Epa-ee nanolipidic compositions, formulations, methods of preparation and uses thereof

Abstract

The present application relates to the field of medical food, health food and medicine, and particularly relates to an EPA-EE nano-lipid composition and preparation thereof, which uses raw material with high content of EPA-EE as main component, and contains high unsaturated phospholipid in emulsifier, can be prepared into nano-grade sub-micro emulsion, used as oral preparation, can maintain long-time effective blood drug concentration of EPA, improve oral absorption and bioavailability of EPA. By adding stabilizer, the high blood drug concentration level of EPA can be maintained; by adding lipoprotein binding promoter, the combination of EPA and lipoprotein can be promoted, the content of EPA in lipoprotein is improved, the effects of reducing blood lipid and reducing arterial plaque are fully exerted; by adding stabilizer and lipoprotein binding promoter at the same time, the high blood drug concentration of EPA can be improved and maintained for a long time, and the effects of reducing blood lipid and reducing arterial plaque are enhanced, which has important significance for the prevention and / or treatment of cardiovascular diseases, especially atherosclerosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oral preparations, in particular to an EPA-EE nano-lipid composition, a preparation thereof, a preparation method and application. BACKGROUND

[0002] With the acceleration of the pace of life, high-sugar and high-fat food has become a popular fast food and stress-relieving snack. On the basis of unbalanced nutrition structure, factors such as smoking, lack of exercise, obesity, etc. make the number of people with abnormal blood lipids in China increase year by year, and the trend of cardiovascular and cerebrovascular diseases is becoming more and more obvious. Related surveys show that the total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels of Chinese residents have increased significantly.

[0003] The blood viscosity of people with high blood lipids will increase, which will slow down the blood flow velocity of the arteries, and thus increase the risk of atherosclerosis, mainly manifested as the deposition of fat and calcium substances on the inner wall of the arteries, and insufficient perfusion of the heart and brain. Apolipoprotein is the protein part of plasma lipoprotein, which can bind and transport blood lipids to various tissues of the body for metabolism and utilization. It plays an important role in the occurrence and development of atherosclerosis. For example, high-density lipoprotein (HDL) can transport the cholesterol deposited in the blood vessels, so that it is excluded from the body through the liver, which plays a positive role in relieving the progression of atherosclerosis. However, other lipoproteins, such as very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), promote the deposition of lipids in blood vessels and accelerate the formation of plaques.

[0004] At present, there is no drug that can directly treat atherosclerosis in clinical practice. Usually, diseases related to the generation of atherosclerosis are treated to prevent the deterioration of plaques. For example, statins are currently commonly used lipid-lowering drugs that limit the cholesterol synthesis pathway based on the liver to reduce blood lipids, stabilize plaques, anti-inflammatory drugs, anticoagulants and antihypertensive drugs are also used for the treatment of advanced atherosclerosis, but they can only alleviate the progression of the disease course, and long-term or large-dose use of various drugs can have obvious toxic and side effects on organs such as liver and kidney, and also cause bleeding risk. Therefore, in the prevention and treatment of arterial plaques, the development of a treatment strategy that can safely and effectively stabilize plaques and reverse plaque formation is still a great challenge for clinical practice.

[0005] It has been shown that Omega-3 polyunsaturated fatty acids (Omega-3 PUFA), mainly including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), alpha-linolenic acid (ALA) and the like, have been confirmed to have the effect of regulating blood lipids and can promote the health of the circulatory system. Omega-3 polyunsaturated fatty acids are derived from deep sea fish oil and have poor water solubility. At present, the commonly seen Omega-3 polyunsaturated fatty acid products on the market mainly exist in the form of soft capsules, such as fish oil soft capsules, eicosapentaenoic acid soft capsules and the like. Patent document CN104856985A provides a composition for use of eicosapentaenoic acid-EE capsule (Vascepa), which uses high-purity -EE type EPA, but the soft capsule preparation has poor absorption (especially in the fasting state), and the bioavailability is lower than 20%, which cannot realize the efficient absorption of EPA and good atherosclerosis treatment effect. The reported EPA preparations have low purity, poor absorption effect, low blood drug concentration, fast metabolism, and cannot maintain the blood drug concentration required to exert the therapeutic effect, resulting in poor hypolipidemic or atherosclerosis treatment effect.

[0006] Therefore, it is necessary to develop an EPA preparation which can improve bioavailability and prolong the maintenance time of effective blood drug concentration, so as to provide a potential therapeutic drug for cardiovascular diseases, especially atherosclerosis. SUMMARY

[0007] Based on this, one object of the present application is to provide an EPA-EE nano-lipid composition having the effects of lowering blood lipids and reducing arterial plaque, which can be used as an oral preparation and applied to the prevention and / or treatment of cardiovascular diseases.

[0008] The above object can be achieved by the following technical solutions.

[0009] According to a first aspect of the present application, an EPA-EE nano-lipid composition is provided, which comprises the following components in the weight percentage as follows based on the total weight of the EPA-EE nano-lipid composition:

[0010]

[0011] In the EPA-EE raw material, the mass content of EPA-EE is ≥60%;

[0012] The first emulsifier is a high-unsaturated phospholipid, and the iodine value of the high-unsaturated phospholipid is ≥70;

[0013] In the high-unsaturated phospholipid, the mass percentage of phosphatidylcholine is ≥50%;

[0014] The second emulsifier is composed of components different from the first emulsifier, and the second emulsifier is selected from food and / or pharmaceutically acceptable raw and auxiliary materials;

[0015] The stabilizer is a non-ionic high molecular polymer;

[0016] The first adjuvant is an adjuvant that promotes the binding of EPA to lipoprotein;

[0017] The second adjuvant is a food and / or pharmaceutically acceptable raw and auxiliary material, and is different from the first emulsifier, the second emulsifier, the stabilizer and the first adjuvant;

[0018] The minimum weight percentage of water in the EPA-EE nanolipid composition is 65% (w / w).

[0019] It should be understood that the total weight of the EPA-EE nanolipid composition is 100%, that is, the sum of the weight percentages of the above-mentioned EPA-EE raw material, the first emulsifier, the second emulsifier, the stabilizer, the first adjuvant, the second adjuvant and water does not exceed 100%, and preferably is 100%.

[0020] In some embodiments of the present application, the content of the stabilizer is 0.1% to 5% (w / w), and / or the content of the first adjuvant is 0.1% to 5% (w / w), based on the total weight of the EPA-EE nanolipid composition.

[0021] In some embodiments of the present application, the EPA-EE nanolipid composition contains the following components with the following weight percentages, based on the total weight of the EPA-EE nanolipid composition:

[0022]

[0023] In some embodiments of the present application,

[0024] The EPA-EE raw material is selected from the ethylated product of the oil of one or more of deep-sea fish oil, seaweed oil, krill oil, etc.; and / or,

[0025] The mass content of EPA-EE in the EPA-EE raw material is ≥70%; and / or,

[0026] The iodine value of the first emulsifier (highly unsaturated phospholipid) is ≥90; and / or,

[0027] In the highly unsaturated phospholipid, the mass percentage of phosphatidylcholine is ≥70%; and / or,

[0028] The first emulsifier is selected from one or more of soybean phospholipid, sunflower seed phospholipid, polyene phosphatidylcholine; and / or,

[0029] The stabilizer is an amphiphilic non-ionic high molecular polymer selected from one or more of a vitamin lipid high molecular derivative, a phospholipid high molecular derivative, a fatty acid ester high molecular derivative, and a polyoxyethylene polyoxypropylene ether block copolymer;

[0030] The vitamin lipid high molecular derivative is vitamin E polyethylene glycol succinate;

[0031] The phospholipid high molecular derivative is a polyethylene glycol modified synthetic phospholipid;

[0032] The fatty acid ester high molecular derivative is a polyethylene glycol modified fatty acid ester;

[0033] The PEG unit in the phospholipid high molecular derivative has a molecular weight of 400 Da to 6000 Da; and / or,

[0034] The PEG unit in the fatty acid ester high molecular derivative has a molecular weight of 200 Da to 4000 Da; and / or,

[0035] The first adjuvant is selected from one or more of a side chain with a negatively charged group amino acid, a negatively charged group amino acid derivative, and a side chain with a negatively charged group small peptide; and / or,

[0036] The second adjuvant is selected from one or more of an antioxidant, a base oil, a co-emulsifier, a flavoring agent, an interfacial film stabilizer, a thickening agent, and a pH adjuster; and / or,

[0037] The EPA-EE nano-lipid composition is a sub-micro emulsion with an average particle size of ≤ 500 nm.

