Liposome compound and application thereof

Abstract

The present invention provides compositions and related methods for reducing local fat, reducing body weight, preventing and treating lightening skin color and melanin precipitation, and slowing symptoms of arthritis. The composition comprises at least one active component and a liposome, and the liposome is used as a carrier of the active component to coat the active component so as to form a liposome compound.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition, in particular to a pharmaceutical composition formed by encapsulating an active agent with liposomes to form a liposome complex, and the pharmaceutical composition is used for reducing local fat, reducing body weight, preventing or treating lightening of skin color and reducing melanin deposition, reducing skin wrinkles or reducing skin roughness, preventing or treating tumors, preventing or treating fat metabolism-related diseases, and alleviating arthritis symptoms. Background Art

[0002] I. Regarding reducing local fat, reducing body weight, and treating obesity:

[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 more emphasis on reducing local fat or sculpting curves to achieve a healthier and more aesthetic body shape. Moreover, general weight loss methods, whether it is 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 fat embolism and anesthesia allergy that cannot be prevented in advance. In addition, after liposuction surgery, there are also 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 number of people actually 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 produce 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, reduce body weight, treat obesity, and has lower side effects, better safety, and a shorter recovery period.

[0006] In the situation where there is a high demand from both consumers and physicians, it will be an urgent topic to be explored and solved to develop pharmaceutical compositions that can break through the current technological limitations for local fat loss, weight loss, and the treatment of obesity.

[0007] II. Regarding the prevention and treatment of skin color lightening and melanin precipitation:

[0008] It is known that the color of the skin is mainly determined by the amount of melanin. Normally, melanin in the skin can serve as a natural protection barrier for the human body. The presence of melanin helps humans resist ultraviolet radiation and avoid the risk of photocarcinogenesis. On the contrary, excessive formation of melanin precipitation will cause many troubles, such as age spots, freckles, dark spots, melasma and other pigment precipitations that the skin does not need, or the pigment precipitation generated during the wound healing process. Melanin is produced by melanocytes present in the epidermis and is transferred to keratinocytes through the dendritic structure of melanocytes in the form of melanosomes. There is not much difference in the number of melanocytes among different ethnic groups, and the main factors affecting skin color differences are the proportion of melanin, the amount and distribution of melanosomes present in keratinocytes.

[0009] Factors that stimulate melanin production include direct exposure to ultraviolet radiation or the action of melanocyte-stimulating hormone (α-MSH) secreted by keratinocytes, which then promotes the enzymatic reaction of tyrosinase to produce melanin. The currently well-known melanin production mechanism is through the action of the enzyme tyrosinase to convert tyrosine through multiple-step reactions. In the whole reaction process, each step involves tyrosinase. First, tyrosinase converts tyrosine in the cell into L-DOPA, and then converts L-DOPA into L-dopaquinone, and continues to form intermediate products at each stage through the action of tyrosinase, including dopachrome, 5,6-dihydroxyindole, and indole-5,6-quinone. After a series of reaction processes, melanin is finally produced. Therefore, tyrosinase can be regarded as the key enzyme responsible for regulating human melanization and melanin production. At the same time, regulating the activity of tyrosinase is also the main research and development direction currently used to reduce dark spots and promote skin whitening. In addition, pigmentation caused by skin injury and inflammation (post-inflammatory hyperpigmentation, PIH) is another reason that promotes the production and transfer of melanin to keratinocytes. Generally speaking, PIH can be divided into epidermal pigmentation (epidermal PIH) and dermal pigmentation (dermal PIH). When an inflammatory reaction occurs in the epidermis, arachidonic acid is acted upon by cyclooxygenase and lipoxygenase to produce prostaglandins E2 (PGE2) and leukotrienes (LTC4). When melanocytes are stimulated by such inflammatory factors, they will promote the massive production of melanin and transfer it to the surrounding keratinocytes. On the other hand, when the basal cell layer at the junction of the epidermis and dermis is damaged by inflammation, a large amount of melanin will be swallowed by macrophages in the dermis, resulting in dark brown or blue-gray precipitation. In daily life, acne, chickenpox, herpes zoster, dermatitis or any skin inflammation caused by wounds may be the cause of PIH.

[0010] Currently, whitening ingredients that are used to inhibit melanin production or lighten melanin include magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbyl glucoside, kojic acid, arbutin, ellagic acid, chamomile ET, 5,5'-dipropyl-biphenyl-2,2'-diol, tranexamic acid, potassium methoxysalicylate, and hydroquinone, among many others that have been proposed to inhibit melanin formation or reduce skin melanin deposition or lighten skin color.

[0011] Metal salts and glycosylated derivatives of vitamin C inhibit the production of melanin by reducing the intermediate product L-dopaquinone of melanin during the reaction of tyrosinase through antioxidant methods. Vitamin C itself is highly safe and can prevent the formation of free radicals, but its stability is poor and it is easily oxidized. Therefore, its stability can be increased through metal binding or glycosylation. Such ingredients include magnesium ascorbyl phosphate, sodium ascorbyl phosphate, and ascorbyl glycoside. Since tyrosinase is an oxidase with divalent copper ions as its active center, structures that can bind to or compete with copper ions have also been developed to inhibit tyrosinase, thereby blocking melanin production. The representative substance of this type is kojic acid. Kojic acid binds to the copper ions of tyrosinase through its own structure, reducing its enzyme activity, and thus affecting the reaction process of tyrosine conversion to L-DOPA and L-dopaquinone. Another type is to competitively replace the substrate of tyrosinase, such as arbutin. Such substances can compete with tyrosine, substantially reducing the action of tyrosinase. Or the free radicals generated by hydroquinone directly cause cytotoxicity to melanocytes, but improper use may cause side effects such as skin irritation, dermatitis, abnormal pigmentation, and skin darkening. Hydroquinone has also been listed as a medicinal ingredient that cannot be added to cosmetics, and the medicinal content cannot exceed 5%.

[0012] Each ingredient achieves the effect of whitening and lightening the skin tone through different mechanisms. However, in terms of the whitening mechanism, it generally includes blocking tyrosinase to reduce melanin production, blocking the transfer of melanosomes from melanocytes to keratinocytes, inhibiting the activity of tyrosinase, promoting the metabolism of melanin in keratinocytes, or using preventive methods to isolate ultraviolet rays. In the past, various ingredients such as vitamin C, tranexamic acid, and vitamin B group have been used in intravenous injection attempts to achieve the treatment effects of removing spots or whitening. However, such administration methods and whitening uses have not been legally marketed in any country, and their whitening effects are not obvious, and there may also be a risk of allergies. In addition, if the product is not sterilized completely or the disinfection is incomplete during injection, there is a very high risk of more serious side effects such as phlebitis, cellulitis, and sepsis.

[0013] Although the development of whitening-related products in the market has never stopped in the past, consumers' demands for removing spots and lightening skin tone have not ceased, and the market for whitening-related technologies or products has gradually increased. However, the effects of various ingredients currently used in lightening or removing melanin still have room for improvement. Therefore, finding safer and more effective ingredients that can inhibit melanin activity is highly anticipated and indispensable in the current medical beauty market.

[0014] In addition, in the past, when developing ingredients or products related to whitening, most experiments would first add drugs and then stimulate melanogenesis by administering melanocyte-stimulating hormone (α-MSH) to compare the melanin inhibition effects of the groups with added drugs; or only conduct in vitro tube reactions, directly mix the drugs with tyrosine, and then add tyrosinase to compare the degree of its activity inhibition. Since this approach is different from the actual sequence of melanin production in the body, even if the experimental results are good, the whitening efficacy after subsequent actual application to whitening products is often far less than expected.

[0015] III. Regarding the part of alleviating arthritis symptoms:

[0016] Degenerative arthritis most commonly occurs in middle-aged and elderly people, long-term overloaded athletes, or obese individuals. This disease can occur in any joint of the body, with the knee joint and hip joint being the most frequently affected. In addition, the causative factors of degenerative arthritis have not been fully clarified, and age and obesity are the main risk factors. Due to the extension of lifespan in modern people, almost everyone will be troubled by degenerative arthritis to some extent as they age. In the long run, patients threatened by this disease will suffer from swelling, pain, and even loss of labor function.

[0017] Degenerative arthritis is a common joint disease that is affected by multiple factors on synovial joints and the extracellular matrix (ECM), resulting in cartilage fibrosis and loss of elasticity. The cartilage covering the outermost bone of the joint wears thin, then fragments, and even completely disappears due to abrasion, the joint space becomes narrow, increasing the stress on the bone under the cartilage, and then causing bone sclerosis, necrosis, turning into cystic cavities, and ultimately leading to pain and severely affecting the ability to move. Degenerative arthritis can be divided into primary arthritis and secondary arthritis. Primary arthritis is mainly related to aging, while secondary arthritis is related to mechanical compression (obesity), injury, metabolic diseases, or congenital abnormalities. The main symptoms of degenerative arthritis include pain, stiffness, swelling, and deformation. When moving the joint, abnormal friction sounds can often be heard. Patients usually seek medical treatment only when the pain in the joint affects their daily life to a certain extent. Diagnosis is carried out through the inquiry of clinical history and pathological examination, and imaging medicine, such as X-ray or ultrasound, can also be used to diagnose the changes in soft tissues, joints, and bones for accurate diagnosis and treatment.

[0018] Degenerative arthritis can be graded according to the degree of joint degeneration based on the patient's X-ray findings, with a total of 0-4 grades (Kellgren-Lawrence Grading Scale). Currently, the primary treatment for degenerative arthritis is to relieve joint pain and stiffness, and then to slow down the progression of the disease. Mild patients only need to change their lifestyle and combine it with physical therapy and other methods to improve. When necessary, or for moderate patients, medication or hyaluronic acid injections are required. Severe patients need to undergo surgery. Therefore, how to effectively improve the incidence of degenerative arthritis and provide effective treatment methods is an important issue.

[0019] When degenerative arthritis occurs, inflammation of the synovial cavity is accompanied by cartilage loss. In more severe arthritis, there may even be swelling and deformation of the limbs, and a large number of macrophages can be observed infiltrating the cartilage and synovial tissues, continuously promoting the inflammatory response. Therefore, an increase in various cytokines such as TNF-α and NF-κB, as well as a large expression of COX-2 enzyme, can be detected at the lesion site. COX-2 is an inducible enzyme that is only induced during tissue inflammation. Under normal physiological conditions, most tissues do not overexpress COX-2; however, when cells are stimulated by inflammatory factors such as IL-1, TNF-α, and NF-κB, the production of COX-2 will increase significantly, and a large amount of prostaglandins involved in the inflammatory response will be metabolized, resulting in a severe pain response in the joint tissue. In addition, arthritis is accompanied by abnormal proliferation of synovial cells and a large production of RANKL (receptor for activation of nuclear factor kappa B ligand). When RANKL binds to the RANK receptor on the surface of macrophages at the inflamed site, it will stimulate the differentiation of macrophages into osteoclasts, releasing the minerals in bone calcium, ultimately leading to the loss of hard bone.

