plurality of drug-containing micelles (micelle) and second liposoluble drug micelles; the steps of the first method for preparing a curcumin-other liposoluble drug compound pharmaceutical composition are as follows:

[0385] (A) The step of preparing a drug-containing micelle sub-composition for preparing a drug-containing micelle sub-composition;

[0386] (B) Steps for preparing a second lipid-soluble drug micelle sub-composition for preparing a second lipid-soluble drug micelle sub-composition: and

[0387] (C) Mixing the drug-containing micelle sub-composition with the second lipid-soluble drug micelle sub-composition to prepare the curcumin-other lipid-soluble drug compound pharmaceutical composition;

[0388] Wherein, the step (A) of preparing the drug-containing micelle sub-composition includes the following steps (a2) to (d2):

[0389] (a2) Mix curcumin with a first solvent and stir at 150 - 500 rpm at room temperature until the curcumin is completely dissolved;

[0390] (b2) Add a pharmaceutically acceptable first surfactant and stir evenly at a rotation speed of 100 - 300 rpm to volatilize the first solvent. Among them, the hydrophilic-lipophilic balance value (HLB value) of the first surfactant is greater than 10;

[0391] (c2) After the first solvent is completely volatilized, obtain a plurality of drug-containing micelles; and

[0392] (d2) After filtering with a 0.2 μm filter membrane, the filtrate is the drug-containing micelle sub-composition containing the drug-containing micelles;

[0393] Moreover, the step (B) of preparing the second lipid-soluble drug micelle sub-composition includes the following steps (a3) to (d3):

[0394] (a3) Mix a second lipid-soluble drug with a second solvent and stir at 200 - 500 rpm at room temperature until the second lipid-soluble drug is completely dissolved;

[0395] (b3) Add a pharmaceutically acceptable second surfactant and stir evenly at a rotation speed of 100 - 300 rpm to volatilize the second solvent. Among them, the hydrophilic-lipophilic balance value (HLB value) of the second surfactant is greater than 10;

[0396] (c3) After the second solvent is completely volatilized, obtain a plurality of second lipid-soluble drug micelles; and

[0397] (d3) After filtering with a 0.2 μm filter membrane, the filtrate is the second lipid-soluble drug micelle sub-composition containing the second lipid-soluble drug micelles.

[0398] Among them, in step (c2), the drug-loaded micelle is a micro-structure formed by a first surfactant, and curcumin is encapsulated in the drug-loaded micelle. In step (c3), the second lipid-soluble drug micelle is a micro-structure formed by a second surfactant, and the second lipid-soluble drug is encapsulated in the second lipid-soluble drug micelle.

[0399] Preferably, the operation steps of step (c2) are as follows: after the first solvent is completely volatilized, a pharmaceutically acceptable aqueous solution is slowly added and stirred evenly to form a plurality of drug-loaded micelles.

[0400] Preferably, the operation steps of step (c3) are as follows: after the second solvent is completely volatilized, a pharmaceutically acceptable aqueous solution is slowly added and stirred evenly to form a plurality of second lipid-soluble drug micelles.

[0401] Preferably, the second lipid-soluble drug is at least one or a combination of other lipid-soluble drugs other than quercetin, synephrine, puerarin, resveratrol, and curcumin.

[0402] Preferably, in step (a2) or / and step (a3), the boiling point of the first solvent or / and the second solvent is less than the boiling point of pure water.

[0403] Preferably, in step (a2) or / and step (a3), the first solvent or / and the second solvent is a hydrophilic solvent.

[0404] Preferably, the hydrophilic solvent is at least one or a combination of methanol, ethanol, acetone, and other hydrophilic solvents.

[0405] Preferably, the first solvent or / and the second solvent in step (a2) or / and (a3) is a lipophilic solvent.

[0406] Preferably, the lipophilic solvent is at least one or a combination of ether, benzene, chloroform, ethyl acetate, dichloromethane, hexane, and other lipophilic solvents.

[0407] Preferably, in step (b2) or / and (b3), the first surfactant or / and the second surfactant is a non-ionic surfactant.

[0408] Preferably, the non-ionic surfactant is at least one or a combination of polysorbate 80 (Tween 80), polyethylene glycol 15-hydroxystearate (Kolliphor HS 15), polyoxyethylene castor oil derivatives, and other non-ionic surfactants.

[0409] Preferably, the polyoxyethylene castor oil derivative is at least one or a combination of polyoxyethylene 35 castor oil (Cremophor ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives.

[0410] Preferably, the weight ratio of curcumin to the second fat-soluble drug is 30:1 to 1:10.

[0411] Preferably, in steps (a2) and (b2), the weight ratio of curcumin to the first surfactant is 1:4 to 1:500.

[0412] Preferably, in steps (a3) and (b3), the weight ratio of the second fat-soluble drug to the second surfactant is 1:4 to 1:500.

[0413] Preferably, in step (c2) or / and (c3), the pharmaceutically acceptable aqueous solution is water for injection, an aqueous injection solution, or physiological saline.

[0414] Preferably, in step (c2) or / and (c3), the pharmaceutically acceptable aqueous solution contains a local anesthetic.