[0038] In some embodiments of the present application,

[0039] The first emulsifier has an iodine value greater than 90 and is selected from one or more of a soybean phospholipid, a sunflower seed phospholipid, and a polyene phosphatidylcholine; and / or,

[0040] The second emulsifier is selected from one or more of a phospholipid, a sucrose ester, a citric acid fatty acid glyceride, a fatty acid glyceride, a polysorbate, a fatty acid sorbitan, a polyoxyethylene fatty acid ester, a span, an alginate, and a caseinate different from the first emulsifier; and / or,

[0041] The PEG unit in the stabilizer provides a terminal group, and the terminal group is OH or methoxy; and / or,

[0042] The vitamin lipid macromolecular derivative is selected from one or more of d-a-tocopherol polyethylene glycol 200 succinate, d-a-tocopherol polyethylene glycol 400 succinate, d-a-tocopherol polyethylene glycol 1000 succinate, d-a-tocopherol polyethylene glycol 1500 succinate, d-a-tocopherol polyethylene glycol 2000 succinate, and d-a-tocopherol polyethylene glycol 4000 succinate; and / or,

[0043] The phospholipid macromolecular derivative is selected from one or more of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000, dipalmitoyl phosphatidyl ethanolamine-methoxy polyethylene glycol 2000, dipalmitoyl phosphatidyl ethanolamine-methoxy polyethylene glycol 5000, soybean phosphatidyl ethanolamine-polyethylene glycol monomethylether 2000, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000, dilauric acid phospholipid-polyethylene glycol 2000, and dioleoyl phosphatidyl ethanolamine-polyethylene glycol; and / or,

[0044] The fatty acid ester macromolecular derivative is selected from one or more of polyethylene glycol 400 oleate, polyethylene glycol 600 oleate, polyethylene glycol 4000 oleate, polyethylene glycol 6000 oleate, polyethylene glycol 400 dioleate, polyethylene glycol 600 dioleate, polyethylene glycol 200 laurate, polyethylene glycol 200 dilaurate, polyethylene glycol 400 laurate, polyethylene glycol 400 dilaurate, polyethylene glycol 400 stearate, and polyethylene glycol 400 distearate; and / or,

[0045] The polyoxyethylene polyoxypropylene ether block copolymer is selected from one or more of Pluronic L65, Pluronic F68; and / or,

[0046] The amino acid in the first excipient having a side chain with a negatively charged group is selected from one or more of aspartic acid, glutamic acid, taurine; and / or,

[0047] The amino acid derivative in the first excipient having a side chain with a negatively charged group is selected from one or more of phosphatidylserine, hexacosanoyl-glutamic acid-glutamine, hexacosanoyl-glutamic acid-glutamic acid, hexacosanoyl-glutamic acid-asparagine; and / or,

[0048] The small peptide in the first excipient having a side chain with a negatively charged group is glutathione; and / or,

[0049] The antioxidant in the second adjuvant is selected from one or more of vitamin E, alpha-tocopherol, alpha-tocopherol, gamma-tocopherol, mixed tocopherols, alpha-tocopherol acetate, alpha-tocopherol acetate, gamma-tocopherol acetate, mixed tocopherols acetate, ascorbic acid, ascorbic acid palmitate, ascorbic acid stearate, ascorbic acid myristate, ascorbic acid sodium, butylated hydroxyanisole, dibutylated hydroxytoluene, propyl gallate, tertiary butyl hydroquinone, and the like; and / or,

[0050] The base oil in the second adjuvant is selected from one or more of soybean oil, olive oil, jojoba oil, sweet almond oil, grape seed oil, corn oil, walnut oil, sea buckthorn oil, olive oil, coix seed oil, grape seed oil, ginger oil, coconut oil, camellia oil, rose oil, peppermint oil, lemon oil, medium-chain triglycerides, and the like; and / or,

[0051] The average particle size of the EPA-EE nanolipid composition is 100-300 nm.

[0052] According to a second aspect of the present application, there is provided an EPA-EE nanolipid preparation comprising the EPA-EE nanolipid composition according to the first aspect of the present application, and further, the EPA-EE nanolipid preparation is an oral preparation.

[0053] According to a third aspect of the present application, there is provided a preparation method of an EPA-EE nanolipid preparation, which can prepare the EPA-EE nanolipid preparation according to the second aspect of the present application.

[0054] In some embodiments of the present application, the preparation method comprises the following steps:

[0055] Mixing the oil phase components including the EPA-EE raw material under heating conditions to prepare an oil phase matrix;

[0056] Dissolving the water phase components in an aqueous solvent to prepare a water phase matrix, or using water as the water phase matrix;

[0057] Mixing the oil phase matrix and the water phase matrix, and shearing and stirring to prepare an oil-in-water primary emulsion;

[0058] Subjecting the oil-in-water primary emulsion to high-pressure homogenization treatment to prepare a sub-microemulsion;

[0059] After the sub-microemulsion is prepared, optional filtration, optional packaging, and optional sterilization are further performed.

[0060] According to a fourth aspect of the present application, the EPA-EE nanolipid composition of the first aspect of the present application, or the EPA-EE nanolipid preparation of the second aspect of the present application, or the preparation method of the third aspect of the present application is used, in particular, the use includes the use in the preparation of a medicine for preventing and / or treating cardiovascular diseases, and the use in medical food, health food.

[0061] In some preferred embodiments of the present application, the cardiovascular disease is atherosclerosis.

[0062] The EPA-EE nanolipid composition provided by the present application contains eicosapentaenoic acid-EE (EPA-EE) as the main component, and contains EPA-EE, an emulsifier and water. The raw material of EPA-EE contains a high content of EPA-EE (for example, ≥60% by mass), and the proportion of inactive fatty acids is reduced. The composition can be prepared into a sub-micro emulsion with a nanometer scale (preferably, the average particle size is ≤500 nm), and is used as an oral preparation, which can maintain an effective blood drug concentration of eicosapentaenoic acid for a long time, and improve the oral absorption and bioavailability of eicosapentaenoic acid (EPA). The emulsifier used in the EPA-EE nanolipid composition contains a high unsaturation degree of phospholipid (denoted as a first emulsifier, preferably, the iodine value is ≥70), which can achieve a better treatment effect of arterial plaques. The introduction of a stabilizer in the EPA-EE nanolipid composition can maintain a high blood drug concentration of EPA and improve the efficacy. The introduction of a lipoprotein binding promoter (denoted as a first auxiliary material) in the EPA-EE nanolipid composition can promote the binding of eicosapentaenoic acid with lipoprotein, increase the content of eicosapentaenoic acid in lipoprotein, and play a role in reducing blood lipids and reducing arterial plaques, thereby promoting the prevention and / or treatment of cardiovascular diseases, in particular, the prevention and / or treatment of atherosclerosis. The simultaneous addition of the stabilizer and the first auxiliary material (lipoprotein binding promoter) in the EPA-EE nanolipid composition can achieve a synergistic effect. The oral nanolipid preparation prepared by the simultaneous addition can promote the binding of lipoprotein with eicosapentaenoic acid, increase the content of eicosapentaenoic acid in lipoprotein, accelerate the metabolism of saturated fatty acids in the body, enhance the efficacy of reducing blood lipids and reducing arterial plaques, and has important significance for the efficient reduction of blood lipids and the prevention and / or treatment of atherosclerosis. The above-mentioned active ingredients of EPA-EE, the first emulsifier, the stabilizer, the first auxiliary material and other components can cooperate with each other to promote a better treatment effect of arterial plaques.

[0063] The EPA-EE nanolipid composition and the preparation (preferably, an oral preparation) thereof provided by the present application can be used in the fields of medical food, health food, medicine and the like. BRIEF DESCRIPTION OF DRAWINGS

[0064] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0065] Figure 1 A bar chart showing the EPA content in low-density lipoprotein in rats after continuous oral administration of different formulations for 8 weeks, with eicosapentaenoic acid at a dose of 400 mg / kg;

[0066] Figure 2 Bar chart showing the ratio of plaque area to vessel area after APOE- / - mice were orally administered different formulations for 8 weeks;

[0067] In the figure, ns represents P>0.05, "**" represents P<0.01, and "***" represents P<0.001. Detailed Implementation

[0068] The present invention will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the invention. The purpose of providing these embodiments and examples is to enable a more thorough and complete understanding of the disclosure of the present invention. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for descriptive purposes only and is not intended to be limiting of the invention.

[0070] Terminology

[0071] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0072] The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or all relevant listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B, and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, and D, i.e., includes the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (i.e., the technical solution connected by "logical and").

[0073] In the present application, "multiple", "plurality", etc. refer to more than two or equal to two in number, unless otherwise specified. For example, "one or more" means one or more than two.

[0074] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof", etc. include all suitable combination manners of any two or more listed items.

[0075] As used herein, "suitable combination manner", "suitable manner", "any suitable manner", etc. refer to the ability to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0076] As used herein, "preferably", "better", "better", "suitable", etc. are only used to describe the implementation manner or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0077] In the present application, "further", "more further", "particularly", etc. are used for description purposes, indicating differences in content, but should not be understood as a limitation on the protection scope of the present application.