[0020] There is currently no complete cure for degenerative arthritis, and it is mostly treated by maintenance and medications to control the condition and relieve pain. Different treatment methods are given according to the severity of the disease, including various types of anti-inflammatory painkillers and hyaluronic acid joint injections. If the joint pain of the patient cannot be improved by the above-mentioned medications or treatments, surgery such as artificial joint replacement must be performed to improve the symptoms. In addition, by changing lifestyle and diet, and maintaining exercise and controlling weight, it is also an effective way to reduce the risk factors of degenerative arthritis. Currently, the painkillers commonly used to improve arthritis include three major categories: acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), and COX-2 inhibitors. Acetaminophen is the first-line medication for relieving pain and can inhibit the secretion of prostaglandins. However, prostaglandins are also related to blood pressure regulation, blood clotting, gastric acid secretion, and renal function regulation. Therefore, interfering with prostaglandins will also affect the normal functioning of the body. On the other hand, when the dosage of acetaminophen is too large, it will increase the risk of gastrointestinal bleeding and is also prone to hepatotoxicity. Nonsteroidal anti-inflammatory drugs are used to inhibit cyclooxygenase COX-1 and COX-2, and have anti-inflammatory and analgesic effects. When COX-2 is inhibited, it will cause a decrease in prostacyclin I2 (PGI2) and produce anti-inflammatory and analgesic effects, but it will also promote platelet aggregation. When COX-1 is inhibited, it will damage the integrity of the gastrointestinal mucosa and affect renal blood flow, and then cause side effects such as gastric ulcers or renal failure. COX-2 inhibitors inhibit prostaglandins by inhibiting COX-2, achieving the effect of reducing inflammation and pain, and can reduce the problems of gastric perforation, bleeding, and ulcers in nearly 50% of patients. However, recent studies have found that COX-2 inhibitors can cause cardiovascular toxicity and risks, so they are recommended to be used in patients with high gastrointestinal risks and low cardiovascular risks.

[0021] It can be seen from this that the commonly used drugs for degenerative arthritis only inhibit pain, but cannot improve the causative factors or sources of pain in the joints. They will not only cause side effects such as liver toxicity, gastrointestinal bleeding, ulcers, and renal failure, but also pose a great burden on the elderly, who are the main group suffering from degenerative arthritis. Although the specific COX-2 inhibitor can reduce COX-2 and achieve a better pain relief effect, it will increase the risk of cardiovascular toxicity, and the improvement of symptoms is not obvious for patients with severely worn or aged joints. The pain relief and anti-inflammatory effects of glucosamine are not good, especially for elderly patients. As for hyaluronic acid joint injection, patients need to be injected once a week on average. If injected continuously for more than five weeks, it will also greatly increase the discomfort and infection risk of patients. The hyaluronic acid joint injection mainly relieves pain by increasing joint lubrication, but cannot directly reduce joint inflammatory factors. At present, the drugs or treatment methods for degenerative arthritis can only relieve the symptoms but not address the root causes. Therefore, there is still a lack of arthritis treatment methods or drugs in the market that can simultaneously reduce inflammation, relieve pain, and have low side effects, being safer and more reliable.

[0022] IV. Regarding reducing skin wrinkles or skin roughness:

[0023] Skin, like other living tissues, gradually ages with increasing age, and aging is further promoted by ultraviolet exposure. From recent research, it can be seen that there are some important molecular biological commonalities between age-related aging and skin aging caused by ultraviolet rays.

[0024] The skin is composed of, from the outside, the stratum corneum, the epidermis, the dermis, and the subcutaneous tissue. The epidermal cells are born in the basal layer, the deepest part of the epidermis, and move toward the outside. New cells often enter to replace them (this is called the turnover of epidermal cells). The main causes of skin aging are known to be a decrease in the turnover of epidermal cells that make up the epidermis and a decrease in collagen, which exists between fibroblasts in the dermis (Arch Dermato 1.2002; 138:1462-1470). Collagen is known to be the main component of the dermis. Due to age or damage from ultraviolet rays, the amount of collagen in the skin decreases, resulting in a loss of skin elasticity, which promotes the formation of wrinkles and skin aging. Furthermore, it is also known that dryness causes the stratum corneum and the epidermis to thicken, and the peeling of the stratum corneum is slow, which is a cause of the formation of fine wrinkles (J.Dermatol.Sci.2001; 27Suppl 1:S19-25). Skin spots and sagging are the main symptoms of skin aging, but the formation of wrinkles is the most important factor among them. Therefore, various methods have been used to prevent wrinkles. For example, methods to promote the synthesis of collagen fibers that support the skin structure and prevent the reduction of collagen. Furthermore, considering promoting skin moisturization maintenance or the regeneration of the stratum corneum and epidermal cells related to the barrier function, that is, promoting the turnover (epidermal renewal) of the epidermis and the stratum corneum (epidermal cell activation), is also an effective method for preventing wrinkles.

[0025] Currently, common means for preventing wrinkles on the market include external cosmetics formulated with ingredients that impart moisture to the skin or are responsible for maintaining elasticity. These cosmetics contain, for example, mucopolysaccharides such as hyaluronic acid and chondroitin sulfate, or collagen, vitamins, amino acids, ceramides, etc.

[0026] In recent years, beauty health foods beneficial to the skin have also been developed. For example, collagen production promoters containing enzyme decomposition products of lactoferrin or enzyme decomposition products of lactoperoxidase, or beauty health foods characterized by containing mucopolysaccharides can be cited.

[0027] Since it is considered particularly effective for preventing and improving skin aging with wrinkles as the main symptom by increasing the amount of collagen in the dermis or promoting epidermal renewal, there is a lack on the market of a treatment method or drug that can simultaneously promote epidermal renewal, skin cell proliferation, inhibit skin aging, and has low side effects and is safer.

[0028] V. Regarding the prevention and treatment of tumors:

[0029] According to global statistics, malignant tumors (cancers) are the second leading cause of death after heart disease. Cancer is characterized by an increase in the number of abnormal or neoplastic cells derived from normal tissue, which proliferate to form a tumor mass; these neoplastic tumor cells invade adjacent tissues; and produce malignant cells that eventually spread to regional lymph nodes via the blood or lymphatic system and to distant sites via a process called metastasis. In a cancerous condition, cells proliferate under conditions where normal cells would not grow. Cancer itself presents in a variety of forms characterized by varying degrees of invasiveness and aggressiveness.

[0030] In an attempt to identify effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or membrane-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells as compared to their expression on one or more normal non-cancerous cells. Typically, these membrane-associated polypeptides are expressed in greater amounts on the surface of cancer cells as compared to their expression on the surface of non-cancerous cells. Identification of these tumor-associated cell surface antigen polypeptides provides the ability to specifically target cancer cells for destruction via antibody-based therapies. In this regard, it has been noted that antibody-based therapies have proven highly effective in treating certain cancers. For example, and (both from Genentech Inc., South San Francisco, California) are antibodies that have been successfully used to treat breast cancer and non-Hodgkin's lymphoma, respectively. More specifically, is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. Overexpression of the HER2 protein is observed in 25 - 30% of primary breast cancers. is a genetically engineered chimeric murine / human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both of these antibodies are recombinantly produced in CHO cells.

[0031] Despite the above-described progress in mammalian cancer therapies, there remains an urgent need for therapeutic methods that effectively inhibit tumor and neoplastic cell growth.

[0032] VI. Regarding the prevention and treatment of fat metabolism-related diseases:

[0033] Obesity is not only a cosmetic problem; it is also an important risk factor for lifestyle-related diseases such as diabetes, fatty liver, hyperlipidemia, and hypertension. As methods for treating and preventing obesity, dietary therapy, exercise therapy, drug therapy, etc. have been proposed or implemented. However, all of these treatments bring side effects such as malnutrition and motor dysfunction, as well as physical and mental distress such as hunger and stress, making it difficult to maintain the therapeutic effect and even damaging health.

[0034] To prevent obesity, many attempts have been made not only through medical treatments and prevention but also through daily food intake. For example, materials aimed at promoting fat metabolism and burning and inhibiting the accumulation of body fat include algal extracts, fruit polyphenols, conjugated polyunsaturated fatty acids, and their mixtures. Specific amino acids and xanthine derivatives, phospholipids from soybeans and egg yolks, diterpene compounds, etc. have been proposed.

[0035] However, even when used alone, their anti-obesity or fat metabolism effects may actually be small, or when used in large amounts in a normal diet, they may not be effective and cannot exhibit a satisfactory obesity prevention effect or fat metabolism improvement effect.

[0036] In summary, there is still a great lack in the market of a pharmaceutical composition that can effectively reduce local fat, reduce body weight, treat obesity, prevent or treat skin color fading and reduce melanin deposition, reduce skin wrinkles or reduce skin roughness, prevent or treat tumors, prevent or treat fat metabolism-related diseases, alleviate arthritis symptoms, and has low side effects and good safety.

Summary of the Invention

[0037] The present invention provides a composition and a preparation method thereof for reducing local fat, reducing body weight, treating obesity, preventing or treating skin color fading and reducing melanin deposition, reducing skin wrinkles or reducing skin roughness, preventing or treating tumors, preventing or treating fat metabolism-related diseases, and alleviating arthritis symptoms. The composition contains at least one active agent and liposome, and the liposome is used as a carrier for the active agent to coat the active agent to form a liposome complex.

[0038] Wherein, the composition may further contain at least one PEGylated component.

[0039] The present invention further provides a method for reducing local fat, losing weight, treating obesity, preventing and treating lightening of skin color and melanin precipitation, reducing skin wrinkles or reducing skin roughness, preventing and treating tumors, preventing and treating fat metabolism-related diseases, and / or alleviating arthritis symptoms. The method comprises administering to an individual in need of treatment an effective dose of a composition comprising (a) an active agent (such as a resveratrol compound, a curcumin compound, or a combination thereof); and (b) a pharmaceutically acceptable carrier. In some embodiments, the resveratrol compound may be resveratrol. In other examples, the curcumin compound may be curcumin.