[0415] Preferably, the local anesthetic is at least one or a combination of amides, para-aminobenzoate esters, and amino ethers.

[0416] Preferably, the amides are at least one or a combination of dibucaine, lidocaine, mepivacaine HCl, bupivacine HCl, pyrrocaine HCl, prilocaine HCl, digammacaine, and oxethazaine.

[0417] Preferably, the para-aminobenzoate esters are at least one or a combination of butacaine, dimethocaine, and tutocaine.

[0418] Preferably, the amino ethers are at least one or a combination of quinisocaine and pramocaine.

[0419] Preferably, in step (c2) or / and (c3), the pharmaceutically acceptable aqueous solution contains an antioxidant.

[0420] Preferably, the antioxidant is at least one of or a combination of beta-carotene, lutein, lycopene, bilirubin, vitamin A, vitamin C (also known as ascorbic acid), vitamin E, uric acid, nitric oxide, nitroxide, pyruvate, catalase, superoxide dismutase, glutathione peroxidases, N-acetyl cysteine, and naringenin.

[0421] The present invention provides a second method for preparing a curcumin-other lipophilic drug composite pharmaceutical composition, and the preparation method of the second curcumin-other lipophilic drug composite pharmaceutical composition is more concise than that of the first curcumin-other lipophilic drug composite pharmaceutical composition; the steps for preparing the second curcumin-other lipophilic drug composite pharmaceutical composition are as follows:

[0422] (a4) Mix curcumin, a second lipophilic drug, and a solvent, and stir at 200-500 rpm at room temperature until the curcumin is completely dissolved;

[0423] (b4) Add a pharmaceutically acceptable surfactant, stir evenly at a rotation speed of 100-300 rpm to volatilize the solvent, wherein the hydrophilic-lipophilic balance value (HLB value) of the surfactant is greater than 10;

[0424] (c4) After the solvent is completely volatilized, slowly add a pharmaceutically acceptable aqueous solution and stir evenly to form a plurality of drug-loaded micelles and a plurality of second lipophilic drug micelles; and

[0425] (d4) After filtering with a 0.2 μm filter membrane, store the filtrate containing a plurality of drug-loaded micelles and a plurality of second lipophilic drug micelles in the dark and refrigerate.

[0426] The solvents, surfactants, pharmaceutically acceptable aqueous solutions, and types of the second fat-soluble drug used in the preparation method of the second curcumin-fat-soluble drug compound pharmaceutical composition are the same as those used in the preparation method of the first curcumin-fat-soluble drug compound pharmaceutical composition. Moreover, the range of the proportional relationship between the components in the preparation method of the second curcumin-fat-soluble drug compound pharmaceutical composition is also the same as that in the preparation method of the first curcumin-fat-soluble drug compound pharmaceutical composition.

[0427] Preferably, the pharmaceutically acceptable aqueous solution contains a local anesthetic or / and an antioxidant.

[0428] Preferably, the types of the local anesthetic or / and antioxidant used in the preparation method of the second curcumin-fat-soluble drug compound pharmaceutical composition are the same as those used in the preparation method of the first curcumin-fat-soluble drug compound pharmaceutical composition.

[0429] Experiment 9: Effect of curcumin-green tea extract compound subcutaneous injection on subcutaneous fat mass in rats

[0430] Experiment 9-1: Effect of green tea extract single-component subcutaneous injection on subcutaneous fat mass in rats

[0431] Preparation method of green tea extract subcutaneous injection:

[0432] Mix the green tea extract with an appropriate amount of physiological saline for injection to obtain the green tea extract subcutaneous injection.

[0433] The rats were divided into a high-fat control group and a green tea extract group, with 6 rats in each group. The rats were fed in the same way as in Experiment 2. The green tea extract subcutaneous injection was injected into the subcutaneous fat layer of the lower groin of the rats in the green tea extract group, and the injection amount per time was 8 mg of green tea extract per kilogram of body weight (8 mg / kg). The high-fat control group was given the same volume of injection water by the same injection method as above.

[0434] The above injection site was the lower groin fat of the rats, and the injection was evenly distributed on the left and right sides. The injection was given once on the 1st, 3rd, and 5th days of the experiment. During the experiment, the rats were continuously fed with a high-fat diet, and the body weight change was recorded daily, and the water intake and food intake were recorded once a week. The experiment was carried out for 20 days, and the rats were sacrificed by carbon dioxide on the 21st day.

[0435] Please refer to Figure 11 . Figure 11 It is a bar graph showing the effect of the green tea extract subcutaneous injection without excipients on the local subcutaneous fat mass in rats.

[0436] Figure 11The results showed that the relative weight of the subcutaneous fat in the lower inguinal region of the rats in the high-fat control group was 100.00 ± 21.51%, and that of the rats in the green tea extract group was 99.50 ± 13.14%. There was no significant difference in the relative weight of the subcutaneous fat in the lower inguinal region between the rats in the green tea extract group and those in the high-fat control group, indicating that the water-soluble plant extract without excipients - green tea extract could not reduce the fat at the administration site (local fat).