[0078] In the present application, "optionally", "optional", "option" means optional, that is, selected from "have" or "no" two parallel schemes. If there are multiple "optional" in a technical solution, unless otherwise specified, and there is no contradiction or mutual restriction, each "optional" is independent of each other. In the present application, "optionally contains", "optionally contains" and the like, means "contains or does not contain". "Optional component X" means that component X exists or does not exist.

[0079] In the present application, in the terms "first", "second", "third", "fourth", etc. in "first aspect", "second aspect", "third aspect", "fourth aspect", "first adjuvant", "second adjuvant" and the like, the terms "first", "second", "third", "fourth" are only for description purposes and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implying the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0080] In the present application, the technical features described in an open manner include both closed technical solutions consisting of the listed features and open technical solutions containing the listed features.

[0081] In the present application, with respect to numerical intervals (i.e. numerical ranges), unless otherwise specified, the optional numerical distribution within the above numerical interval is considered to be continuous and includes the two numerical endpoints (i.e. the minimum and maximum values) of the numerical range and every numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range and every integer between the two endpoints. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein should be understood to include any and all sub-ranges encompassed therein.

[0082] In the present application, with respect to approximations, unless otherwise specified, the fluctuation range is generally ±10%, and can further refer to ±8%, ±5%, ±3%, etc. In the present application, the approximation provides both the listed numerical value and the numerical interval represented by the approximation. For example, approximately 200 nm provides a technical solution of "200 nm", and also provides a technical solution of the numerical interval "200 nm ± fluctuation range".

[0083] In the present application, unless otherwise specified, the temperature parameter allows for constant temperature treatment and allows for fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C.

[0084] In the present application, the term "room temperature" generally refers to 4°C to 35°C, preferably 20°C ± 5°C. In some embodiments of the present application, room temperature refers to 20°C to 30°C.

[0085] In the present application, % (w / w) and wt% both refer to weight percentage.

[0086] All the documents mentioned in the present application are incorporated by reference in the present application as if each document is incorporated by reference individually. Unless and to the extent conflicting with the purpose and / or technical solution of the present application, the documents referred to in the present application are incorporated by reference in their entirety. When the present application refers to the documents, the definitions of the relevant technical features, terms, names, phrases, etc. in the documents are also incorporated by reference. When the present application refers to the documents, the examples and preferred modes of the relevant technical features incorporated by reference can also be incorporated by reference in the present application, provided that the present application can be implemented. It should be understood that when the incorporated content conflicts with the description in the present application, the present application is the priority or is amended according to the description in the present application.

[0087] The abbreviations used herein, unless otherwise specified, have the following meanings: PUFA refers to polyunsaturated fatty acid, Omega-3 PUFA refers to Omega-3 polyunsaturated fatty acid, EPA refers to eicosapentaenoic acid, DHA refers to docosahexaenoic acid, and PC refers to phosphatidylcholine.

[0088] In the present application, the unsaturation degree of phospholipids is mainly characterized by iodine value, unless otherwise specified, which refers to average iodine value.

[0089] In the present application, "highly unsaturated phospholipids", unless otherwise specified, refer to phospholipids with iodine value ≥ 70, which can be phospholipids with higher iodine value, such as phospholipids with iodine value ≥ 90.

[0090] In the present application, "submicron emulsion" refers to an emulsion with an average droplet size in the range of 100 nm to 1000 nm. Preferably, the average droplet size of any submicron emulsion herein is independently less than or equal to 500 nm.

[0091] In the present application, when referring to molecular weight, unless otherwise specified, it refers to average molecular weight, which can be number average molecular weight or weight average molecular weight, unless otherwise specified, which refers to weight average molecular weight.

[0092] In the present application, "small peptides" refer to peptides with 2 to 3 amino acid units and a molecular weight below 1000 daltons.

[0093] The "medium chain triglyceride" used in the present application, also known as "medium chain triglyceride", English full name medium chain triglycerides, abbreviated as MCT. Medium chain refers to the chain of their fatty molecule is medium length (i.e. containing 6, 8 or 12 carbon atoms). In the national food safety standard, medium chain triglyceride can be used as food raw material or emulsifier.

[0094] In the present application, the "long chain group" in the stabilizer refers to the polyethylene glycol segment for the vitamin lipid high polymer derivative, the phospholipid high polymer derivative, and the fatty acid ester high polymer derivative.

[0095] In the present application, polyethylene glycol (PEG) and polyoxyethylene (POE) have the same meaning and can be used interchangeably. The molecular weight related to PEG, such as the average molecular weight, can be number average molecular weight or weight average molecular weight, such as the weight average molecular weight, unless otherwise specified.

[0096] In the present application, "above" and "below" each independently contain the number, unless otherwise specified.

[0097] In the present application, "≥" and "greater than or equal to" have the same meaning and can be used interchangeably, both indicating greater than or equal to. "≤" and "less than or equal to" have the same meaning and can be used interchangeably, both indicating less than or equal to.

[0098] First aspect of the invention

[0099] According to the first aspect of the present application, an EPA-EE nano-lipid composition capable of prolonging the maintenance time of effective blood drug concentration while improving the bioavailability is provided, which is mainly composed of raw materials with high content of EPA-EE, and can be prepared into sub-micro emulsion with nano-scale (preferably, the average particle size is ≤500 nm), used as oral preparation, capable of maintaining long-time EPA effective blood drug concentration, improving the oral absorption and bioavailability of EPA.

[0100] According to the first aspect of the present application, an EPA-EE nano-lipid composition is provided, which comprises the following components with the weight percentage as follows, based on the total weight of the EPA-EE nano-lipid composition:

[0101]

[0102] Among the EPA-EE raw materials, the mass content of EPA-EE is ≥60%;

[0103] The first emulsifier is high unsaturated phospholipid, and the iodine value of the high unsaturated phospholipid is ≥70;

[0104] In the high unsaturated phospholipid, the mass fraction of phosphatidylcholine is ≥50%.

[0105] The second emulsifier is composed of different components from the first emulsifier, and is selected from food and / or pharmaceutically acceptable excipients. The stabilizer is a non-ionic high molecular polymer for maintaining the stability of the emulsion, which can effectively prolong the blood concentration of EPA;

[0106] The first excipient is an excipient for promoting the binding of EPA to lipoprotein.

[0107] The second excipient is a food and / or pharmaceutically acceptable excipient, and is different from the first emulsifier, the second emulsifier, the stabilizer, and the first excipient.

[0108] It should be understood that the water in the EPA-EE nanolipid composition is an appropriate amount of water, preferably the minimum weight percentage of water in the EPA-EE nanolipid composition is 65% (w / w), but the sum of the weight percentages of the components does not exceed 100%.

[0109] In some embodiments, the sum of the weight percentages of the EPA-EE raw material, the first emulsifier, the second emulsifier, the stabilizer, the first excipient, the second excipient, and water does not exceed 100%, preferably one of them is 100%.

[0110] In some embodiments of the present application, the EPA-EE nanolipid composition comprises 1% to 30% (w / w) of EPA-EE raw material, 0.1% to 10% (w / w) of high unsaturated phospholipid (as the first emulsifier), 0% to 10% of other emulsifiers (as the second emulsifier), 0 to 10% of excipients for promoting the binding of EPA to lipoprotein (as the first excipient), 0 to 40% of other food and / or pharmaceutically acceptable excipients (as the second excipient), and an appropriate amount of water, wherein the EPA-EE provides a high content of active EPA-EE, the first emulsifier provides a high iodine value of unsaturated phospholipid, the stabilizer is used to improve the stability of the nanolipid composition system, the first excipient provides a lipoprotein binding promoter for EPA, and various components cooperate to improve the oral absorption and bioavailability of eicosapentaenoic acid (EPA).

[0111] The EPA-EE nanolipid composition provided by the present application comprises EPA-EE, emulsifier, and water, and a high content of EPA-EE is provided by the EPA-EE raw material (for example, the mass percentage of the EPA-EE raw material is ≥60%); the EPA-EE nanolipid composition can be made into a sub-micro emulsion of nanometer scale (preferably, the average particle size is ≤500 nm), which is used as an oral preparation, can maintain an effective blood drug concentration of eicosapentaenoic acid for a long time, and improves the oral absorption and bioavailability of eicosapentaenoic acid (EPA).

[0112] The inventors have found, through extensive exploration and research, that EPA is a key active fatty acid for treating cardiovascular diseases, and the binding of eicosapentaenoic acid to low-density lipoprotein helps prevent the formation of oxidized low-density lipoprotein, which is the key to the hypolipidemic effect of EPA. In addition, EPA preparations aiming to use EPA as an active ingredient need to provide a relatively high blood concentration (or exposure) of EPA and maintain a relatively long exposure time, so as to achieve good binding of eicosapentaenoic acid to low-density lipoprotein, and significant reduction of oxidized low-density lipoprotein is conducive to alleviating inflammation and endothelial cell damage at the atherosclerotic site, and achieving and promoting the therapeutic effect of atherosclerosis.