[0040] The pharmaceutically acceptable carrier may be a pharmaceutically acceptable non-ionic surfactant that is either polyethylene glycolated or non-polyethylene glycolated (such as polysorbate 80 (Tween 80), polyoxyl 15 hydroxystearate (also known as polyethylene glycol 15 hydroxystearate (Solutol HS15)), a polyoxyethylene castor oil derivative, or a combination thereof). Some preferred non-ionic surfactants have a hydrophilic-lipophilic balance value (HLB value) greater than 10. In some embodiments, the weight ratio of the active agent to the non-ionic surfactant may be from 1:5 to 1:500. The pharmaceutical composition formed by the active ingredient and the polyethylene glycolated non-ionic surfactant may be in the form of a liposome complex.

[0041] In other embodiments, the pharmaceutical composition may further comprise additional liposomes formed from a polyethylene glycolated or non-polyethylene glycolated nonionic surfactant (such as those described above), and one or more hydrophilic therapeutic agents (such as green tea extract, epicatechin, epicatechin gallate, epigallocatechin, gallocatechin gallate, gallocatechin, catechin gallate, catechin, epigallocatechin gallate (EGCG), caffeine, carnitine, L-carnitine, synephrine, chlorogenic acid, and other hydrophilic drugs, or combinations thereof). The weight ratio of the hydrophilic therapeutic agent to the nonionic surfactant in the additional liposomes may also be from 1:5 to 1:500.

[0042] Any liposomes included in the compositions disclosed herein (such as those containing active agents as disclosed herein) may have a diameter of less than 50 nm (e.g., about 1 to about 50 nm, or about 10 - 25 nm), and / or a polydispersity index (PDI) value of less than 0.4. In some embodiments, the compositions may be formulated for parenteral administration, such as for injection, implantation, or transdermal administration. Compositions formulated for injection may be in the form of a powder (e.g., a lyophilized powder), a sterilized suspension, an injectable solution, an injectable emulsion, or an intravenous injection solution. In some embodiments, the composition may be placed in a microneedle device for injection. Alternatively, the composition may be formulated for transdermal administration, such as in the form of an ointment, a lotion, a liniment, a cream, a gel, a dressing, an emulsion, a film, a patch, a poultice, a cataplasm, a topical powder, or a topical solution.

[0043] In some embodiments, any method disclosed herein may be implemented by administering any composition disclosed herein via a parenteral route (e.g., topical application, or local injection). In some embodiments, the composition may be administered to a local site. In some embodiments, the local site may be in the thigh, buttocks, lower limbs, pelvic region, or abdomen, where fat, lipomas, or liposarcomas often occur, and the composition may be administered to an individual once or multiple times.

[0044] The present invention provides a composition comprising an active agent and a liposome; the liposome is used to coat the active agent to jointly form a liposome complex with the active agent

[0045] Wherein, the composition may further comprise at least one polyethylene glycolated component.

[0046] In one embodiment, the active agent comprises at least one of curcumin, curcumin derivatives, curcumin metabolites, resveratrol, oxyresveratrol, resveratrol derivatives, and resveratrol metabolites.

[0047] In one embodiment, the active ingredient is not artificially made.

[0048] In one embodiment, the active ingredient is synthetic.

[0049] In one embodiment, the liposome complex is a cationic liposome complex.

[0050] In one embodiment, the liposome comprises dimyristoyl phosphatidylcholine (DMPC), cholesterol, and DMPE-PEG-200, and the weight ratio of dimyristoyl phosphatidylcholine (DMPC), cholesterol, and DMPE-PEG-200 is 90:10:2 to 90:10:9.

[0051] In one embodiment, the particle size of the liposome complex is 20 - 500 nanometers.

[0052] The present invention further provides a use of a composition for preparing a drug for administration to a local subcutaneous site of an individual to reduce the amount of fat at the local subcutaneous site.

[0053] The present invention further provides a use of a composition for preparing a drug for administration to a local subcutaneous site of an individual to reduce the body weight of the individual.

[0054] The present invention further provides a use of a composition for preparing a drug for administration to a local subcutaneous site of an individual to treat obesity.

[0055] The present invention further provides a use of a composition for preparing a drug for inhibiting melanogenesis or precipitation.

[0056] The present invention further provides a use of a composition for preparing a drug for reducing skin lines or reducing skin roughness.

[0057] The present invention further provides a use of a composition for preparing a drug for alleviating the degree of joint inflammation. In one embodiment, the joint inflammation is arthritis. In one embodiment, the joint inflammation is degenerative arthritis.

[0058] In one embodiment, the dosage form of the drug is a subcutaneous injection or a subcutaneous adipose layer injection, and the dosage of the drug is 0.02 - 20 milligrams of the active ingredient per square centimeter of injection; alternatively, the dosage form of the drug is a subcutaneous injection or a subcutaneous adipose layer injection, and the dosage of the drug is 0.01 - 500 milligrams of the active ingredient per kilogram of body weight. Details of one or more embodiments of the present invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following several embodiments of the drawings and detailed description, as well as the appended claims. Detailed Embodiments

[0059] Disclosed herein are pharmaceutical compositions and their therapeutic uses for reducing local fat, reducing body weight, preventing or treating lightening of skin color and reducing melanin deposition, reducing skin wrinkles or reducing skin roughness, preventing or treating tumors, preventing or treating fat metabolism-related diseases, and alleviating arthritis symptoms. The pharmaceutical compositions disclosed herein may comprise one or more active agents, which may be a curcumin, a curcumin derivative, a curcumin metabolite, a resveratrol, an oxidized resveratrol, a resveratrol derivative, or a resveratrol metabolite, or a combination thereof. The one or more active agents may form a first plurality of liposome complexes with at least one suitable pharmaceutically acceptable non-ionic surfactant, such as those disclosed herein. In some embodiments, the pharmaceutical composition may further comprise a second plurality of liposome complexes. In some embodiments, the pharmaceutical composition may further comprise a third plurality of liposome complexes. Among them, at least one or a combination of the curcumin, the curcumin derivative, and the curcumin metabolite is included in the first liposome complexes; at least one or a combination of the resveratrol, the oxidized resveratrol, the resveratrol derivative, and the resveratrol metabolite is included in the second liposome complexes; and the third liposome complexes include (1) at least one or a combination of the curcumin, the curcumin derivative, and the curcumin metabolite, and (2) at least one or a combination of the resveratrol, the oxidized resveratrol, the resveratrol derivative, and the resveratrol metabolite.

[0060] In some embodiments, the pharmaceutical composition may be formed by a suitable pharmaceutically acceptable non-ionic surfactant, an active agent, and a hydrophilic agent as disclosed herein.

[0061] Pharmaceutical Composition

[0062] The pharmaceutical composition disclosed herein comprises one or more of curcumin, a curcumin derivative, a curcumin metabolite, resveratrol, oxyresveratrol, a resveratrol derivative, or a resveratrol metabolite as an active agent, for reducing local fat, reducing body weight, preventing or treating lightening of skin color and reducing melanin deposition, reducing skin wrinkles or reducing skin roughness, preventing or treating tumors, preventing or treating fat metabolism-related diseases, and alleviating arthritis symptoms. The active agent as disclosed herein can form a first liposome complex, a second liposome complex, or / and a third liposome complex with suitable nonionic surfactants such as those disclosed herein.

[0063] (A) Active ingredient

[0064] Exemplary active agents provided herein include resveratrol compounds and curcumin compounds. Resveratrol compounds may have the structure of formula (I) or a salt thereof:

[0065]

[0066] wherein R1, R2, R3, and R4 are each independently H, a halogen (such as F, Cl, or Br), a hydroxyl group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, a thiol group, or an amine. In some embodiments, the resveratrol compound may have the structure of formula (Ia) or a salt thereof:

[0067]

[0068] wherein R 1 , R 2 and R 3 are each as defined above. In one example, the resveratrol compound is resveratrol (with R 1 –R 3 both being hydroxyl groups and R 4 being -H). Alternatively, as is well known to those of ordinary skill in the art, the resveratrol compound may have suitable substitutions at one or more suitable positions of resveratrol.

[0069] The curcumin compounds as disclosed herein may have the structure of formula (II) or a salt thereof:

[0070]

[0071] wherein R 1 , R 2 , R 3 and R 4Each is independently H, a halogen group (such as F, Cl, or Br), a hydroxyl group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, a thiol group, or an amine; Z is CH2, NH, O, or S; and Z' is CH2, NH, O, or S. In some embodiments, the curcumin compound is a curcuminoid, such as curcumin, demethoxycurcumin, or bisdemethoxycurcumin. In a particular embodiment, the curcumin compound is curcumin. Alternatively, as is well known to those of ordinary skill in the art, the curcumin compound may have suitable substitutions at one or more suitable positions of curcumin.

[0072] In some embodiments, the curcumin compound may have the structure of formula (IIa) or a salt thereof:

[0074]

[0075] Wherein R 1 , R 2 , R 3 and R 4 Each is independently H, a halogen group (such as F, Cl, or Br), a hydroxyl group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, a thiol group, or an amine; X is CH2, NH, O, or S; and Y is methyl (CH3), amino (NH2), hydroxyl (OH), or thiol (SH). In some embodiments, the curcumin compound is a curcuminoid, such as curcumin, demethoxycurcumin, or bisdemethoxycurcumin. In a particular embodiment, the curcumin compound is curcumin. Alternatively, as is well known to those of ordinary skill in the art, the curcumin compound may have suitable substitutions at one or more suitable positions of curcumin.

[0076] "Alkyl" refers to a straight-chain, saturated, acyclic, monovalent hydrocarbon radical, or a branched-chain, saturated, acyclic, monovalent hydrocarbon radical having one to three carbon atoms attached to the remainder of the molecule by a single bond, such as methyl, ethyl, n-propyl, or 1-methylethyl (isopropyl). Optionally substituted alkyl is alkyl that may be optionally substituted, where valence permits, by one, two, three, four, or five substituents independently selected from the group consisting of halogen, cyano, nitro, oxo, hydroxyl, thio, or amino.

[0077] "Alkenyl" refers to a straight-chain, acyclic, monovalent hydrocarbon group, or a branched-chain, acyclic, monovalent hydrocarbon group, which contains a carbon-carbon double bond and has two or three carbon atoms attached to the remainder of the molecule by a single bond, such as vinyl or propenyl. Optionally substituted alkenyl is an alkenyl that may optionally be substituted by one, two, or three substituents independently selected from the group consisting of halo, cyano, nitro, hydroxy, thio, or amino groups, provided that the substitution is permitted by the valence.