[0437] Experiment 9-2: Effect of Administration Frequency on Subcutaneous Fat Content and Body Weight of Rats

[0438] In this experiment, when the curcumin-green tea extract compound pharmaceutical composition was administered to each group of rats, the total dose administered was the same but the administration frequency was different, so as to evaluate the effect of the administration frequency on the subcutaneous fat content and body weight of rats. In addition, the main component of the commercially available local lipolytic injection was administered to the rats, so as to simultaneously compare the effects of the curcumin-green tea extract compound pharmaceutical composition of the present invention and the commercially available local lipolytic injection on the subcutaneous fat content and body weight of rats.

[0439] The sodium deoxycholate solution and the curcumin-green tea extract compound pharmaceutical composition were prepared in the following manner.

[0440] Preparation method of the sodium deoxycholate solution: The same as the preparation method of the sodium deoxycholate solution in Experiment 7-4.

[0441] Preparation method of the curcumin-green tea extract compound pharmaceutical composition: 0.8 g of curcumin and 150 - 200 mL of dichloromethane were mixed, and stirred at 150 - 500 rpm at room temperature until the curcumin was completely dissolved. 40 g of polyoxyl 35 castor oil (Kolliphor ELP, abbreviated as ELP) was added, and stirred evenly at a rotation speed of 100 - 300 rpm to volatilize the dichloromethane. After the dichloromethane was completely volatilized, injectable physiological saline was slowly added to make the final volume reach 200 mL, wherein the injectable physiological saline contained 0.2 g of green tea extract. Stir evenly to obtain the curcumin-green tea extract compound solution containing ELP. The curcumin-green tea extract compound solution containing ELP contained drug-loaded micelles, the total concentration of curcumin and green tea extract was 5 mg / mL, and the weight ratio of curcumin to green tea extract was 4:1.

[0442] The rats were randomly divided into 4 groups, namely the high-fat control group, the sodium deoxycholate group, the high administration frequency group of curcumin-green tea extract (abbreviated as the high administration frequency group in this experiment), and the low administration frequency group of curcumin-green tea extract (abbreviated as the low administration frequency group in this experiment), and the rats were fed in the same way as in Experiment 2.

[0443] The following describes the method of administering the drug:

[0444] Sodium deoxycholate group: Inject the sodium deoxycholate solution into the subcutaneous fat layer of the lower inguinal region of the rats in the sodium deoxycholate group. The injection volume each time is 4 mL per kilogram of body weight (4 mL / kg), so that the drug dosage each time is 10.3 mg (10.3 mg / kg; the calculation method is 2.575 mg / mL × 4 mL / kg = 10.3 mg / kg). Inject once on each of the 1st, 3rd, and 5th days of the experiment, for a total of 3 injections, so that the total dose is 30.9 mg / kg (10.3 mg / kg × 3 times = 30.9 mg / kg).

[0445] High dosing frequency group: Inject the curcumin - green tea extract compound pharmaceutical composition into the subcutaneous fat layer of the lower inguinal region of the rats in the high dosing frequency group. The injection volume each time is 4 mL per kilogram of body weight (4 mL / kg), so that the drug dosage each time is 20 mg (20 mg / kg; the calculation method is 5 mg / mL × 4 mL / kg = 20 mg / kg). Inject once on each of the 1st, 3rd, 5th, 7th, 9th, and 11th days of the experiment, for a total of 6 injections, so that the total dose is 120 mg / kg (20 mg / kg × 6 times = 120 mg / kg).

[0446] Low dosing frequency group: Inject the curcumin - green tea extract compound pharmaceutical composition into the subcutaneous fat layer of the lower inguinal region of the rats in the low dosing frequency group. The injection volume each time is 8 mL per kilogram of body weight (8 mL / kg), so that the drug dosage each time is 40 mg (40 mg / kg; the calculation method is 5 mg / mL × 8 mL / kg = 40 mg / kg). Inject once on each of the 1st, 3rd, and 5th days of the experiment, for a total of 3 injections, so that the total dose is 120 mg / kg (40 mg / kg × 3 times = 120 mg / kg).

[0447] High - fat control group: Administer the same volume of injection water by the same injection method as above.

[0448] During the experiment, continuously feed the high - fat diet. The experiment lasts for 20 days, and sacrifice the rats with carbon dioxide on the 21st day.

[0449] Please refer to Figure 12A and 12B . Figure 12A is the bar graph of the effect of the dosing frequency of the curcumin - green tea extract compound pharmaceutical composition on the local subcutaneous fat mass of rats, Figure 12B is the bar graph of the effect of the dosing frequency of the curcumin - green tea extract compound pharmaceutical composition on the total weight gain of rats.

[0450] Figure 12AThe results showed that the relative weight of the subcutaneous fat in the lower inguinal region of the rats in the high-fat control group was 100.0 ± 22.6%, that of the rats in the sodium deoxycholate group was 88.8 ± 16.7%, that of the rats in the high drug administration frequency group was 57.6 ± 7.4%, and that of the rats in the low drug administration frequency group was 60.7 ± 4.0%.

[0451] Compared with the high-fat control group, both the high drug administration frequency group and the low drug administration frequency group could significantly reduce the fat at the administration site (local fat) (p < 0.05). It can be seen that if the concentration of the curcumin-green tea extract compound drug is sufficient, the low drug administration frequency can achieve the local fat-dissolving effect.