[0113] In the above-mentioned EPA-EE nanolipid composition, the first emulsifier is used to achieve emulsification of the preparation. In addition, the unsaturation degree of the phospholipid also affects the drug efficacy, and a phospholipid with a higher unsaturation degree is better for treating atherosclerosis, and a phospholipid with an iodine value of 70 or higher is conducive to cardiovascular health. In some embodiments of the present application, the emulsifier component in the EPA-EE nanolipid can include other emulsifiers (referred to as second emulsifiers) in addition to the first emulsifier (highly unsaturated phospholipid), which can be used to flexibly control emulsification. The content of the second emulsifier can be 0 (i.e., no second emulsifier is contained).

[0114] In the above-mentioned EPA-EE nanolipid composition, the content of the stabilizer can be 0 (i.e., no stabilizer is contained). When the content of the stabilizer is not 0, by introducing a stabilizer (which can also be referred to as an EPA stabilizer) into the EPA-EE nanolipid composition, a relatively high blood concentration level of EPA can be maintained, and the drug efficacy can be improved.

[0115] In the above-mentioned EPA-EE nanolipid composition, the content of the first auxiliary material (also referred to as a lipoprotein binding promoter) can be 0 (i.e., no first auxiliary material is contained). When the content of the first auxiliary material is not 0, by introducing a lipoprotein binding promoter into the EPA-EE nanolipid composition, the binding of eicosapentaenoic acid to lipoprotein can be promoted, the content of eicosapentaenoic acid in lipoprotein can be increased, the hypolipidemic and atherosclerotic plaque reducing effects can be exerted, and the application in the prevention and / or treatment of cardiovascular diseases, in particular the prevention and / or treatment of atherosclerosis, can be promoted.

[0116] When the content of the stabilizer and the first adjuvant are both not 0, i.e. both the stabilizer and the first adjuvant are contained, the oral nano-lipid preparation prepared from the above-mentioned EPA-EE nano-lipid composition can achieve synergistic effect, can improve and maintain high EPA blood concentration for a long time, promote the binding of lipoprotein and eicosapentaenoic acid, increase the content of eicosapentaenoic acid in apolipoprotein, accelerate the metabolism of saturated fatty acids in vivo, enhance the effects of reducing blood lipids and reducing arterial plaque, and has important significance for the prevention and / or treatment of high blood lipids and atherosclerosis.

[0117] In some embodiments of the present application, the EPA-EE nano-lipid composition comprises a stabilizer and / or a first adjuvant. That is, the EPA-EE nano-lipid composition comprises at least one of a stabilizer and a first adjuvant.

[0118] In some embodiments of the present application, the EPA-EE nano-lipid composition comprises an EPA-EE raw material, a first emulsifier, an optional second emulsifier, water, a stabilizer, an optional first adjuvant, and an optional second adjuvant.

[0119] In some embodiments of the present application, the EPA-EE nano-lipid composition comprises an EPA-EE raw material, a first emulsifier, an optional second emulsifier, water, an optional stabilizer, a first adjuvant, and an optional second adjuvant.

[0120] In some embodiments of the present application, the EPA-EE nano-lipid composition comprises an EPA-EE raw material, a first emulsifier, an optional second emulsifier, water, a stabilizer, a first adjuvant, and an optional second adjuvant.

[0121] In some embodiments of the present application, the EPA-EE nano-lipid composition comprises an EPA-EE raw material, a first emulsifier, an optional second emulsifier, water, a stabilizer, a first adjuvant, and a second adjuvant.

[0122] EPA-EE raw material

[0123] In the present application, the EPA-EE nanolipid composition contains an EPA-EE raw material. In some embodiments of the present application, the content of the EPA-EE raw material in the EPA-EE nanolipid composition is 1% to 30% by weight, and further can be 4% to 20%, and specifically can be any one of the following percentages or a percentage range formed by any two of the following percentages: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, and the like, and for example, 15% to 30%.

[0124] The EPA-EE raw material of the present application can provide a high content of EPA. In the present application, the mass percentage of EPA-EE in the EPA-EE raw material (i.e., the purity) is preferably ≥ 60%, and further preferably ≥ 70%. If the content of EPA-EE in the EPA-EE raw material is low (e.g., < 40%), the active substance exposure required for treatment cannot be achieved after administration. The composition of the present application can encapsulate a high concentration of EPA-EE, which can meet the required EPA dose after formulation.

[0125] In some embodiments of the present application, the EPA-EE raw material is derived from an oil selected from one or more of deep sea fish oil, seaweed oil, krill oil, and the like.

[0126] In some embodiments of the present application, the EPA-EE raw material is an ethyl esterization product of an oil selected from one or more of deep sea fish oil, seaweed oil, krill oil, and the like. Taking the case where the EPA-EE is derived from deep sea fish oil, in some embodiments of the present application, the EPA-EE raw material is obtained by subjecting the deep sea fish oil to ethyl esterization (EE treatment). Eicosapentaenoic acid in fish oil mainly exists in the form of a triglyceride. The deep sea fish oil is concentrated, hydrolyzed, and separated to obtain eicosapentaenoic acid, which is then reacted with ethanol and concentrated sulfuric acid to perform pre-esterification, and then subjected to an ester exchange reaction to achieve EE of eicosapentaenoic acid glyceride. The resulting raw material, i.e., the EPA-EE raw material of the present application, is obtained by separation. In the resulting raw material, the purity of eicosapentaenoic acid-EE is preferably ≥ 60% by mass, and more preferably > 60%. In some preferred embodiments, the purity of eicosapentaenoic acid-EE in the resulting raw material is ≥ 70%, and more preferably > 70%.

[0127] In some embodiments, the EPA-EE in the EPA-EE raw material has a mass percentage of any one of the following percentages or a percentage range formed by any two of the following percentages: 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 97%, etc.

[0128] In some embodiments of the present application, the EPA-EE raw material is selected from any one of the following products or any combination thereof from KinOmega: 6015 EE EPA 60%+DHA 12%, KinOmega 7010 EE EPA 70%+DHA 8%, K85 EE Omega-3-acids-EE (EPA EE 86227-47-6), Maxomega EPA 97 EE, etc.

[0129] Emulsifier

[0130] In the present application, the EPA-EE nanolipid composition contains an emulsifier, and the composition can be prepared into an emulsion, especially an oral emulsion. The EPA-EE nanolipid composition of the present application can encapsulate a high concentration of eicosapentaenoic acid-EE in a unique nanolipid prescription to meet the dosage of eicosapentaenoic acid required for high-efficiency lipid-lowering and treatment of atherosclerosis in the body after oral administration.

[0131] In some embodiments of the present application, the emulsifier has a content of 0.1% to 10% by weight in the EPA-EE nanolipid composition, and further can be 0.5% to 5%, and specifically can be any one of the following percentages or a percentage range formed by any two of the following percentages: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc., and the percentage range can be, for example, 0.5% to 10%.

[0132] Highly unsaturated phospholipid (first emulsifier)

[0133] The EPA-EE nano-lipid composition of the present application contains high unsaturated phospholipids, denoted as the first emulsifier, which are phospholipids with iodine value ≥ 70. In some embodiments, the first emulsifier can be a mixture of two or more phospholipids, in which the iodine value of any one phospholipid component satisfies ≥ 70, further ≥ 80, and further ≥ 90, and further ≥ 100. In some embodiments of the present application, the iodine value of the high unsaturated phospholipids is 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 92, 94, 95, 96, 98, 99, 100, 101, 102, 103, etc. When the iodine value is greater than 80, the treatment effect on arterial plaques is better.

[0134] In some embodiments of the present application, the first emulsifier (high unsaturated phospholipids) is selected from one or more of soybean phospholipids, sunflower seed phospholipids, polyene phosphatidylcholine, etc.

[0135] In some embodiments of the present application, the mass fraction of phosphatidylcholine in the phospholipid component of the first emulsifier (in high unsaturated phospholipids) is ≥ 50%, further ≥ 60%, and further ≥ 70%.

[0136] In some specific embodiments, the mass content of phosphatidylcholine in the first emulsifier (high unsaturated phospholipids) is, for example, any one of the following percentages or the percentage interval formed by any two of the following percentages 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 98%, etc.

[0137] The inventors have also found that the unsaturation of the phosphatidylcholine also has an impact on the efficacy, and the PC with higher unsaturation is better for the treatment of atherosclerosis. The unsaturation of the phosphatidylcholine can also be characterized by iodine value, and the higher the iodine value, the higher the unsaturation. When the iodine value is greater than 80, the treatment effect on the arterial plaque is better. In some embodiments, the iodine value of the phosphatidylcholine is ≥ 80, further can be ≥ 90, and further can be ≥ 100. In some embodiments of the present application, the iodine value of the phosphatidylcholine is 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 92, 94, 95, 96, 98, 99, 100, 101, 102, 103, etc. In some embodiments of the present application, the content of phosphatidylcholine (PC) in the phospholipid is greater than or equal to 50%, and the iodine value is greater than 80, at which time the treatment effect on the arterial plaque is better. In some preferred embodiments of the present application, the phosphatidylcholine is one or more of soybean phospholipid S75, S100, sunflower seed phospholipid H100, and polyene phosphatidylcholine.