[0078] "Alkynyl" refers to a straight-chain, acyclic, monovalent hydrocarbon group, or a branched-chain, acyclic, monovalent hydrocarbon group, which contains a triple bond and has two or three carbon atoms attached to the remainder of the molecule by a single bond, such as ethynyl or propynyl. Optionally substituted alkynyl is an alkynyl that may optionally be substituted by a substituent selected from the group consisting of halo, cyano, nitro, hydroxy, thio, or amino groups.

[0079] "Alkoxy" refers to a group of the formula -ORa, where Ra is hydrogen or an alkyl group containing one to three carbon atoms as defined above. The alkyl portion of the optionally substituted alkoxy is optionally substituted as defined above for alkyl.

[0080] "Amino" refers to a group of the formula -NRbRc, where Rb and Rc are each hydrogen or an alkyl group containing one to three carbon atoms as defined above. The alkyl portion of the optionally substituted amino is optionally substituted as defined above for alkyl.

[0081] "Mercapto" refers to a group of the formula -SRd, where Rd is hydrogen or an alkyl group containing one to three carbon atoms as defined above. The alkyl portion of the optionally substituted mercapto is optionally substituted as defined above for alkyl.

[0082] As used herein, "hydrophilic" refers to one or more molecules having a hydrophilic-lipophilic balance greater than 10, for example, as determined using the Griffin method.

[0083] (B) Hydrophilic therapeutic agent

[0084] In some embodiments, the pharmaceutical compositions disclosed herein may further comprise one or more hydrophilic therapeutic agents as described herein, which may enhance the efficacy of the active agent and / or reduce the side effects associated with the active agent. Hydrophilic therapeutic agents are agents that are soluble in water. Exemplary hydrophilic therapeutic agents include, but are not limited to, green tea extract, flavonoids, including those based on catechins (e.g., catechin, epicatechin gallate), gallocatechins (e.g., gallocatechin, gallocatechin gallate), epicatechins (e.g., epicatechin, epicatechin gallate), epigallocatechins (e.g., epigallocatechin, epigallocatechin gallate), xanthines, including methylxanthines (e.g., caffeine), carnitine, L-carnitine, synephrine, and chlorogenic acid. Combinations of the foregoing agents may also serve as the hydrophilic therapeutic agents used herein. Alternatively, other hydrophilic therapeutic agents may be used in combination with the present technology to assist with the various effects of the present invention.

[0085] In some embodiments, the weight ratio of the active ingredient to the hydrophilic therapeutic agent ranges from 30:1 to 1:10, for example: from 30:1 to 1:5, from 30:1 to 1:1, from 30:1 to 5:1, from 30:1 to 10:1, from 30:1 to 20:1, from 20:1 to 1:10, from 10:1 to 1:10, from 5:1 to 1:10, from 1:1 to 1:10, from 1:5 to 1:10, from 20:1 to 1:5, from 10:1 to 1:1, or from 5:1 to 1:1.

[0086] (C) Nonionic surfactants

[0087] The pharmaceutical compositions disclosed herein may comprise one or more nonionic surfactants, which may form liposome complexes with the active agent and / or hydrophilic therapeutic agents as disclosed herein. For example, the nonionic surfactant is a PEGylated nonionic surfactant, and the PEGylated nonionic surfactant forms liposome complexes with the active agent and / or hydrophilic therapeutic agents as disclosed herein.

[0088] Alternatively, the pharmaceutical composition disclosed herein comprises one or more PEGylated ingredients, and the PEGylated ingredient forms a liposome complex with the active agent and / or hydrophilic therapeutic agent as disclosed herein.

[0089] The nonionic surfactant used in the present technology at least facilitates the formation of liposomes in the composition. In some embodiments, the liposomes have the function of encapsulating the active ingredient and / or hydrophilic therapeutic agent.

[0090] The active ingredient disclosed in the present invention may comprise the following ingredients or a combination thereof: a resveratrol compound, a curcumin compound, or a combination thereof. In some examples, the resveratrol compound may be resveratrol or oxyresveratrol. In other instances, the curcumin compound may be curcumin. In short, the active ingredient may comprise one or a combination of curcumin, curcumin derivatives, curcumin metabolites, resveratrol, oxyresveratrol, resveratrol derivatives, and resveratrol metabolites.

[0091] The nonionic surfactant used in any of the pharmaceutical compositions disclosed herein preferably has a hydrophilic-lipophilic balance (HLB) value greater than 10. The nonionic surfactant is used in the present technology in the above proportions. Exemplary nonionic surfactants include but are not limited to polysorbate 80 (also known as Tween 80 80), polyoxyl 15 hydroxystearate (also known as polyethylene glycol 15 hydroxystearate (solutol HS15)), polyoxyethylene castor oil derivatives (such as polyoxyl 35 castor oil (also known as polyethylene glycol 35 castor oil) ELP), polyoxyl 40 hydrogenated castor oil (also known as polyethylene glycol 40 hydrogenated castor oil) RH40), and polyoxyl 60 hydrogenated castor oil (also known as polyethylene glycol 60 hydrogenated castor oil)( RH60), polyoxyethylene (12) glyceryl laurate (also known as polyethylene glycol (12) glyceryl laurate) (UNIGLY ML-212), polyoxyl 20 stearate (also known as polyethylene glycol 20 stearate) (Myrj TM S20)), polyoxyl 40 stearate (also known as polyethylene glycol 40 stearate) (Myrj TM S40)), polyoxyl 12 cetostearyl ether (also known as polyethylene glycol 12 cetostearyl ether)( CS12)), and polyoxyl 20 cetostearyl ether (also known as polyethylene glycol 20 cetostearyl ether)( CS20)).

[0092] In some embodiments, the nonionic surfactant can be a polyoxyethylene derivative, also known as a Pegylated excipient. In some embodiments, the polyoxyethylene derivative is a polyethylene glycol castor oil derivative, which is a material obtained by reacting different amounts of ethylene oxide with castor oil or hydrogenated castor oil. Examples include polyoxyethylene-35 castor oil (PEG-35 castor oil), and polyoxyethylene-40 hydrogenated castor oil (PEG-40 hydrogenated castor oil). In other embodiments, the polyoxyethylene derivative is a polyethylene glycol ester, which can be prepared by reacting polyethylene glycol with a fatty acid. Examples include polyoxyethylene 40 stearate (PEG-40 stearate), and polyoxyethylene-15 hydroxystearate (PEG-15 hydroxystearate). In some embodiments, the ester can be a sorbitan fatty acid ester (e.g., polyoxyethylene-20 sorbitan monooleate (PEG-20 sorbitan monooleate), polyoxyethylene-40 sorbitan monooleate (PEG-40 sorbitan monooleate), polyoxyethylene-60 sorbitan monooleate (PEG-60 sorbitan monooleate), polyoxyethylene-80 sorbitan monooleate (PEG-80 sorbitan monooleate), polyoxyethylene-20 sorbitan isostearate (PEG-20 sorbitan isostearate), polyoxyethylene-30 sorbitan tetraoleate (PEG-30 sorbitan tetraoleate), polyoxyethylene-40 / -60 sorbitan tetraoleate (PEG-40,-60 sorbitan tetraoleate), polyoxyethylene-40 sorbitan diisostearate (PEG-40 sorbitan diisostearate), or polyoxyethylene-60 sorbitan tetrastearate (PEG-60 sorbitan tetrastearate)), an alkyl glyceride (e.g., polyoxyethylene-8 caprylic / capric glycerides (PEG-8 caprylic / capric glycerides), polyoxyethylene-32 hydrogenated palm glycerides (PEG-32 hydrogenated palm glycerides), or polyoxyethylene-32 lauroyl glyceride (PEG-32 lauroylglycerides)), or an alkyl ether (such as polyoxyethylene-6 cetostearyl ether (PEG-6 cetostearyl ether), polyoxyethylene-12 cetostearyl ether (PEG-12 cetostearyl ether), polyoxyethylene-20 cetostearyl ether (PEG-20 cetostearyl ether), polyoxyethylene-10 cetyl ether (PEG-10 cetyl ether), polyoxyethylene-20 cetyl ether (PEG-20 cetyl ether), polyoxyethylene-4 lauryl ether (PEG-4 lauryl ether), polyoxyethylene-23 lauryl ether (PEG-23 lauryl ether), polyoxyethylene-2 oleyl ether (PEG-2 oleyl ether), polyoxyethylene-10 oleyl ether (PEG-10 oleyl ether), polyoxyethylene-20 oleyl ether (PEG-20 oleyl ether), polyoxyethylene-2 stearyl ether (PEG-2 stearyl ether), polyoxyethylene-10 stearyl ether (PEG-10 stearyl ether), polyoxyethylene-21 stearyl ether (PEG-21 stearyl ether), or polyoxyethylene-100 stearylether)). In some other embodiments, the ester can be a monolaurate (e.g., polyethylene glycol-2 laurate (PEG-2 laurate), polyethylene glycol-4 laurate (PEG-4 laurate), polyethylene glycol-6 laurate (PEG-6 laurate), polyethylene glycol-8 laurate (PEG-8 laurate), polyethylene glycol-9-14 laurate (PEG-9-14 laurate), polyethylene glycol-20 laurate (PEG-20 laurate), polyethylene glycol-32-150 laurate (PEG-32-150 laurate), or polyethylene glycol-12 glyceryl laurate (PEG-12 glyceryl laurate)), a dilaurate (e.g., polyethylene glycol-2 dilaurate (PEG-2 dilaurate), polyethylene glycol-4 dilaurate (PEG-4 dilaurate), or polyethylene glycol-6-150 dilaurate (PEG-6-150 dilaurate)), or a monostearate (e.g., polyethylene glycol-2 stearate (PEG-2 stearate), polyethylene glycol-3 stearate (PEG-3 stearate), polyethylene glycol-4 stearate (PEG-4 stearate), polyethylene glycol-4 isostearate (PEG-4 isostearate), polyethylene glycol-5-7 stearate (PEG-5-7 stearate), polyethylene glycol-6-8 isostearate (PEG-6-8 isostearate), polyethylene glycol-8 stearate (PEG-8 stearate), polyethylene glycol-9 stearate (PEG-9 stearate), polyethylene glycol-10 stearate (PEG-10 stearate), polyethylene glycol-10 isostearate (PEG-10-isostearate), polyethylene glycol-12 isostearate (PEG-12 isostearate), polyethylene glycol-12-18 stearate (PEG-12-18 stearate), polyethylene glycol-20 stearate (PEG-20 stearate), polyethylene glycol-23-45 stearate (PEG-23-45 stearate), polyethylene glycol-40 stearate (PEG-40 stearate), polyethylene glycol-50 stearate (PEG-50 stearate), polyethylene glycol-100 stearate (PEG-100 stearate), polyethylene glycol-75-150 stearate (PEG-75-150 stearate), or polyethylene glycol-6 palmitostearate and polyethylene glycol-32 palmitostearate (PEG-6 andPEG-32 palmitostearate), glyceryl stearate (such as glyceryl stearate / PEG-40 stearate, glyceryl stearate / PEG-100 stearate, PEG-120 glyceryl stearate, PEG-20 methyl glucose sesquistearate, or PEG-25 propylene glycol stearate), distearate (such as PEG-2 distearate, PEG-3-120 distearate, PEG-150 distearate, or PEG-175 distearate), hydroxystearate (such as PEG-15 hydroxystearates).