[0452] Compared with the low drug administration frequency group, the high drug administration frequency group had a better local fat-dissolving effect. It can be seen that although there was no significant difference between the high drug administration frequency and the low drug administration frequency, the local fat-dissolving effect of the high drug administration frequency had a better trend.

[0453] Figure 12B The results showed that the relative total weight gain of the rats in the high-fat control group was 100.0 ± 11.6%, that of the rats in the sodium deoxycholate group was 100.2 ± 12.6%, that of the rats in the high drug administration frequency group was 58.7 ± 9.0%, and that of the rats in the low drug administration frequency group was 74.9 ± 9.0%. Compared with the relative total weight gain of the rats in the high-fat control group, the relative total weight gain of the rats in the low drug administration frequency group and the high drug administration frequency group both decreased significantly (p < 0.05), and the relative total weight gain decreased by 25.1% and 41.3% respectively, and the weight loss effect was quite significant.

[0454] It can be seen that the curcumin-green tea extract compound pharmaceutical composition of the present invention can significantly reduce the weight, and the weight loss effect of the high drug administration frequency is significantly better than that of the low drug administration frequency (p < 0.05).

[0455] According to the inventor's experience, when the drug administration frequency applicable to rats is 3 to 6 times, the drug administration frequency applicable to humans is 1 to 12 times. Preferably, the drug administration frequency applied to humans is 1 to 6 times.

[0456] Preferably, the frequency applied to humans is 1 to 12 times per administration at intervals of 1 to 30 days. Preferably, the frequency applied to humans is 1 to 6 times per administration at intervals of 1 to 30 days. Or, preferably, the frequency applied to humans is 3 to 60 times per administration at intervals of 1 to 20 days; preferably, the frequency applied to humans is 6 to 42 times per administration at intervals of 1 to 14 days.

[0457] Experiment Ten: The Effect of the Curcumin Compound Pharmaceutical Composition on Fat Dissolving

[0458] In this experiment, compound pharmaceutical compositions were prepared using water-soluble drugs other than green tea extract to evaluate the fat-dissolving effects of various water-soluble compound pharmaceutical compositions on mature adipocytes.

[0459] In this experiment, caffeine and L-carnitine were selected to prepare various water-soluble compound pharmaceutical compositions.

[0460] Experiment 10-1 Cytotoxicity Test

[0461] The cell viability assay (MTT assay) was used to evaluate whether 50 ppm of caffeine and L-carnitine were toxic to cells other than adipocytes. If they were not toxic, the fat-dissolving test was then carried out.

[0462] The experimental results showed that 50 ppm of caffeine and L-carnitine were not cytotoxic to normal somatic cells other than rat adipocytes, so this dose would not affect normal somatic cells.

[0463] Experiment 10-2 Fat-Dissolving Effect on Mature Adipocytes

[0464] The cell culture media for the sterile water control group, curcumin cell culture media, caffeine cell culture media, L-carnitine cell culture media, curcumin-caffeine compound cell culture media, and curcumin-L-carnitine compound cell culture media were prepared in the following manner.

[0465] Cell culture media for the sterile water control group: Sterile water was mixed with cell culture media to prepare the cell culture media for the sterile water control group. Among them, the volume ratio of sterile water to cell culture media was 1:1000.

[0466] Curcumin cell culture media: The preparation method was the same as that of the curcumin cell culture media in Experiment 8-2.

[0467] Caffeine cell culture media: Caffeine (purchased from Sigma-Aldrich) was mixed with an appropriate amount of sterile water to prepare a caffeine solution. The caffeine solution was then mixed with cell culture media to prepare caffeine cell culture media containing 50 ppm of caffeine. Among them, the volume ratio of the caffeine solution to cell culture media was 1:1000.

[0468] L-carnitine cell culture medium: L-carnitine (purchased from Sigma-Aldrich) was mixed with an appropriate amount of sterile water to prepare an L-carnitine solution. The L-carnitine solution was mixed with the cell culture medium to prepare an L-carnitine cell culture medium containing 50 ppm of L-carnitine, wherein the volume ratio of the L-carnitine solution to the cell culture medium was 1:1000.

[0469] Curcumin-caffeine compound cell culture medium: Curcumin, caffeine, and an appropriate amount of sterile water were mixed to prepare a curcumin-caffeine compound solution. Among them, the weight ratio of curcumin to caffeine was 2:3. The curcumin-caffeine compound solution was mixed with the cell culture medium to prepare a curcumin-caffeine compound cell culture medium containing 50 ppm of the curcumin-caffeine compound drug, wherein the concentration of curcumin was 20 ppm, the concentration of caffeine was 30 ppm, and the volume ratio of the curcumin-caffeine compound solution to the cell culture medium was 1:1000.