[0138] In some embodiments, the mass percentage of phosphatidylcholine in the highly unsaturated phospholipid is ≥ 50%, and the iodine value of the phosphatidylcholine is ≥ 80.

[0139] In some embodiments, the iodine value of the first emulsifier (highly unsaturated phospholipid) is ≥ 80 (further can be ), and in the highly unsaturated phospholipid, the mass percentage of phosphatidylcholine (PC) is ≥ 50%, at which time the treatment effect on the arterial plaque is better.

[0140] In some embodiments, the iodine value of the first emulsifier is greater than 90, and is selected from one or more of soybean phospholipid, sunflower seed phospholipid, and polyene phosphatidylcholine.

[0141] Any phospholipid component in the present application can be an independent phospholipid molecule, or can be a derivative or modified phospholipid of the phospholipid molecule.

[0142] The phospholipid component in the EPA-EE nano-lipid composition is not limited to being provided by the first emulsifier, but can also be provided by the second emulsifier. However, the phospholipid component provided by the second emulsifier is not a phospholipid with an iodine value ≥ 70. The phospholipid component in the second emulsifier can also be the modified phospholipid described above.

[0143] The first emulsifier in the EPA-EE nano-lipid composition can also simultaneously perform other functions, such as, it can also simultaneously serve as the first adjuvant, such as, it can be a liver targeting molecule modified with highly unsaturated phospholipid, or can be a PEG modified highly unsaturated phospholipid.

[0144] In some embodiments, among all the phospholipid components in the EPA-EE nano-lipid composition, the mass percentage of phospholipid with an iodine value ≥ 70 is greater than 90%.

[0145] In some embodiments, the mass percentage of phospholipids with iodine value ≥ 90 in all phospholipid components of the EPA-EE nanolipid composition is greater than 90%.

[0146] Second emulsifier

[0147] The second emulsifier in the present application does not contain the same component as the first emulsifier. That is, the second emulsifier is composed of a component different from the first emulsifier.

[0148] In some embodiments of the present application, the emulsifier component in the EPA-EE nanolipid composition can include other emulsifiers (denoted as the second emulsifier) in addition to the first emulsifier, which plays a role in flexible control of emulsification. In some embodiments of the present application, the second emulsifier is selected from one or more of other phospholipids (different from the phospholipids in the first emulsifier, such as egg yolk phospholipids), sucrose esters, citric acid fatty acid glycerides, fatty acid glycerides, polysorbates, fatty acid sorbitans, polyoxyethylene fatty acid esters, spans, alginates, and caseinates. In some embodiments of the present application, the content of the second emulsifier in the EPA-EE nanolipid is 0% to 10% by weight, and further can be 0% to 5%, and specific examples include 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.5%, 4%, 4.2%, 4.5%, 4.6%, 4.8%, 5%, and the like.

[0149] In some embodiments, the second emulsifier is an emulsifier that does not contain high-unsaturated phospholipids.

[0150] In some embodiments, the second emulsifier contains phospholipid components, but these phospholipid components are different from the first emulsifier, that is, they are not high-unsaturated phospholipids.

[0151] In some embodiments, the second emulsifier contains phospholipid components, but they are all saturated phospholipids.

[0152] In some embodiments, the second emulsifier does not contain phosphatidylcholine components.

[0153] In some embodiments, the second emulsifier does not contain phospholipid components.

[0154] Stabilizer

[0155] In the present application, the EPA-EE nano-lipid composition optionally comprises a stabilizer, which can stabilize EPA and has the effect of maintaining the blood concentration of EPA, and therefore can also be referred to as an EPA stabilizer. Taking oral preparations as an example, after oral absorption, the stabilizer becomes a component of chylomicrons together with eicosapentaenoic acid. The stabilizer used in the present application can form a hydrated film on the surface of chylomicrons, covering the hydrophobic binding sites that interact with opsonins; the long-chain groups (high-molecular-chain) in the stabilizer can form steric hindrance on the surface of chylomicrons, effectively avoiding recognition and phagocytosis by the endothelial reticular system. Therefore, the prepared nano-lipid preparation can prolong the blood circulation time of eicosapentaenoic acid, maintain the effective drug concentration in the blood, and have the effect of long-acting to exert the hypolipidemic effect of EPA, thereby achieving a significant therapeutic effect on atherosclerotic plaques, while ordinary EPA preparations have a poor therapeutic effect on atherosclerotic plaques.

[0156] In some embodiments of the present application, the EPA-EE nano-lipid composition does not contain the stabilizer.

[0157] In some embodiments of the present application, the EPA-EE nano-lipid composition contains a stabilizer, and the stabilizer is a non-ionic high-molecular-weight polymer, and further, the stabilizer is an amphiphilic non-ionic high-molecular-weight polymer.

[0158] In some embodiments of the present application, the stabilizer is selected from one or more of vitamin lipid high-molecular-weight derivatives, phospholipid high-molecular-weight derivatives, fatty acid ester high-molecular-weight derivatives, polyoxyethylene polyoxypropylene ether block copolymers, etc., all of which can achieve the aforementioned effect of stabilizing EPA.

[0159] When the PEG unit provides a terminal group in the stabilizer, the terminal group provided by the PEG unit can be OH or methoxy.

[0160] In some embodiments of the present application, the non-ionic high molecular polymer is a polyethylene glycol derivative, further an amphiphilic polyethylene glycol derivative, wherein the molecular weight of the PEG unit is mainly considered in the comprehensive consideration of various factors such as stabilization, formulation particle size, drug release, etc. In some embodiments of the present application, the non-ionic high molecular polymer is selected from one or more of vitamin lipid high molecular derivative, phospholipid high molecular derivative, fatty acid ester high molecular derivative, etc., and further, the molecular weight of the PEG unit is 200 Da to 6000 Da, and further can be 400 Da to 6000 Da, and specific examples are about 200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 1000 Da, 1200 Da, 1300 Da, 1400 Da, 1500 Da, 1600 Da, 1800 Da, 2000 Da, 2200 Da, 2400 Da, 2500 Da, 2600 Da, 2800 Da, 3000 Da, 3200 Da, 3300 Da, 3400 Da, 3500 Da, 4000 Da, 4200 Da, 4400 Da, 4500 Da, 5000 Da, 5500 Da, 6000 Da, etc., and "about" means that it can vary within a certain range, such as ±10%, and taking about 1000 as an example, it can be 1000 ±10% (numerically equivalent to 9000-1100). Among them, 2000, 200, 400, 600, 4000, 6000, etc. represent the molecular weight of the PEG block, which can be the number average molecular weight or the weight average molecular weight.

[0161] In some embodiments of the present application, the vitamin unit in the vitamin lipid high molecular derivative is independently preferably vitamin E. In some embodiments of the present application, the vitamin lipid high molecular derivative is a vitamin lipid polyethylene glycol derivative. In some embodiments of the present application, the vitamin lipid high molecular derivative is vitamin E polyethylene glycol succinate. In some embodiments of the present application, the molecular weight of the PEG unit in the vitamin lipid high molecular derivative is 200 Da to 4000 Da. In some embodiments, specific examples of the vitamin lipid high molecular derivative are d-α-tocopherol polyethylene glycol 200 succinate, d-α-tocopherol polyethylene glycol 400 succinate, d-α-tocopherol polyethylene glycol 1000 succinate, d-α-tocopherol polyethylene glycol 1500 succinate, d-α-tocopherol polyethylene glycol 2000 succinate, d-α-tocopherol polyethylene glycol 4000 succinate, etc.

[0162] In some embodiments of the present application, the phospholipid high molecular weight derivative is a polyethylene glycol modified synthetic phospholipid. Further, the PEG unit in the phospholipid high molecular weight derivative can have a molecular weight of 400 Da to 6000 Da, such as 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, 1500 Da, 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, 5000 Da, 6000 Da, etc.

[0163] In some embodiments of the present application, the phospholipid unit in the phospholipid high molecular weight derivative independently preferably comprises a phosphatidylethanolamine unit.

[0164] In some embodiments of the present application, the phospholipid high molecular weight derivative is selected from the group consisting of phosphatidylethanolamine-polyethylene glycol (PE-PEG, preferably containing C 12-20 fatty acyl (such as stearoyl), further preferably C 12-20 fatty acyl phosphatidylethanolamine-polyethylene glycol). In some embodiments of the present application, the phospholipid high molecular weight derivative is selected from the group consisting of one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 5000, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, soybean phosphatidylethanolamine-polyethylene glycol monomethylether 2000, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, dilauric acid phospholipid-polyethylene glycol 2000, dioleoylphosphatidylethanolamine-polyethylene glycol.