[0093] In some embodiments, the non-ionic surfactant can be a castor oil derivative (such as PEG-35 castor oil or PEG-40 castor oil), or a hydrogenated castor oil (such as PEG-40 hydrogenated castor oil, PEG-54 hydrogenated castor oil, or PEG-60 hydrogenated castor oil).

[0094] Other embodiments of the non-ionic surfactant include PEG-15 cocamine, vitamin E polyethylene glycol succinate, PEG-75 lanolin, and PEG-120 methylglucose dioleate.

[0095] In some embodiments, the nonionic surfactant is further subjected to a polyethylene glycolylation process to become a polyethylene glycolylated nonionic surfactant, which together with the active ingredient forms the liposome complex.

[0096] (D) Liposome and its preparation

[0097] Liposomes are a type of targeted drug carrier and a new dosage form belonging to the targeting drug delivery system, with advantages such as targeting, stability, long-acting properties, and reduced drug toxicity. Liposomes are also the most commonly studied nanocarriers in targeted drug delivery systems. Liposomes are spherical lipid vesicles (usually with a particle size of 50 - 500 nanometers, and a more general diameter range of 20 - 500 nanometers), composed of one or more lipid bilayers, which is the result of emulsifying natural or synthetic lipids in an aqueous medium. The structure of liposomes can be classified into four categories according to size and the number of bilayers: small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), multilamellar vesicles (MLV), and multivesicular vesicles (MVV).

[0098] The encapsulation efficiency of liposomes increases with the increase in liposome size and decreases with the increase in the number of bilayers, but this is only limited to hydrophilic compounds. The size of the vesicles is an important factor controlling the circulation half-life of liposomes. Both the size and the number of bilayers of liposomes affect the amount of drug encapsulated. When liposomes are used for drug administration, the required vesicle size is usually from 50 nanometers to 150 nanometers.

[0099] Structurally, liposomes are spherical or multi-layered spherical vesicles formed by the self-assembly of diacyl chain phospholipids (lipid bilayers) in an aqueous solution. The lipid bilayer membrane has hydrophobic tails and hydrophilic heads, which results in the formation of an amphiphilic structure. Liposomes can be made from natural and synthetic phospholipids. The lipid composition strongly affects the properties of liposomes, including: particle size, rigidity, fluidity, stability, and charge. For example, liposomes made from natural unsaturated phosphatidylcholine, such as egg or soy phosphatidylcholine, exhibit characteristics of high permeability and low stability. However, liposomes based on saturated phospholipids such as dipalmitoyl phosphatidylcholine result in a rigid and almost impermeable bilayer structure. The hydrophilic groups in lipids can carry a negative charge, a positive charge, or an amphoteric charge (both negative and positive charges in the same molecule). The charge of the hydrophilic group provides stability through electrostatic repulsion. The hydrophobic groups in lipids vary in acyl chain length, symmetry, and saturation.

[0100] According to the composition and application of liposomes, they can be classified into conventional liposomes, charged liposomes, stealth stable liposomes, actively targeted liposomes, stimulus-responsive liposomes, and bubble liposomes.

[0101] Conventional liposomes are the first-generation liposomes made from natural or synthetic phospholipids with / without cholesterol. Cholesterol is added to improve the fluidity of liposomes, change the rigidity of the bilayer, and the stability of liposomes. Due to the easy elimination by the mononuclear phagocyte system (MPS), conventional liposomes show a short blood circulation time and rapidly accumulate in the liver and spleen. Therefore, MPS hinders the delivery of conventional liposomes to the target area and limits their distribution to other tissues of the body. Conventional liposomes also show relatively limited stability in vitro. Therefore, stealth stable liposomes were invented to increase blood circulation and enhance the stability of liposomes in vivo.

[0102] Charged liposomes show higher liposome stability during storage because charged particles repel each other, reducing the aggregation ability. Cationic liposomes are used in gene therapy because they can successfully encapsulate nucleic acids through electrostatic attraction. Cationic liposomes are suitable for delivering various negatively charged macromolecules such as DNA, RNA, and oligonucleotides because their negative charge and relatively large size limit their passive diffusion into cells. Cationic liposomes can also selectively target angiogenic endothelial cells in tumors. Cationic liposomes are considered potential tools for delivering therapeutic agents to the brain. Cationic liposomes can cross the blood-brain barrier through receptor-mediated transcytosis or absorptive-mediated transcytosis. The relatively high positive charge on the surface of cationic liposomes may affect their blood circulation and lead to increased liposome aggregation due to electrostatic interactions with anionic species in the blood, reducing their localization at the site of action. Decorating the surface of these liposomes with polyethylene glycol (PEG) can protect them from circulating proteins, improving the systemic circulation time and reducing immunogenicity to enhance drug efficiency.

[0103] Stealth liposomes are characterized by the surface decorated with synthetic polymers, glycoproteins, polysaccharides, or specific receptor ligands to achieve a reduced distribution range and accumulation at a predetermined site. Hyaluronic acid, polyvinyl alcohol (PVA), and polyethylene glycol (PEG) are considered the best models for liposome steric protection. And pegylated liposomes are called stealth liposomes. Stealth stable liposomes show a longer circulation time, resulting in better targeted accumulation than conventional liposome drugs.

[0104] Active targeting liposomes represent the third generation of liposomes. The active targeting of liposomes increases the selective interaction of liposomes with diseased cells and triggers receptor-mediated endocytosis to deliver liposomes and their payloads to the desired cellular targets. Liposome targeting can be enhanced by conjugating molecular recognition moieties, which may result in better efficiency and lower side effects in drug delivery. For example, liposome targeting strategies utilize simple peptides, proteins (including antibodies) or protein fragments, carbohydrates, nucleic acids, or vitamins. Active targeting of nanocarriers can be achieved by non-covalently or covalently conjugating targeting ligands to drug molecules or to the surface of nanocarriers to selectively bind to overexpressed target biomarkers on tumor cells. Direct conjugation of drugs to targeting ligands may disrupt receptor / ligand recognition and may alter the potency of the drug. Active targeting of nanocarriers enables drugs to be more efficiently localized at the site of action to reduce drug dosage, reduce drug side effects, and reduce fluctuations in drug concentration in the blood. Stealth and conventional liposomes typically show slow release of the loaded drug and fail to fuse with endosomes after endocytosis. Therefore, stimuli-responsive liposomes have been introduced to overcome these challenges.

[0105] Stimuli-responsive liposomes are intelligent liposome systems that can rapidly release their drug payloads under physicochemical or biochemical stimuli such as pH, temperature, redox potential, enzyme concentration, ultrasound, electric field, or magnetic field. Stimuli-responsive liposomes should contain some component to control the stability and permeability of the lipid bilayer. There are two basic modes of activation for stimuli-responsive liposomes: remote and local. Remote activation responds to external stimuli, including heat, magnetic field, light, electric field, and ultrasound. Locally triggered release responds to stimuli within the target tissue, such as pH, redox potential, and enzymes. These triggering factors result in a nanosystem for controlled release that enhances intracellular distribution.

[0106] Gas-filled liposomes (liposomes encapsulating gas) are promising for creating new applications in the fields of gene delivery and drug delivery systems. Recently, liposomes have been used to encapsulate bioactive gases and / or drugs to achieve ultrasound-controlled drug release and enhance drug delivery. For example, nitric oxide (NO) gas-filled liposomes provide an alternative intravenous therapy distinct from common microbubbles, enabling liposomes to protect nitric oxide from scavenging by hemoglobin in vitro. Oxygen gas-filled liposomes (OBL) enable high oxygen concentrations and high pO2 conditions in the lungs, which distinguishes OBL from a large number of perfluorocarbons and hemoglobin-based oxygen carriers and maintains their use as a supportive oxygen transport platform.

[0107] Liposomes can be formulated by different methods. The liposome manufacturing process and phospholipid type have a crucial impact on the properties of the final liposomes. The manufacturing procedures of liposomes can be classified as follows.

[0108] The first manufacturing process: Thin-film hydration method (Bangham method). In this method, all lipids and lipophilic drugs are dissolved in a suitable organic solvent, using a round-bottom flask, and then the organic solvent is gently evaporated under reduced pressure to form a thin film. The resulting thin film is then hydrated with an aqueous buffer solution above the transition temperature (Tm) of the lipids used. The hydration solution may contain one or more hydrophilic drugs to be loaded into the aqueous core of the liposome. The hydration rate determines the drug encapsulation efficiency, and the slower the hydration rate, the higher the encapsulation efficiency. By extrusion through a polycarbonate membrane with a specific pore size or using a bath-type or probe-type ultrasonic device, the reformation, lamellar type, and particle distribution of the liposomes can be controlled. The extrusion method ensures more stable liposomes and higher encapsulation efficiency than the ultrasonic method. The ultrasonic method usually produces SUVs liposomes and may also degrade or hydrolyze the encapsulated drugs and / or lipids. Probe-type ultrasonication may subject the liposome suspension to potential metal contamination.

[0109] The second manufacturing process: Reverse-phase evaporation method. The reverse-phase evaporation method is usually used as an alternative to the thin-film hydration method and is carried out by forming a water-in-oil emulsion. First, the lipids are dissolved in an organic solvent and then directly mixed with an aqueous buffer containing hydrophilic drugs. Subsequently, the organic solvent is evaporated under reduced pressure on a rotary evaporator, thereby forming lipid vesicles dispersed in an aqueous solution. The average size and polymorphism of the preformed vesicles can be reduced by extrusion.