[0470] Curcumin-L-carnitine compound cell culture medium: Curcumin, L-carnitine, and an appropriate amount of sterile aqueous solution were mixed to prepare a curcumin-L-carnitine compound solution. Among them, the weight ratio of curcumin to L-carnitine was 2:3. The curcumin-L-carnitine compound solution was mixed with the cell culture medium to prepare a curcumin-L-carnitine compound cell culture medium containing 50 ppm of the curcumin-L-carnitine compound drug, wherein the concentration of curcumin was 20 ppm, the concentration of L-carnitine was 30 ppm, and the volume ratio of the curcumin-L-carnitine compound solution to the cell culture medium was 1:1000.

[0471] The method for preparing mature adipocytes was the same as that in Experiment 8-2.

[0472] The mature adipocytes were divided into 6 groups, namely the sterile water control group, the curcumin group, the caffeine group, the L-carnitine group, the curcumin-caffeine compound group, and the curcumin-L-carnitine compound group.

[0473] The mature adipocytes in the sterile water control group, the curcumin group, the caffeine group, the L-carnitine group, the curcumin-caffeine compound group, and the curcumin-L-carnitine compound group were cultured with the sterile water control group cell culture medium, the curcumin cell culture medium, the caffeine cell culture medium, the L-carnitine cell culture medium, the curcumin-caffeine compound cell culture medium, and the curcumin-L-carnitine compound cell culture medium for 24 hours, respectively.

[0474] After mixing Annexin V protein (purchased from eBioscience) and Propidium iodide stain (abbreviated as PI; purchased from eBioscience) with cells in each group for a period of time, the proportion of cells in each group labeled by Annexin V protein and PI stain was analyzed using flow cytometry to evaluate the proportion of mature adipocytes undergoing apoptosis. Among them, mature adipocytes labeled by both Annexin V protein and PI stain represent those that have entered the apoptosis program; the more mature adipocytes undergoing apoptosis, the better the lipid-dissolving effect of the administered drug, and it represents that lipid-dissolving is through the apoptosis program rather than causing cell necrosis.

[0475] Since the total dose of the drug administered to each group is 50 ppm, and the proportion of curcumin is 40% while that of caffeine is 60%, therefore, the apoptosis effect of the curcumin-caffeine compound group should approach the average value of the curcumin group and the caffeine group. If the apoptosis effect of the curcumin-caffeine compound group is significantly better than the average value of the curcumin group and the caffeine group, it represents that the curcumin-caffeine compound pharmaceutical composition has a synergistic effect in terms of lipid-dissolving efficacy. Similarly, since the total dose of the drug administered to each group is 50 ppm, and the proportion of curcumin is 40% while that of L-carnitine is 60%, therefore, the apoptosis effect of the curcumin-L-carnitine compound group should approach the average value of the curcumin group and the L-carnitine group. If the apoptosis effect of the curcumin-L-carnitine compound group is significantly better than the average value of the curcumin group and the L-carnitine group, it represents that the curcumin-L-carnitine compound pharmaceutical composition has a synergistic effect in terms of lipid-dissolving efficacy.

[0476] Please refer to Figure 13 。 Figure 13 is the effect of curcumin-other water-soluble drug compound pharmaceutical compositions on the apoptosis of mature adipocytes.

[0477] Figure 13 The results showed that the percentage of apoptosis in the sterile water control group was 0.8 ± 0.4%, in the curcumin group was 78.4 ± 5.4%, in the caffeine group was 2.0 ± 1.7%, in the L-carnitine group was 1.7 ± 0.5%, in the curcumin-caffeine compound group was 69.3 ± 4.5%, and in the curcumin-L-carnitine compound group was 74.1 ± 10.2%.

[0478] By comparing the apoptosis effects of the curcumin group, the caffeine group, and the curcumin-caffeine compound group, it can be seen that the curcumin-caffeine compound pharmaceutical composition has a synergistic effect in the efficacy of fat dissolution.

[0479] By comparing the apoptosis effects of the curcumin group, the L-carnitine group, and the curcumin-L-carnitine compound group, it can be seen that the curcumin-L-carnitine compound pharmaceutical composition has a synergistic effect in the efficacy of fat dissolution.

[0480] From this, it can be known that the compound pharmaceutical compositions formed by curcumin and various water-soluble drugs can all achieve the effect of fat dissolution, and curcumin and various water-soluble drugs have a synergistic effect in the efficacy of fat dissolution. Therefore, the present invention uses curcumin and various water-soluble drugs to prepare a curcumin-water-soluble drug compound pharmaceutical composition containing drug-loaded micelles, which can be used as a pharmaceutical composition for local fat dissolution and weight loss.

[0481] The present invention provides a method for preparing a curcumin-water-soluble drug compound pharmaceutical composition, which curcumin-water-soluble drug compound pharmaceutical composition contains a plurality of drug-loaded micelles and a water-soluble drug; the steps for preparing the curcumin-water-soluble drug compound pharmaceutical composition are as follows:

[0482] (a5) Mix curcumin with a solvent, and stir at 150-500 rpm at room temperature until the curcumin is completely dissolved;

[0483] (b5) Add a pharmaceutically acceptable surfactant, and stir evenly at a rotation speed of 100-300 rpm to volatilize the solvent, wherein the hydrophilic-lipophilic balance value (HLB value) of the surfactant is greater than 10;

[0484] (c5) After the solvent is completely volatilized, slowly add a first pharmaceutically acceptable aqueous solution, and stir evenly at a rotation speed of 100-300 rpm to form a plurality of drug-loaded micelles; and

[0485] (d5) After filtering with a 0.2 μm filter membrane, store the filtrate containing the drug-loaded micelles in the dark and refrigerate.