[0165] In some embodiments, the PEG unit in the phospholipid high molecular weight derivative provides a terminal group, and the terminal group is OH or methoxyl. In some embodiments, the fatty acid ester unit in the fatty acid ester high molecular weight derivative independently preferably C 12-20 fatty acid ester unit having 12 to 20 carbon atoms, such as 12, 14, 16, 18, 20; the number of fatty acid chains in one molecule of any one fatty acid ester unit can be 1, 2, or more, independently, depending on the type of ester, etc. In some embodiments, the PEG unit in the fatty acid ester high molecular weight derivative provides a terminal group, and the terminal group is OH or methoxyl.

[0166] In some embodiments of the present application, the fatty acid ester high molecular weight derivative is a polyethylene glycol modified fatty acid ester. Further, the PEG unit of the fatty acid ester high molecular weight derivative can have a molecular weight of 200 Da to 4000 Da, such as 200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, 1500 Da, 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, etc.

[0167] In some embodiments of the present application, the fatty acid ester high molecular weight derivative is selected from the group consisting of polyethylene glycol-C 12-20 fatty acid ester, polyethylene glycol-C 12-20 fatty acid ester, polyethylene glycol-C In some embodiments of the present application, the fatty acid ester high molecular weight derivative is selected from the group consisting of polyethylene glycol 400 oleate, polyethylene glycol 600 oleate, polyethylene glycol 4000 oleate, polyethylene glycol 6000 oleate, polyethylene glycol 400 dioleate, polyethylene glycol 600 dioleate, polyethylene glycol 200 laurate, polyethylene glycol 200 dilaurate, polyethylene glycol 400 laurate, polyethylene glycol 400 dilaurate, polyethylene glycol 400 stearate, polyethylene glycol 400 distearate, etc.

[0168] In some embodiments of the present application, the polyoxyethylene polyoxypropylene ether block copolymer is a two-block copolymer. In some embodiments of the present application, the polyoxyethylene polyoxypropylene ether block copolymer has an average molecular weight of 3000 Da to 10000 Da, such as 3500 Da, 8350 Da, etc. In some embodiments of the present application, the mass content of the polyoxyethylene block is 50% to 80%. In some embodiments of the present application, the polyoxyethylene polyoxypropylene ether block copolymer is a poloxamer, which is commercially available, and further, the poloxamer can be Pluronic L65 (polyoxyethylene content 50%, average molecular weight 3500 Da), Pluronic F68 (polyoxyethylene content 80%, average molecular weight 8350 Da), etc. In some embodiments of the present application, the stabilizer comprises a polyoxyethylene polyoxypropylene ether block copolymer. In some embodiments of the present application, the stabilizer is a polyoxyethylene polyoxypropylene ether block copolymer.

[0169] In some embodiments, the EPA-EE nanolipidic composition is a PEG-modified lipid composition, and further, the PEG-modified raw material can be present in a percentage of 0.01% to 10% by weight of the EPA-EE nanolipidic composition, such as, for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and the like.

[0170] In some embodiments of the present application, the stabilizer is present in a percentage of 0% to 5% (w / w) by weight of the EPA-EE nanolipidic composition, further in a percentage of 0% to 5% (w / w), and even further in a percentage of 0.1% to 3% (w / w), such as, for example, any one of the following percentages or a percentage interval defined by any two of the following percentages: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.5%, 4%, 4.2%, 4.5%, 4.6%, 4.8%, 5%, and the like, such as, for example, 0% to 5% (w / w).

[0171] First excipient

[0172] In the present application, the EPA-EE nanolipidic composition optionally comprises a lipoprotein binding promoter, also referred to as a first adjuvant. The first adjuvant can promote the binding of EPA to lipoproteins, and is a competitive lipoprotein binding adjuvant. The first adjuvant can interact with positively charged residues on the polar-nonpolar interface of the amphipathic helix of lipoproteins, and promote the binding of eicosapentaenoic acid in the lipoproteins to low-density lipoproteins. In the present application, increasing the content of eicosapentaenoic acid in lipoproteins and reducing the content of saturated fatty acids in lipoproteins can both help reduce the formation of oxidized lipoproteins, reduce the accumulation of saturated fatty acids on the inner wall of blood vessels, improve the damage to endothelial cells, and improve the treatment effect of atherosclerosis.

[0173] In some embodiments of the present application, the EPA-EE nanolipidic composition does not comprise the first adjuvant.

[0174] In some embodiments of the present application, the first adjuvant is selected from one or more of the following: a side chain negatively charged amino acid, a negatively charged amino acid derivative, a small peptide with a side chain negatively charged amino acid, etc. In some embodiments, the side chain negatively charged amino acid is selected from one or more of the following: aspartic acid, glutamic acid, taurine, etc. In some embodiments, the side chain negatively charged amino acid derivative is selected from one or more of the following: phosphatidylserine, hexacosanoyl-glutamic acid-glutamine, hexacosanoyl-glutamic acid-glutamic acid, hexacosanoyl-glutamic acid-asparagine, etc. In some embodiments, the small peptide with a side chain negatively charged amino acid is selected from one or more of the following: glutathione.

[0175] In some specific embodiments of the present application, the first adjuvant is selected from one or more of the following: aspartic acid, glutamic acid, taurine, phosphatidylserine, hexacosanoyl-glutamic acid-glutamine, hexacosanoyl-glutamic acid-glutamic acid, hexacosanoyl-glutamic acid-asparagine, glutathione, etc.

[0176] In some embodiments of the present application, the first adjuvant is present in the EPA-EE nanolipid composition in an amount of 0-5% (w / w), further in an amount of 0.1-5% (w / w), and further in an amount of 0.1-3% (w / w). For example, the first adjuvant is present in the EPA-EE nanolipid composition in an amount of any one of the following percentages or in an amount of any two of the following percentages to form a percentage range: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.5%, 4%, 4.2%, 4.5%, 4.6%, 4.8%, 5%, etc. For example, the first adjuvant is present in the EPA-EE nanolipid composition in an amount of 0.1-5% (w / w).

[0177] In some embodiments of the present application, the EPA-EE nanolipid composition contains both the aforementioned stabilizer and the aforementioned first adjuvant. In this case, by the synergy among the raw material with high content of EPA-EE, the stabilizer, and the first adjuvant, the formulation of the composition can maintain the concentration of EPA in the blood plasma, increase the exposure amount, and increase the binding with low-density lipoprotein, thereby improving the hypolipidemic and atherosclerosis treatment effects of EPA and exerting a significant therapeutic effect that cannot be achieved by the existing reported formulations.

[0178] In some embodiments of the present application, the content of the stabilizer is 0.1-5% (w / w) and / or the content of the first adjuvant is 0.1-5% (w / w), based on the total weight of the EPA-EE nanolipid composition. The preferred and exemplary contents of the stabilizer and the first adjuvant can be referred to the foregoing description.

[0179] Second excipient

[0180] In the present application, the EPA-EE nanolipid composition optionally further comprises other adjuvants (denoted as second adjuvants) in addition to the first adjuvant.

[0181] The second adjuvant in the present application is different from the first emulsifier, the second emulsifier, the stabilizer, and the first adjuvant.

[0182] In some embodiments of the present application, the second adjuvant comprises one or more of an antioxidant, a base oil, a flavoring agent, an interfacial film stabilizer, a pH adjuster, etc.

[0183] In some embodiments of the present application, the second adjuvant comprises one or more of a base oil (mainly refers to other oils in addition to eicosapentaenoic acid and its derivatives), an antioxidant, an emulsifying aid, a pH adjuster, a thickening agent, a flavoring agent, etc.

[0184] In some embodiments of the present application, the weight percentage of the second adjuvant in the EPA-EE nanolipid composition is 0-15% (w / w), and further can be 0.01%-10% (w / w), for example, any one of the following percentages or a percentage interval constituted by any two of the following percentages: 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.5%, 4%, 4.2%, 4.5%, 4.6%, 4.8%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, etc., for example, 0.01%-15% (w / w).

[0185] In some embodiments of the present application, the EPA-EE nanolipidic composition optionally comprises an antioxidant. In some embodiments of the present application, the antioxidant is derived from one or more of vitamin E, alpha-tocopherol, alpha-tocopherol, gamma-tocopherol, mixed tocopherols, alpha-tocopherol acetate, alpha-tocopherol acetate, gamma-tocopherol acetate, mixed tocopherols acetate, ascorbic acid (vitamin C), ascorbic acid palmitate, ascorbic acid stearate, ascorbic acid myristate, ascorbic acid sodium, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), tertiary butylhydroquinone (TBHQ), and the like. In some embodiments of the present application, the antioxidant is present in the EPA-EE nanolipidic composition in an amount of 0-1%, such as 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, and the like.