[0110] The third manufacturing process: Solvent injection method. The organic solvent dissolving the lipids and hydrophobic active ingredients is rapidly injected into the aqueous phase. Using ether, the solvent can be directly evaporated during the mixing process at a temperature higher than the boiling point of the solvent used. Using ethanol for injection requires 10 to 20 times the aqueous solution, and the ethanol can be evaporated using a rotary evaporator, dialysis, or filtration under vacuum. This method mostly prepares liposome formulations with a higher polydispersity index (PDI). In addition, continuous exposure to high temperatures and organic solvents may reduce the stability of the drugs and lipids.

[0111] The fourth manufacturing process: Detergent removal method. Lipids and surfactants with a high critical micelle concentration (CMC) are dissolved in a suitable organic solvent and placed in a round-bottom flask. After gently evaporating the solvent, a thin film forms at the bottom of the flask. Then, a solution of mixed micelles is obtained by hydrating the lipid film in an aqueous solution containing drug molecules. Subsequently, the surfactant is removed by dialysis, size exclusion chromatography, adsorption on hydrophobic beads, or dilution. Finally, LUVs liposome vesicles are formed after concentrating the solution. The main disadvantage of the detergent removal method is that most hydrophilic drugs will separate from the liposomes during the detergent removal step.

[0112] The fifth manufacturing process: dehydration-rehydration method. This is a method for generating LUVs using ultrasound without the need for organic solvents. The dehydration-rehydration method is based on directly dispersing lipids at a low concentration into an aqueous solution containing drug molecules and then subjecting them to sonication. First, a dehydration step is carried out, where nitrogen is used to evaporate the water to form a multi-layer film that entraps the drug molecules. Then, a rehydration step is performed to form large vesicles encapsulating the drug molecules. Although the dehydration-rehydration method is simple, it has a relatively high heterogeneity in liposome size.

[0113] The sixth manufacturing process: heating method. This is also a technique without the need for organic solvents. In the heating method, lipids are directly hydrated with an aqueous solution and heated to a temperature not lower than the transition temperature (Tm) of the phospholipids used for more than one hour in the presence of 3-5% hydrating agent (such as glycerol or propylene glycol). When cholesterol is added to the formulation, the suspension can be heated to 100 °C. The hydrating agent acts as a stabilizer and isotonic additive, preventing nanoparticle aggregation and precipitation. In addition, the hydrating agent provides a cryoprotective effect, making the heating method an effective way to prepare inhalable liposome powder.

[0114] The seventh manufacturing process: pH-jump method, which is a solvent-free method for liposome preparation. In the pH-jump method, an aqueous solution of phosphatidic acid and phosphatidylcholine undergoes an almost four-fold increase in pH value within a short period to break down multilamellar vesicles (MLVs) into small unilamellar vesicles (SUVs). The ratio of phosphatidic acid to phosphatidylcholine determines the percentage of SUVs and LUVs produced.

[0100] The eighth manufacturing process: microfluidic channel method. Microfluidic technology provides tools for manipulating liquids within microchannels. In the microfluidic channel method, lipids are dissolved in ethanol or isopropanol, and the resulting solution is then injected into an aqueous medium vertically or in the opposite direction within the microchannel. The microfluidic channel method involves continuous axial mixing of organic and aqueous solutions, resulting in liposome formation. Surfactants are used to stabilize the liposomes to avoid aggregation and separation. The microfluidic channel method controls the mixing process of the organic and aqueous phases to achieve reproducible liposomes with appropriate average size, polymorphism, morphology, and lamellarity.

[0101] The ninth manufacturing process: supercritical fluid method. The supercritical fluid method utilizes a supercritical fluid to dissolve lipids, namely carbon dioxide (CO2), instead of using organic solvents. A high-performance liquid pump provides a continuous inflow of the aqueous phase into a cell containing a supercritical lipid solution, allowing the dissolved phospholipids to undergo a phase change. After a sudden decrease in pressure, liposomes are formed after completely removing carbon dioxide. The supercritical fluid method can achieve an encapsulation efficiency five times higher than usual. However, the disadvantages are high cost and low yield, and even when using environmentally friendly and inexpensive carbon dioxide, special infrastructure is required.

[0102] In some embodiments, the method for preparing cationic liposome curcumin comprises the following steps: Weigh precisely 3.0 grams of egg yolk lecithin, 1.0 gram of cholesterol, 0.2 gram of octadecylamine, and 0.3 gram of vitamin E into a pear-shaped flask, add 10 milliliters of ether and shake, then add 5 milliliters of anhydrous ethanol solution containing 1 milligram of curcumin, evaporate to dryness and form a film at 35 degrees Celsius on a rotary evaporator, then add 20 milliliters of phosphate buffer solution with a pH of 6.6, rotate for twenty minutes to dissolve the film, let it stand for three hours to fully hydrate, perform ultrasonic treatment for three minutes, and filter through a 0.22-μm filter membrane to prepare cationic liposome curcumin. The method for preparing cationic liposome resveratrol is similar to the method for preparing cationic liposome curcumin.

[0103] In some embodiments, the method for preparing cationic liposome curcumin comprises the following steps: Weigh precisely 3.0 grams of egg yolk lecithin, 1.0 gram of cholesterol, 0.05 gram of [2-[[4-[(carboxymethyl)dithio]-1-iminobutyl]amino]ethyl]carbamate cholesterol (CHETA), and 0.3 gram of vitamin E into a pear-shaped flask, add 10 milliliters of ether and shake, then add 5 milliliters of anhydrous ethanol solution containing 1 milligram of curcumin, evaporate to dryness and form a film at 35 degrees Celsius on a rotary evaporator, then add 20 milliliters of phosphate buffer solution with a pH of 6.6, rotate for twenty minutes to dissolve the film, let it stand for three hours to fully hydrate, perform ultrasonic treatment for three minutes, and filter through a 0.22-μm filter membrane to prepare cationic liposome curcumin. The method for preparing cationic liposome resveratrol is similar to the method for preparing cationic liposome curcumin.

[0104] The liposome of the present invention encapsulates an active ingredient to jointly form a liposome complex.

[0105] In some embodiments, the liposome comprises a nonionic surfactant and an active agent in a suitable weight ratio, thereby having a suitable particle size and / or a suitable polydispersity index (PDI) value. For example, the suitable weight ratio between the active agent and the nonionic surfactant is in the range of about 1:5 to about 1:500, such as: between about 1:5 and 1:8, between about 1:5 and 1:10, between about 1:5 and 1:20, between about 1:5 and 1:40, between about 1:5 and 1:100, between 1:5 and 1:150, between 1:5 and 1:200, between 1:5 and 1:300, between 1:5 and 1:400, between 1:5 and 1:500, between about 1:8 and 1:10, between about 1:8 and 1:20, between about 1:8 and 1:40, between about 1:8 and 1:100, between 1:8 and 1:150, between 1:8 and 1:200, between 1:8 and 1:300, between 1:8 and 1:400, between 1:8 and 1:500, between about 1:10 and 1:20, between about 1:10 and 1:40, between about 1:10 and 1:100, between 1:10 and 1:150, between 1:10 and 1:200, between 1:10 and 1:300, between 1:10 and 1:400, between 1:10 and 1:500, between about 1:20 and 1:40, between about 1:20 and 1:100, between 1:20 and 1:150, between 1:20 and 1:200, between 1:20 and 1:300, between 1:20 and 1:400, between 1:20 and 1:500, between about 1:40 and 1:100, between 1:40 and 1:150, between 1:40 and 1:200, between 1:40 and 1:300, between 1:40 and 1:400, between 1:40 and 1:500, between 1:100 and 1:150, between 1:100 and 1:200, between 1:100 and 1:300, between 1:100 and 1:400, between 1:100 and 1:500, or between 1:150 and 1:500, to form a suitable particle size (e.g., less than 50 nm) and a suitable PDI value (e.g., <0.4).

[0106] The results show that the cationic liposome curcumin of the present invention can greatly reduce the dosage of curcumin, significantly increase its biological effect, improve its therapeutic index, and the aqueous solution preparation of cationic liposome curcumin has high stability. Finally, the cationic liposome curcumin of the present invention can better achieve various effects of this case. The cationic liposome curcumin of the present invention benefits from its high stability and solves the problems of poor water solubility and low stability.

[0107] The results show that the cationic liposome resveratrol of the present invention can greatly reduce the dosage of resveratrol, significantly increase its biological effect, improve its therapeutic index, and the aqueous solution preparation of cationic liposome resveratrol has high stability. Finally, the cationic liposome resveratrol of the present invention can better achieve various effects of this case. The cationic liposome resveratrol of the present invention benefits from its high stability and solves the problems of poor water solubility and low stability.

[0108] In some embodiments, the cationic liposome curcumin drug prepared by the present invention is composed of 0.0001-5% curcumin and 95-100% liposome carrier. In some embodiments, the cationic liposome curcumin drug prepared by the present invention is composed of 0.001-3% curcumin and 97-99% liposome carrier. In some embodiments, the cationic liposome curcumin drug prepared by the present invention is composed of 0.01-1% curcumin and 98-99% liposome carrier. In some embodiments, the cationic liposome curcumin drug prepared by the present invention is composed of 0.004% curcumin and 99.996% liposome carrier.

[0109] In some embodiments, the cationic liposome resveratrol drug prepared by the present invention is composed of 0.0001-5% resveratrol and 95-100% liposome carrier. In some embodiments, the cationic liposome resveratrol drug prepared by the present invention is composed of 0.001-3% resveratrol and 97-99% liposome carrier. In some embodiments, the cationic liposome resveratrol drug prepared by the present invention is composed of 0.01-1% resveratrol and 98-99% liposome carrier. In some embodiments, the cationic liposome resveratrol drug prepared by the present invention is composed of 0.004% resveratrol and 99.996% liposome carrier.

[0110] Additional details for preparing the liposomes of the technology of the present invention can be found in the published patent No. CN 103054802 or No. TW I737974, the relevant disclosure of which is incorporated herein by reference for the subject matter and purposes cited herein.