[0486] Wherein, the first pharmaceutically acceptable aqueous solution contains a water-soluble drug.

[0487] Preferably, the first pharmaceutically acceptable aqueous solution contains a local anesthetic.

[0488] Preferably, the local anesthetic is at least one or a combination of amides, para-aminobenzoate esters, and amino ethers.

[0489] Preferably, the amide is at least one of dibucaine, lidocaine, mepivacaine HCl, bupivacine HCl, pyrrocaine HCl, prilocaine HCl, digammacaine, and oxethazaine, or a combination thereof.

[0490] Preferably, the para-aminobenzoate is at least one of butacaine, dimethocaine, and tutocaine, or a combination thereof.

[0491] Preferably, the amino ether is at least one of quinisocaine and pramocaine, or a combination thereof.

[0492] Preferably, the first pharmaceutically acceptable aqueous solution contains an antioxidant.

[0493] Preferably, the antioxidant is at least one of beta-carotene, lutein, lycopene, bilirubin, vitamin A, vitamin C (also known as ascorbic acid), vitamin E, uric acid, nitric oxide, nitroxide, pyruvate, catalase, superoxide dismutase, glutathione peroxidases, N-acetyl cysteine, and naringenin, or a combination thereof.

[0494] Preferably, in step (a5), the boiling point of the solvent is lower than that of pure water.

[0495] Preferably, in step (a5), the solvent is a hydrophilic solvent.

[0496] Preferably, the hydrophilic solvent is at least one of methanol, ethanol, acetone, and other hydrophilic solvents, or a combination thereof.

[0497] Preferably, the solvent in step (a5) is a lipophilic solvent.

[0498] Preferably, the lipophilic solvent is at least one or a combination of diethyl ether, benzene, chloroform, ethyl acetate, dichloromethane, hexane and other lipophilic solvents.

[0499] Preferably, in step (b5), the surfactant is a nonionic surfactant.

[0500] Preferably, the nonionic surfactant is at least one or a combination of polysorbate 80 (Tween 80), polyethylene glycol 15-hydroxystearate (Kolliphor HS 15), polyoxyethylene castor oil derivatives, and other nonionic surfactants.

[0501] Preferably, the polyoxyethylene castor oil derivative is at least one or a combination of polyoxyethylene 35 castor oil (Cremophor ELP), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), and other polyoxyethylene castor oil derivatives.

[0502] Preferably, between step (c5) and step (d5), the method further includes the step of:

[0503] (c51) adding a second pharmaceutically acceptable aqueous solution, stirring evenly to completely dissolve the second pharmaceutically acceptable aqueous solution.

[0504] Preferably, the water-soluble drug is dissolved in the first pharmaceutically acceptable aqueous solution, the drug-loaded micelle is a micro-structure formed by the surfactant, and curcumin is encapsulated in the drug-loaded micelle.

[0505] Preferably, the water-soluble drug in the first pharmaceutically acceptable aqueous solution is at least one of or a combination of green tea extract, epigallocatechin gallate, epicatechin, epicatechin gallate, epigallocatechin, gallocatechin gallate, gallocatechin, catechin gallate, catechin, epigallocatechingallate (EGCG), caffeine, carnitine (also known as carnitine or carnitine), L-carnitine, synephrine, chlorogenic acid, and other water-soluble drugs.

[0506] Preferably, in steps (a5) and (c5), the weight ratio of the curcumin to the water-soluble drug is 30:1 to 1:10.

[0507] Preferably, in steps (a5) to (c5), based on the total weight of the curcumin and the water-soluble drug as one weight unit, the weight of the surfactant is 0.24 to 70 weight units; alternatively, the weight ratio of the total weight of the curcumin and the water-soluble drug to the weight of the surfactant is 4:1 to 1:70.

[0508] Preferably, in steps (a5), (c5), and (c51), based on the total weight of the curcumin and the water-soluble drug as one weight unit, the total weight of the first pharmaceutically acceptable aqueous solution and the second pharmaceutically acceptable aqueous solution is 16 to 400 weight units.

[0509] Preferably, in steps (c5) and (c51), the first pharmaceutically acceptable aqueous solution and the second pharmaceutically acceptable aqueous solution are water for injection, aqueous injection solution, or physiological saline.