[0186] In some embodiments of the present application, the EPA-EE nanolipidic composition optionally comprises a base oil. In some embodiments of the present application, the base oil refers to an oil other than eicosapentaenoic acid and its derivatives. In some embodiments of the present application, the base oil is derived from one or more of soybean oil, olive oil, jojoba oil, sweet almond oil, grape seed oil, corn oil, walnut oil, sea buckthorn oil, olive oil, coix seed oil, grape seed oil, ginger oil, coconut oil, camellia oil, rose oil, peppermint oil, lemon oil, medium-chain triglycerides (e.g., C 8-10 glycerol ester of fatty acid), and the like. In some embodiments of the present application, the base oil is present in the EPA-EE nanolipidic composition in an amount of 0-1%, such as 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, and the like.

[0187] In some embodiments of the present application, the EPA-EE nanolipidic composition optionally comprises a co-emulsifier. In some embodiments of the present application, the co-emulsifier is derived from one or more of casein, sodium caseinate, sodium polyacrylate, and the like.

[0188] In some embodiments of the present application, the EPA-EE nanolipidic composition optionally comprises a pH regulator. The pH regulator is mainly used to adjust the pH environment of the aqueous phase during the preparation of the EPA-EE nanolipidic composition. In some embodiments of the present application, the pH regulator is selected from one or more of citric acid, sodium citrate, potassium citrate, acetic acid, sodium acetate, phosphoric acid, phosphate, hydrochloric acid, citric acid, sodium citrate, lactic acid, tartaric acid, malic acid, DL-malic acid, fumaric acid, metatartaric acid, L(+)-tartaric acid, glacial acetic acid, acetic acid, adipic acid, monosodium fumarate, calcium lactate, sodium acetate, calcium hydroxide, potassium hydroxide, sodium hydroxide, and the like.

[0189] In some embodiments of the present application, the EPA-EE nanolipid composition optionally comprises an interfacial film stabilizer. In some embodiments of the present application, the interfacial film stabilizer is selected from one or more of glycerol, propylene glycol, mannitol, oleic acid, sodium oleate, and cholesterol.

[0190] In some embodiments of the present application, the EPA-EE nanolipid composition optionally comprises a thickening agent. In some embodiments of the present application, the thickening agent is selected from one or more of carrageenan, xanthan gum, carbomer, and the like.

[0191] In some embodiments of the present application, the EPA-EE nanolipid composition optionally comprises a flavoring agent. In some embodiments of the present application, the flavoring agent is selected from one or more of sucrose, fructose, sucralose, neotame, erythritol, mogroside, natural essence, natural flavor, menthol, and the like.

[0192] In some embodiments of the present application, the EPA-EE nanolipid composition comprises eicosapentaenoic acid-EE (high concentration, provided by high purity of EPA-EE raw material), a stabilizer having an effect of maintaining EPA blood concentration, a first excipient having an effect of facilitating the binding of EPA to lipoprotein, an emulsifier, an antioxidant, and other excipients for adjusting the taste and flavor of the nanolipid formulation.

[0193] In some embodiments of the present application, the EPA-EE nanolipid composition comprises, based on the total weight of the EPA-EE nanolipid composition, 4% to 20% (w / w) eicosapentaenoic acid-EE, 0.1% to 10% (w / w) first emulsifier, 0.01% to 10% (w / w) second emulsifier, and water added to 100% (w / w). Further, one or more of 0 to 5% (w / w) stabilizer (having an effect of maintaining EPA blood concentration), 0 to 5% (w / w) first excipient (having an effect of facilitating the binding of EPA to lipoprotein), and 0 to 15% (w / w) second excipient (other food and / or pharmaceutically acceptable excipients, preferably pharmaceutically acceptable excipients) can be further contained. In one specific example, the EPA-EE nanolipid composition contains 0 to 5% (w / w) antioxidant.

[0194] Water

[0195] In the present application, the EPA-EE nanolipid composition necessarily contains water as a solvent in order to be able to produce a water-based formulation that is easy to apply to a patient.

[0196] In the present application, the water in the EPA-EE nanolipidic composition can be deionized water, distilled water, sterile water, etc., as long as it is suitable for the preparation of pharmaceutical preparations. The amount of water used in the EPA-EE nanolipidic composition is an appropriate amount of water, and the minimum amount of water used is 65% (w / w). The appropriate amount of water allows the EPA-EE nanolipidic composition to form a suitable oil phase and water phase ratio, and can form an oil-in-water structure. In some embodiments of the present application, the weight percentage of water in the EPA-EE nanolipidic composition is, for example, any one of the following percentages or a percentage interval formed by any two of the following percentages: 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89.1%, 89.2%, 89.3%, 89.4%, 89.5%, 89.6%, 89.7%, 89.8%, etc.

[0197] Some embodiments

[0198] In some embodiments of the present application, the EPA-EE nanolipidic composition contains the following components in the weight percentage, respectively, based on the total weight of the EPA-EE nanolipidic composition:

[0199]

[0200] Water (an appropriate amount of water, preferably and exemplarily including but not limited to the above).

[0201] In some embodiments of the present application, the EPA-EE nanolipidic composition contains the following components in the weight percentage, respectively, based on the total weight of the EPA-EE nanolipidic composition:

[0202]

[0203]

[0204] Water (an appropriate amount of water, preferably and exemplarily including but not limited to the above).

[0205] Further, the EPA-EE nanolipidic composition preferably has a maximum blood concentration higher than 700 μg / mL within 2 hours after oral administration of 400 mg / kg to rats.

[0206] Examples of the EPA-EE nanolipidic composition include but are not limited to those listed in Example 1, and 1 g therein can be regarded as 1 mass part.

[0207] In some embodiments of the present application, the EPA-EE nanolipid composition comprises: 50-500 parts by mass of EPA-EE (further can be 100-400 parts by mass, and further can be 100-300 parts by mass), 10-100 parts by mass of a first emulsifier (such as 10, 20, 30, 40, 50, etc. parts by mass; which can be selected from soybean phospholipid (such as Lipoid S75, Lipoid S100, etc.), sunflower seed phospholipid, polyene phosphatidylcholine, etc.), 0-100 parts by mass of a second emulsifier (such as 0, 10, 20, 30, 40, 50, etc. parts by mass; which can be selected from egg yolk lecithin (such as E80), polysorbate (such as polysorbate 80), sorbitan oleate 80, etc.), 0-1.2 parts by mass of α-tocopherol (further can be 0.1-1 parts by mass, and further can be 0.5-1 parts by mass), 0-60 parts by mass of a base oil (further can be 30-50 parts by mass; which can be selected from corn oil, olive oil), and water (an appropriate amount of water); further, the total weight of the EPA-EE nanolipid composition can be 900-1100 parts by mass (preferably 1000 parts by mass). The types, specifications / models, and amounts of each component can further refer to the partial section 1.1 of the embodiment 1.

[0208] In some embodiments of the present application, the EPA-EE nanolipid composition comprises: 50-500 parts by mass of EPA-EE (further can be 100-400 parts by mass, and further can be 100-200 parts by mass), 10-100 parts by mass of a first emulsifier (such as 10, 20, 30, 40, 50, etc. parts by mass; which can be selected from soybean phospholipid (such as Lipoid S75), 0-60 parts by mass of a stabilizer (further can be 10-50 parts by mass, and further can be 10-20 parts by mass; which can be selected from TPGS, DSPE-PEG, S40, etc.), 0-50 parts by mass of a first adjuvant (further can be 10-30 parts by mass, and further can be 10-20 parts by mass; which can be selected from phosphatidylserine, sodium glutamate, taurine, etc.), and water (an appropriate amount of water); further, the total weight of the EPA-EE nanolipid composition can be 900-1100 parts by mass (preferably 1000 parts by mass). The types, specifications / models, and amounts of each component can further refer to the partial section 1.2 of the embodiment 1.

[0209] It should be understood that in each of the above examples, the specific examples of the types and amounts of each component can refer to the foregoing, and can be independent of each other.

[0210] It should be understood that in each of the above embodiments, the specific examples of each component can be independent of each other. The "appropriate amount of water" should satisfy the emulsification that can be achieved, and be able to control the appropriate particle size.

[0211] In each of the above embodiments, the total weight of the EPA-EE nanolipid composition can be about 1000 parts by mass (see Formulation Example 1).

[0212] In some embodiments of the present application, the EPA-EE nanolipid composition is a sub-microemulsion, and further, the average particle size is ≤ 500 nm. In some embodiments of the present application, the EPA-EE nanolipid composition is highly dispersed into a drug carrier (nanolipid carrier) with an average particle size of 10 nm to 500 nm, which facilitates the absorption of eicosapentaenoic acid by intestinal epithelial cells into mesenteric capillaries and into the systemic circulation, thereby improving the oral bioavailability and the blood concentration of EPA. In some embodiments of the present application, the average particle size of the droplets in the sub-microemulsion is less than 500 nm, and further, the average particle size can be ≤ 300 nm (e.g., 100 nm to 300 nm), and further, the average particle size can be ≤ 250 nm, and further, the average particle size can be about 200 nm. In some specific embodiments of the present application, the average particle size of the droplets in the sub-microemulsion is about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm.