[0111] In some embodiments, the liposome complexes disclosed in the present invention, such as the first liposome complex, the second liposome complex, the third liposome complex (i.e., liposome curcumin or curcuminoids, liposome resveratrol or resveratroloids, and liposome curcumin or curcuminoids and resveratrol or resveratroloids) can be used to reduce local fat, reduce body weight, prevent and treat skin color fading and melanin deposition, reduce skin wrinkles or skin roughness, prevent and treat tumors, prevent and treat fat metabolism-related diseases or / and alleviate arthritis symptoms, such as 300 mg / m after six hours 2 The dose of the liposome complex therapy is the recommended starting dose for reducing local fat, reducing body weight, preventing and treating skin color fading and melanin deposition, reducing skin wrinkles or skin roughness, preventing and treating tumors, preventing and treating fat metabolism-related diseases or alleviating arthritis symptoms and having good tolerance, or a higher dose such as 325, 350, 375, 400, 425, 450 or 475 mg / m 2 of the liposome complex can be used. In some embodiments, the dose can also include at least two or more treatment cycles of the therapeutically effective amount of the liposome complex of the present invention administered over eight hours or less, where the dose can be 100, 125, 150, 200, 250, 300, 450, 500 or 600 mg / m 2 2 2 . In some embodiments, the liposome comprises at least one of the following: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), DMPC / DMPG liposomes or lysophosphatidylglycerol comprising at least one of the following: lysophosphatidylcholine, lauroyl-lysophosphatidylcholine, myristoyl-lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, stearoyl-lysophosphatidylcholine, arachidoyl-lysophosphatidylcholine, oleoyl-lysophosphatidylcholine, linolenoyl-lysophosphatidylcholine, linoleoyl-lysophosphatidylcholine, or erucoyl-lysophosphatidylcholine. In some embodiments, the liposome complex disclosed in the present invention is administered intravenously to a human in need in a therapeutically effective amount of the liposome complex dose, and the regimen comprises: administering at least two or more treatment cycles of the therapeutically effective amount of the liposome complex of the present invention over eight hours or less, where the dose can be 100, 125, 150, 200, 250, 300, 450, 500 or 600 mg / m 2 2 2. In some embodiments, in the liposome complex, the weight percentage of curcumin is 2-9%. In some embodiments, in the liposome complex, the weight percentage of resveratrol is 2-9%.

[0112] (E) Pharmaceutical composition

[0113] Any active agent, optionally a hydrophilic therapeutic agent, and a non-ionic surfactant that can form a liposome complex with the active agent and optionally the hydrophilic therapeutic agent can be formulated into a pharmaceutical composition for the therapeutic applications disclosed herein.

[0114] The pharmaceutical compositions for the method of the present invention may comprise pharmaceutically acceptable carriers, excipients, or stabilizers in lyophilized formulations or aqueous solution forms. (Remington: The Science and Practice of Pharmacy, 20th Edition (2000), Lippincott Williams and Wilkins, Ed. K. E. Hoover).Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and may include buffering solutions such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as ethylenediaminetetraacetic acid (EDTA); sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counter-ions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as polysorbates (TWEEN). TM ) and pluronics TM) or polyethylene glycol (also known as poly(ethylene glycol); PEG).

[0115] In other embodiments, the pharmaceutical compositions described herein can be formulated in a sustained-release (extended-release) form. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of shaped articles such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0116] Pharmaceutical compositions for in vivo administration must be sterile. This can be readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper that can be pierced by a hypodermic injection needle.

[0117] The pharmaceutical compositions described herein may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation. For example, such pharmaceutical compositions may be formulated in a manner suitable for administration by a suitable route, such as orally, parenterally, topically, rectally, buccally, vaginally, or by an implanted reservoir.

[0118] A sterile injectable composition, such as a sterile aqueous or oily suspension, can be formulated using suitable dispersing, or wetting agents (such as polysorbate 80), and suspending agents according to techniques known in the art. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable carriers and solvents that can be used are mannitol, water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as a solvent or suspending medium (such as synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives, can be used to prepare injectable preparations, and pharmaceutically acceptable natural oils can also be used to prepare injectable preparations, such as olive oil or castor oil, especially in their polyoxyethylated forms. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants, such as Tweens or Spans or other similar emulsifiers or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, can also be used for formulation purposes.

[0119] In some embodiments, the compositions described herein can be formulated as topical formulations, such as creams, emulsions, or gels for topical use (local application). Such creams, emulsions, or gels can be formulated using ingredients known in the art that are suitable for topical medications.

[0120] In the sense of being compatible with the active ingredient of the formulation (and preferably, being able to stabilize the active ingredient) and being harmless to the individual being treated, the carrier in the pharmaceutical composition must be 'acceptable'. For example, solubilizing agents, such as cyclodextrins, which form more soluble complexes with oxadiazole compounds, or more solubilizing agents, can be used as pharmaceutical carriers to deliver oxadiazole compounds. Other examples of carriers include colloidal silicon dioxide, magnesium stearate, sodium lauryl sulfate, and D&C Yellow #10.

[0121] In some embodiments, the pharmaceutical compositions disclosed herein may further comprise antioxidants. Examples include but are not limited to beta-carotene, lutein, lycopene, bilirubin, vitamin A, vitamin C (ascorbic acid), vitamin E, uric acid, nitric oxide, nitroxide, pyruvate, catalase, superoxide dismutase, glutathione peroxidases, N-acetyl cysteine, and naringenin, or combinations thereof.

[0122] The kits of the present invention are in suitable packaging. Suitable packaging includes but is not limited to vials, bottles, jars, flexible packages (such as sealed polyester resin or plastic bags), and the like. Packaging used in conjunction with a particular device is also contemplated, such as an inhaler, a nasal delivery device (such as a nebulizer), or an infusion device, like a minipump. The kit may have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle).

[0123] The kit may optionally provide additional components, such as buffers and explanatory messages, etc. Generally speaking, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the present invention provides an article comprising the contents of the above-described kit. General technology

[0124] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the relevant art. Such techniques are well explained in the literature, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis ed., 1989) Academic Press; Animal Cell Culture (R.I. Freshney ed. 1987); Introuction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths and D.G. Newell eds 1993-8) J.Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell eds); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos eds, 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al. eds 1987); PCR: The Polymerase Chain Reaction, (Mullis et al. eds 1994); Current Protocols in Immunology (J.E. Coligan et al. eds, 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practice approach (D. Catty, ed., IRL Press, 1988–1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover, ed., 1985); Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins, eds., (1985)); Transcription and Translation (B. D. Hames and S. J. Higgins, eds., (1984)); Animal Cell Culture (R. I. Freshney, ed., (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); and B. Perbal, A practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.).

[0125] Without further elaboration, it is believed that those of ordinary skill in the art can make the fullest use of the present invention based on the above description. Therefore, the following specific embodiments should be considered illustrative only and should not limit the remainder of the disclosure in any way. All publications cited herein are hereby incorporated by reference for the purposes or subject matter cited herein. Example 1: Effects of Liposome Complexes on Subcutaneous Fat Mass and Body Weight in Rats

[0126] Prepare an active ingredient physiological saline solution, an active ingredient PEG solution, and an active ingredient liposome solution in the following manner.

[0127] Method for preparing the active ingredient physiological saline solution: Mix the active ingredient with an appropriate amount of physiological saline for injection, stir evenly to completely dissolve the active ingredient, and an active ingredient physiological saline solution can be obtained.

[0128] Preparation method of the active ingredient PEG solution: Mix polyethylene glycol 400 (abbreviated as PEG400), glycerol, and an appropriate amount of physiological saline for injection, stir evenly to completely dissolve PEG400 and glycerol, and obtain a polyethylene glycol and glycerol mixture. Mix the active ingredient with an appropriate amount of the polyethylene glycol and glycerol mixture, stir evenly to completely dissolve the active ingredient, and an active ingredient PEG solution can be obtained.

[0129] Preparation method of the active ingredient liposome: Using the cationic liposome preparation method disclosed in this case, prepare the active ingredient liposome solution of this example from the active ingredient and non-ionic surfactants (dimyristoylphosphatidylcholine (DMPC), cholesterol, and DMPE-PEG-200). Then, confirm the presence and size of the liposomes through a particle size analyzer.

[0130] The active ingredient is curcumin and resveratrol.

[0131] Male Sprague-Dawley rats at 6 weeks of age were used for the experiment. First, 20 rats were fed with a high-fat diet (brand: Research Diets, Inc.; model: #D12492) to induce an increase in subcutaneous fat. After continuous feeding until the body weight of the rats reached 330 ± 10 g, the rats were randomly divided into 4 groups, namely the control group, the physiological saline group, the PEG group, and the liposome group, with 5 rats in each group, so that there was no statistical difference in the body weight of the rats in each group. Record the body weight of each rat, which is defined as the "pre-test body weight" of each rat. Then, the drugs were administered in the following manner.

[0132] Inject the active ingredient physiological saline, the active ingredient PEG solution, and the active ingredient liposome solution into the subcutaneous fat layer of the lower groin of the rats in the physiological saline group, the PEG group, and the liposome group, respectively. The injection volume each time is 4 mL per kilogram of body weight (4 mL / kg), so that the injection dose each time is 20 mg of the active ingredient 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 physiological saline for injection in the same injection manner as above.

[0133] The injection site described above was the subinguinal fat of rats, and the injection was evenly distributed on the left and right sides, with one injection on each of the 1st, 2nd, 3rd, and 4th days of the experiment. During the experiment, the rats were continuously fed a high-fat diet, and the body weight changes were recorded daily, and the water intake and food intake were recorded once a week. The experiment lasted for 14 days, and the rats were sacrificed by carbon dioxide on the 15th day.

[0134] Record the body weight of each rat, which is defined as the "post-experiment body weight" of each rat. Subtract the "pre-experiment body weight" from the "post-experiment body weight" of each rat to obtain the "total weight gain". Divide the total weight gain of the rats in each group by the total weight gain of the control group rats to obtain the "relative total weight gain".

[0135] Weigh the subcutaneous fat of the left and right subinguinal regions of the rats, and add up the subcutaneous fat amounts of the left and right subinguinal regions to calculate the subcutaneous fat amount of the subinguinal region. Divide the subcutaneous fat amount of the subinguinal region of the rats in each group by the subcutaneous fat amount of the subinguinal region of the control group rats to obtain the "relative weight of subcutaneous fat in the subinguinal region".

[0136] The data are presented in the form of mean ± SD, and statistical analysis is performed using one-way analysis of variance (one-way ANOVA). 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).

[0137] The results of the local fat experiment showed that directly injecting the active ingredient into the subcutaneous fat layer of the application site could not reduce the fat (local fat) at the application site. There was no significant difference in the relative weight of subcutaneous fat in the subinguinal region between the rats in the PEG group and the control group rats; there was a significant difference in the relative weight of subcutaneous fat in the subinguinal region between the rats in the liposome group and the control group rats (p < 0.05).