[0510] As can be seen from the embodiments of the present invention, the curcumin single-component pharmaceutical composition, curcumin-resveratrol compound pharmaceutical composition, curcumin-green tea extract compound pharmaceutical composition, curcumin-other liposoluble drug compound pharmaceutical composition, curcumin-water-soluble drug compound pharmaceutical composition, and other pharmaceutical compositions provided by the present invention can all reduce the local fat mass and can also reduce body weight. Therefore, the curcumin single-component pharmaceutical composition, curcumin-resveratrol compound pharmaceutical composition, curcumin-green tea extract compound pharmaceutical composition, curcumin-other liposoluble drug compound pharmaceutical composition, curcumin-water-soluble drug compound pharmaceutical composition, and other pharmaceutical compositions provided by the present invention can be used to prepare subcutaneous implant devices, subcutaneous implants, implantable infusion fluids, ointments, or patches, and can be administered to the sites in need of reducing subcutaneous fat through subcutaneous implantation, implantable infusion, ointment, or patch application. Or, it can be used to prepare subcutaneous implant devices, subcutaneous implants, implantable infusion fluids, ointments, or patches, and can be administered to an individual through subcutaneous implantation, intravenous injection, implantable infusion, ointment, or patch application to reduce the body weight of the individual.

[0511] Preferably, the curcumin single-component pharmaceutical composition, curcumin-resveratrol compound pharmaceutical composition, curcumin-green tea extract compound pharmaceutical composition, curcumin-other liposoluble drug compound pharmaceutical composition, curcumin-water-soluble drug compound pharmaceutical composition, and other pharmaceutical compositions provided by the present invention can reduce the fat at the administration site or reduce the body weight through subcutaneous fat injection. Therefore, the curcumin single-component pharmaceutical composition, curcumin-resveratrol compound pharmaceutical composition, curcumin-green tea extract compound pharmaceutical composition, curcumin-other liposoluble drug compound pharmaceutical composition, curcumin-water-soluble drug compound pharmaceutical composition, and other pharmaceutical compositions provided by the present invention can be used to prepare subcutaneous fat layer injection needles or subcutaneous injection needles for reducing local subcutaneous fat.

[0512] Preferably, the curcumin single-component pharmaceutical composition, curcumin-resveratrol compound pharmaceutical composition, curcumin-green tea extract compound pharmaceutical composition, curcumin-other liposoluble drug compound pharmaceutical composition, curcumin-water-soluble drug compound pharmaceutical composition, and other pharmaceutical compositions provided by the present invention can reduce body weight through subcutaneous fat injection or intravenous injection. Therefore, the curcumin single-component pharmaceutical composition, curcumin-resveratrol compound pharmaceutical composition, curcumin-green tea extract compound pharmaceutical composition, curcumin-other liposoluble drug compound pharmaceutical composition, curcumin-water-soluble drug compound pharmaceutical composition, and other pharmaceutical compositions provided by the present invention can be used to prepare subcutaneous fat layer injection needles, intravenous injection needles, or subcutaneous injection needles for reducing body weight.

[0513] The above are only the preferred embodiments of the present invention and are not intended to limit the claims of the present invention. Therefore, all other changes or modifications made without departing from the spirit disclosed by the present invention shall be included in the claims of the present invention.

Claims

1. A subcutaneous preparation, comprising: A first plurality of drug-containing microcells; and A curcuminoid substance encapsulated in the first plurality of drug-containing microcells, wherein the curcuminoid substance is curcumin; Among them, The first plurality of drug-containing microcells are a first micro-structure formed by a first nonionic surfactant, and the hydrophilic-lipophilic balance (HLB) value of the first nonionic surfactant is greater than 10; wherein The first nonionic surfactant includes polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil or a combination thereof, and the weight ratio of the curcuminoid substance to the first nonionic surfactant is 1:8 to 1:500; or The first nonionic surfactant is polyethylene glycol 15-hydroxy stearate, and the weight ratio of the curcuminoid substance to the first nonionic surfactant is 1:

40.

2. The subcutaneous preparation according to claim 1, wherein the first nonionic surfactant includes polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil or a combination thereof, and the weight ratio of the curcuminoid substance to the first nonionic surfactant is 1:20 to 1:

150.

3. The subcutaneous preparation according to claim 1, wherein the concentration of the curcuminoid substance in the subcutaneous preparation is 0.3 to 120 mg / g.

4. The subcutaneous preparation according to claim 3, wherein the concentration of the curcuminoid substance in the subcutaneous preparation is 2 to 91 mg / g.

5. The subcutaneous preparation according to claim 1, wherein the particle size of the first plurality of drug-containing microcells is 3 to 50 nm.

6. The subcutaneous preparation according to claim 5, wherein the particle size of the first plurality of drug-containing microcells is 5 to 20 nm.

7. The subcutaneous preparation according to claim 1, which further comprises a pharmaceutically acceptable aqueous solution.

8. The subcutaneous preparation according to claim 1, which further comprises a second plurality of lipid-soluble drug microcells and a lipid-soluble drug encapsulated in the second plurality of lipid-soluble drug microcells; Wherein the second plurality of lipid-soluble drug microcells are a second micro-structure formed by a second nonionic surfactant, and the second nonionic surfactant includes polyethylene glycol 15-hydroxy stearate, polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil or a combination thereof.

9. The subcutaneous preparation according to claim 8, wherein the HLB value of the second nonionic surfactant is greater than 10.

10. The subcutaneous preparation according to claim 8, wherein the lipid-soluble drug includes quercetin, synephrine, puerarin, resveratrol or a combination thereof.

11. The subcutaneous preparation according to claim 8, wherein the weight ratio of the curcuminoid substance to the lipid-soluble drug in the preparation is 30:1 to 1:

10.