[0138] The results of the weight loss experiment showed that directly applying the active ingredient could not reduce the body weight. There was no significant difference in the body weight between the rats in the PEG group and the control group rats; there was a significant difference in the body weight between the rats in the liposome group and the control group rats (p < 0.05).

[0139] From the above experiments, it can be seen that directly injecting the active ingredient into the subcutaneous fat layer of the application site could not reduce the fat (local fat) at the application site, nor could it reduce the body weight. Injecting the active ingredient composition with the excipient PEG (a commonly used co-solvent) into the subcutaneous fat layer of the application site could not reduce the fat (local fat) at the application site, nor could it reduce the body weight; however, after encapsulating the active ingredient with liposomes and injecting it into the subcutaneous fat layer of the application site, it could not only significantly reduce the fat (local fat) at the application site, but also reduce the body weight. Thus, it can be seen that liposome encapsulation can significantly enhance the effect of the active ingredient in reducing subcutaneous fat (local fat) at the application site and reducing body weight. Example 2: Effect of Liposome Complex on Preventing and Treating Skin Color Fading and Melanin Deposition Example 2-1: Inhibiting Melanin Production Experiment (Inducing Melanin Production First and Then Administering Drugs)

[0140] This example tests the effects of the physiological saline solution, active ingredient PEG solution, and active ingredient liposome solution of Example 1 on the ability to inhibit melanin production. First, using mouse melanoma cells B16-F10, there are a total of five experimental groups, namely the control group (α-MSH), arbutin, physiological saline group, PEG group, and liposome group. Each experiment is repeated three times, and the melanin content of each group is measured.

[0141] Seed 2x10 5 cells into a 6-well plate. After culturing for 24 hours, add 10 ng / ml α-MSH per milliliter and act for 30 minutes. Except for the control group, add 250 ppm arbutin, physiological saline solution with a concentration of 8 ppm, active ingredient PEG solution, or active ingredient liposome solution to each group, and culture for 48 hours. Then centrifuge at 20 °C for 5 minutes to collect the cells, and resuspend the cells in 1N sodium hydroxide (NaOH) (containing 10% DMSO). After mixing evenly and thoroughly, place the sample on a heating plate and heat at 80 °C for 1.5 hours. After the sample cools, use a M2e Multimode Microplate Reader to measure the absorbance value at a wavelength of 475 nm and calculate the inhibition rate of melanin production.

[0142] From the experimental results, it was observed that the active ingredient liposome has a significantly better inhibitory effect on melanin than the physiological saline solution and the active ingredient PEG solution, and its inhibitory effect is also significantly better than arbutin. That is, the active ingredient liposome can effectively reduce melanin production. Example 2-2: Inhibiting Tyrosinase Activity Experiment (Inducing Melanin Production First and Then Administering Drugs)

[0143] This example evaluates and compares the ability of the physiological saline solution, active ingredient PEG solution, and active ingredient liposome solution of Example 1 to inhibit tyrosinase activity by measuring the amount of L-DOPA converted into dopaquinone. This example uses mouse melanoma cells B16-F10. There are a total of five experimental groups, namely the control group (α-MSH), arbutin, physiological saline group, PEG group, and liposome group, and the enzyme activity of tyrosinase in each group is measured.

[0144] Seed 2x105 Cells were seeded in a 6-well plate. After culturing for 24 hours, 10 ng / ml α-MSH was added and the cells were incubated for 30 minutes. Then, except for the control group, arbutin at a concentration of 250 ppm, physiological saline solution at a concentration of 8 ppm, active ingredient PEG solution, or active ingredient liposome solution was added to each group and cultured for 48 hours. After that, the cells were collected with Trypsin-EDTA, washed with PBS, and the tyrosinase protein in the cells was extracted with 0.1 M PBS (pH 7.0) containing 1% Triton X-100 and 0.1 mM phenylmethanesulfonylfluoride (PMSF), and then quantified. After quantification, 30 μg was mixed with 0.4 mg / ml L-DOPA, and the change in absorbance was measured at a wavelength of 405 nm using a microplate analyzer for one hour, and the absorbance was recorded every 10 minutes.

[0145] The results showed that the active ingredient liposome solution had a significantly stronger ability to inhibit tyrosinase activity than the physiological saline solution and the active ingredient PEG solution, and was also significantly better than arbutin. Example 3: Effect of Liposome Complex on Arthritis

[0146] The purpose of this example was to observe the effects of the physiological saline solution, active ingredient PEG solution, and active ingredient liposome solution in Example 1 on inhibiting the inflammatory response under the condition of inducing the inflammatory response first and then administering the drugs. The groups in this example included a DMSO control group, an LPS (lipopolysaccharide) control group, a physiological saline group, a PEG group, and a liposome group.

[0147] The macrophage cell line RAW264.7 was cultured in a 6-cm culture dish at 3x10 5 cells / well. After culturing for 24 hours, the cell culture medium was removed and the cells were washed with PBS. Except for the DMSO control group, the other groups were first induced with 100 ng / ml LPS for 30 minutes to cause an inflammatory response, and then a physiological saline solution at a concentration of 20 ppm, an active ingredient PEG solution, and an active ingredient liposome solution were added to each test group respectively, and each experiment was repeated at least three times. After culturing for six hours, the cell fluid in each group was drained, washed clean with PBS, and then 1 mL TRIzol was added for RNA extraction, and the gene expression level of COX-2 was measured.

[0148] In terms of the results, the active ingredient liposome solution can significantly reduce the gene expression of COX-2, and its anti-inflammatory effect is significantly better than that of the physiological saline solution and the active ingredient PEG solution.

[0149] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature that achieves the same, equivalent or similar purpose. Therefore, unless otherwise clearly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0150] As described above, those of ordinary skill in the art can easily grasp the important features of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and situations. Therefore, other embodiments are also within the scope of the claims. EQUIVALENTS

[0151] Although several embodiments of the invention have been described and illustrated herein, those of ordinary skill in the art will readily conceive of various other methods and / or structures for performing the functions described herein and / or obtaining the results described herein and / or one or more of the advantages described herein, and each such variation and / or modification is considered to be within the scope of the embodiments of the invention described herein. More generally, those of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be illustrative, and that the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications of the teachings of the present invention. Those skilled in the art will recognize or be able to ascertain many equivalents of the specific embodiments of the invention described herein using only routine experimentation. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that the embodiments of the invention may be practiced in a manner different from that specifically described and claimed within the scope of the claims and their equivalents. The embodiments of the invention disclosed herein are directed to each feature, system, object, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, objects, materials, kits, and / or methods is included within the scope of the invention disclosed herein if such features, systems, objects, materials, kits, and / or methods are not mutually incompatible.

[0152] All definitions defined and used herein shall be understood to govern the definitions in the dictionary, the definitions in the documents incorporated by reference, and / or the ordinary meaning of the defined terms.

[0153] All references, patents, and patent applications disclosed herein are incorporated by reference as to the subject matter for which each is cited, and in some cases may include the entire document.

[0154] As used in the specification and claims, the indefinite articles "a" and "an" shall be understood to mean "at least one" unless clearly indicated to the contrary.

[0155] As used in the specification and claims, the phrase "and / or" shall be understood to mean "either or both" of the components so joined, i.e., in some cases the components may be present jointly and in other cases separately. Multiple components listed with "and / or" shall be construed in the same manner, i.e., "one or more" of the components so joined. Other components may optionally exist in addition to the components specifically identified by the "and / or" subclause, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising", reference to "A and / or B" may in one embodiment contain only A (optionally including components other than B); in another embodiment, only B (optionally including components other than A); in yet another embodiment, both A and B (optionally including other components), etc.

[0156] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one, but also including more than one or some or all of the listed components, as well as optionally additional unlisted items. Only words that clearly indicate the contrary, such as "only one" or "exactly one", or, when used in claims, "consisting of" will refer to exactly one of the components comprising some or all of the listed components. In general, the word "or" as used herein shall be interpreted as indicating an exclusive alternative (i.e., "one or the other, but not both") only when preceded by exclusive terms such as "either", "one of", "only one of", or "exactly one of". When used in claims, "consisting essentially of" shall have the ordinary meaning as used in the field of patent law.

[0157] As used in this specification and the claims, when referring to a list of one or more components, the phrase "at least one" shall be understood to mean at least one component selected from any one or more of the components in the list, but not necessarily including at least one of each and every component specifically listed in the list of components, and not excluding any combinations of components in the list. This definition also allows that there may optionally be additional components other than those specifically identified in the list of components referred to by the phrase "at least one", whether related or unrelated to those specifically identified components. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B"), in one embodiment, may mean at least one, optionally including more than one, A, and no B (and optionally including components other than B); in another embodiment, it may mean at least one, optionally including more than one, B, and no A (and optionally including components other than A); in yet another embodiment, it may mean at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other components), and so on.

[0158] It should also be understood that, unless the contrary is explicitly indicated, in any method in the claims herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order recited in the steps or acts of the method.

Claims

1. A composition, comprising: an active ingredient; and liposomes for coating the active ingredient to jointly form a liposome complex with the active ingredient.

2. The composition according to claim 1, wherein the active ingredient comprises at least one of curcumin, curcumin derivatives, curcumin metabolites, resveratrol, oxyresveratrol, resveratrol derivatives, and resveratrol metabolites; wherein, The active ingredient is non-artificially made or synthetic.

3. The composition according to claim 1 or 2, wherein the liposomes comprise dipalmitoyl phosphatidylcholine, cholesterol, and DMPE-PEG-200, and the weight ratio of dipalmitoyl phosphatidylcholine, cholesterol, and DMPE-PEG-200 is 90:10:2 to 90:10:

9.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a product for reducing the amount of fat in a subcutaneous region.

5. Use of the composition according to any one of claims 1 to 3 in the preparation of a product for treating obesity.

6. Use of the composition according to any one of claims 1 to 3 in the preparation of a product for reducing melanin.

7. Use of the composition according to any one of claims 1 to 3 in the preparation of a product for reducing skin wrinkles or reducing skin roughness.

8. Use of the composition according to any one of claims 1 to 3 in the preparation of a drug for relieving joint inflammation.

9. Use of the composition according to any one of claims 1 to 3 in the preparation of a drug for preventing or treating tumors.

10. Use of the composition according to any one of claims 1 to 3 in the preparation of a drug for preventing or treating fat metabolism-related diseases.

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