12. The subcutaneous preparation according to claim 8, which further comprises a water-soluble drug.

13. The subcutaneous preparation according to claim 12, wherein the water-soluble drug comprises green tea extract, epigallocatechin gallate, epicatechin, epicatechin gallate, epigallocatechin, gallocatechin gallate, gallocatechin, catechin gallate, catechin, caffeine, carnitine, L-carnitine, synephrine, chlorogenic acid or a combination thereof.

14. The subcutaneous preparation according to claim 13, wherein the weight ratio of the curcuminoid substance to the water-soluble drug in the preparation is 30:1 to 1:

10.

15. The subcutaneous preparation according to claim 1, further comprising a co-solvent, a suspending agent, an oil-phase excipient or a combination thereof.

16. The subcutaneous preparation according to claim 15, wherein the first micro-structure is jointly formed by the first non-ionic surfactant, the oil-phase excipient and the co-solvent.

17. The subcutaneous preparation according to claim 1, wherein the subcutaneous preparation is a subcutaneous injection or a subcutaneous implant.

18. The subcutaneous preparation according to claim 1, wherein the subcutaneous preparation is a subcutaneous adipose tissue injection.

19. Use of a pharmaceutical composition in the preparation of a drug for reducing subcutaneous fat in a local part of an individual, the pharmaceutical composition comprising: a first plurality of drug-containing micro-cells; and a curcuminoid substance encapsulated in the first plurality of drug-containing micro-cells, wherein the curcuminoid substance is curcumin; Among them, the first plurality of drug-containing micro-cells is a micro-structure formed by a first non-ionic surfactant, and the hydrophilic-lipophilic balance (HLB) value of the first non-ionic surfactant is greater than 10, wherein the first non-ionic surfactant comprises polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil or a combination thereof, and the weight ratio of the curcuminoid substance to the first non-ionic surfactant is 1:8 to 1:500; or the first non-ionic surfactant is polyethylene glycol 15-hydroxystearate, and the weight ratio of the curcuminoid substance to the first non-ionic surfactant is 1:

40.

20. The use according to claim 19, wherein the first non-ionic surfactant comprises polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil or a combination thereof, and the weight ratio of the curcuminoid substance to the first non-ionic surfactant is 1:20 to 1:

150.

21. The use according to claim 19, wherein the drug is the subcutaneous preparation according to any one of claims 1 to 18.

22. The use according to claim 19, wherein the drug is a subcutaneous injection, an ointment dosage form or a patch dosage form.

23. The use according to claim 19, wherein the drug is a subcutaneous adipose tissue injection.

24. The use according to claim 19, wherein the pharmaceutical composition is administered to the local part of the individual.

25. The use according to claim 24, wherein the dosage of the pharmaceutical composition administered to the local part of the individual is 0.02 to 20 mg per square centimeter of injection.

26. The use according to claim 24, wherein the dosage of the pharmaceutical composition administered to the local part of the individual is 0.01 to 40 mg per kg of injection.

27. The use according to claim 24, wherein the frequency of administering the pharmaceutical composition to the local part of the individual is 1 to 12 times every 1 to 30 days.

28. The use of a pharmaceutical composition in the preparation of a medicament for reducing the body weight of an individual, the pharmaceutical composition comprising: a first plurality of drug-containing micelles; and curcuminoids encapsulated in the first plurality of drug-containing micelles, wherein the curcuminoids are curcumin; Among them, the first plurality of drug-containing micelles is a micro-structure formed by a first non-ionic surfactant, and the hydrophilic-lipophilic balance (HLB) value of the first non-ionic surfactant is greater than 10, wherein the first non-ionic surfactant includes polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil or a combination thereof, and the weight ratio of the curcuminoids to the first non-ionic surfactant is 1:8 to 1:500; or the first non-ionic surfactant is polyethylene glycol 15-hydroxy stearate, and the weight ratio of the curcuminoids to the first non-ionic surfactant is 1:

40.

29. The use according to claim 28, wherein the first non-ionic surfactant includes polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil or a combination thereof, and the weight ratio of the curcuminoids to the first non-ionic surfactant is 1:20 to 1:

150.

30. The use according to claim 28, wherein the pharmaceutical composition is administered to a local part of the individual in the form of a subcutaneous injection, an intravenous injection, an implantable infusion solution, an ointment, or a patch.

31. The use according to claim 28, wherein the pharmaceutical composition is administered to a local part of the individual in the form of a subcutaneous injection or an intravenous injection.

32. The use according to claim 28, wherein the pharmaceutical composition is administered to a local part of the individual in the form of a subcutaneous fat layer injection.

33. The use according to claim 31 or 32, wherein the dosage of the pharmaceutical composition administered to the local part of the individual is 0.2 to 16 mg per square centimeter of injection.

34. The use according to claim 31 or 32, wherein the dosage of the pharmaceutical composition administered to the local part of the individual is 0.4 to 40 mg per kg of injection.

35. The use according to any one of claims 30 to 32, wherein the frequency of administering the pharmaceutical composition to the local part of the individual is 3 to 60 times every 1 to 20 days.

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