[0213] In some embodiments of the present application, the particle size of the sub-microemulsion is 100 nm to 300 nm, which can effectively reduce blood lipids and reduce atherosclerotic plaques.

[0214] Second aspect of the invention

[0215] According to a second aspect of the present application, there is provided an EPA-EE nanolipid formulation comprising the EPA-EE nanolipid composition according to the first aspect of the present application. It should be understood that the EPA-EE nanolipid formulation is one of the forms of the EPA-EE nanolipid composition.

[0216] The EPA-EE nanolipid formulation encapsulates a high concentration of eicosapentaenoic acid-EE in a unique nanolipid formulation, which can meet the dosage of eicosapentaenoic acid required for high-efficiency lipid-lowering and atherosclerosis treatment in the body after oral administration.

[0217] The lipid preparation can be highly dispersed into nanometer scale droplets (e.g., droplets with an average particle size of 10 nm to 500 nm), which can facilitate the absorption of eicosapentaenoic acid by intestinal epithelial cells into the mesenteric capillary and reach the systemic circulation, thereby improving the oral bioavailability and the blood concentration of EPA. In some embodiments of the present application, the average particle size of the droplets in the EPA-EE nanolipid preparation is about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm.

[0218] Preferably, the EPA-EE nanolipid preparation is an oral preparation.

[0219] In some embodiments of the present application, the EPA-EE nanolipid preparation is an oral emulsion. In some embodiments of the present application, the average particle size of the droplets in the oral emulsion is less than 500 nm, further, the average particle size can be ≤ 300 nm, and more further, the average particle size can be ≤ 250 nm, and more further, the average particle size can be about 200 nm. For example, the particle size in the above-mentioned lipid preparation can be, for example, 100 nm to 300 nm.

[0220] In some embodiments of the present application, the EPA-EE nanolipid preparation has an oil-in-water structure. In the oil-in-water structure, the oil phase component such as EPA-EE ester is located in the oil phase.

[0221] The EPA-EE nanolipid preparation can be obtained by the preparation method of the third aspect of the present application.

[0222] In some embodiments of the present application, the EPA-EE nanolipid preparation preferably has the following effect: after oral administration of 400 mg / kg to rats, the maximum blood concentration is reached within 2 hours, and is higher than 550 μg / mL, and further preferably, the maximum blood concentration is higher than 700 μg / mL within 2 hours after oral administration of 400 mg / kg to rats.

[0223] In some embodiments of the present application, the EPA-EE nanolipid preparation preferably has the following effect: after oral administration of 400 mg / kg to rats, the total EPA concentration in the blood (including serum or plasma) is maintained at a range higher than 200 μg / mL for a time period of 2.5 h or more, and 100 μg / mL for a time period of 9 h or more. Further, the EPA concentration is maintained at a range higher than 200 μg / mL for a time period of 3 h or more, and 100 μg / mL for a time period of 10 h or more.

[0224] Third aspect of the invention

[0225] According to a third aspect of the present application, there is provided a method for preparing the EPA-EE nanolipid preparation, which can prepare the EPA-EE nanolipid preparation according to the second aspect of the present application.

[0226] The method for preparing can be selected from any one of emulsification, high-pressure homogenization, high-shear method, ultrasonic emulsification, microfluidization, etc.

[0227] In some embodiments of the present application, the method for preparing comprises the following steps:

[0228] S100, preparing an oil phase matrix (preferably under inert gas protection): mixing the oil phase components including the EPA-EE raw material under heating conditions (preferably at a heating temperature of 50-70°C, for example 50°C, 55°C, 60°C, 65°C, or 70°C) to uniformity to prepare an oil phase matrix;

[0229] S200, preparing a water phase matrix (preferably under inert gas protection): dissolving the water phase components in an aqueous solvent to clarity to prepare a water phase matrix, or using water as the water phase matrix, preferably preheating the water phase to a certain temperature (preferably the same or similar temperature as the oil phase, for example 50-70°C, for example 50°C, 55°C, 60°C, 65°C, or 70°C);

[0230] S300, preparing a primary emulsion: mixing the oil phase matrix and the water phase matrix (the mixing can be performed under heating conditions, further at 50-70°C, for example 50°C, 55°C, 60°C, 65°C, or 70°C), shearing and stirring, and adding water to a preset volume to prepare an oil-in-water primary emulsion;

[0231] S400, preparing a sub-micro emulsion: performing high-pressure homogenization on the oil-in-water primary emulsion to prepare a sub-micro emulsion, preferably with an average particle size of ≤500 nm (further preferably ≤300 nm, and further for example 100-300 nm).

[0232] The oil phase components refer to lipid-soluble components. The water phase components refer to water-soluble components. It should be understood that some components have amphiphilic properties and can be used as both oil phase components and water phase components.

[0233] It should be understood that the primary emulsion and the sub-micro emulsion are both exemplary forms of the EPA-EE nanolipid composition of the present application.

[0234] In some embodiments of the present application, the sub-micro emulsion is further filtered after being prepared.

[0235] In some embodiments of the present application, the sub-micro emulsion is further packaged after being prepared.

[0236] In some embodiments of the present application, the sub-microemulsion is sterilized after being prepared.

[0237] In some embodiments of the present application, the sub-microemulsion is packaged after being prepared.

[0238] The above filtration, packaging, sterilization and packaging steps are each independently optional. Optionally, after step S400, step S500 is further performed, post-processing (preferably under inert gas protection): the sub-microemulsion is filtered, packaged and sterilized to obtain a sterilized EPA-EE nano-lipid preparation, which can be used as an oral emulsion.

[0239] The inert gas protection used in the above preparation process can be nitrogen protection.

[0240] It should be understood that the above steps of the preparation method have no order restrictions unless otherwise specified. For example, the steps of S100 and S200 have no order restrictions, but both are performed before S300. For another example, the order between packaging and sterilization is not limited.

[0241] As used herein, "aqueous solvent" refers to a solvent that provides a pharmaceutically acceptable aqueous phase. It can be water or a mixed solvent of water and other solvents.

[0242] In some embodiments, the pH adjusting agent is mixed with water (to adjust the pH of the aqueous solution with the pH adjusting agent) to obtain an aqueous solvent for subsequent preparation. The pH of the aqueous solvent is adjusted according to the needs of the final preparation of the emulsion, and then a suitable pH adjusting agent is selected. In some embodiments, the pH of the final emulsion is 7-8.

[0243] In some embodiments, the pH adjusting agent is included in the preparation raw materials, and the pH is adjusted after the dispersion treatment in S300 is completed. The pH of the system is preferably suitable for obtaining stable and appropriate particle size, not affecting the play of drug activity, and being suitable for pharmaceutical use (especially oral pharmaceutical use). For example, the pH can be adjusted to pH 7-8.

[0244] In some embodiments of the present application, the EPA-EE nano-lipid preparation can be prepared by using a stator-rotor shear machine in combination with a high-pressure homogenizer or a microfluidizer. The operating parameters can be adjusted according to the particle size requirement. In some embodiments, the rotation speed of the stator-rotor shear machine is 7000 rpm to 10000 rpm, for example, 7000 rpm, 8000 rpm, 9000 rpm, or 10000 rpm. The stirring time can be 3 min to 5 min, for example, 3 min, 4 min, or 5 min. In some embodiments, the pressure of the high-pressure homogenization is 200 bar to 800 bar, for example, 200 bar, 300 bar, 400 bar, 500 bar, 600 bar, 700 bar, or 800 bar. The homogenization can be performed once or multiple times, preferably multiple times, for example, 3 to 10 times, or for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.

[0245] In some specific embodiments of the present application, the preparation method comprises the following steps: mixing eicosapentaenoic acid-EE and other lipid-soluble ingredients as an oily base (also referred to as an oil phase) and preheating to a certain temperature (for example, 50°C to 70°C); adding the water-soluble ingredients in the EPA-EE nano-lipid composition into water, preheating to a certain temperature (preferably the same or similar temperature as the preparation of the oil phase, for example, 50°C to 70°C) to obtain an aqueous solution as an aqueous base (also referred to as an aqueous phase); mixing the preheated oily base and the aqueous solution uniformly under the stirring of a stator-rotor stirring shear machine to obtain a primary emulsion, and then passing the formed primary emulsion through a high-pressure homogenizer to prepare a sub-micro emulsion with a certain particle size; the obtained sub-micro emulsion can be filtered or not filtered; then, under the protection of nitrogen filling, it is filled into an oral liquid bottle and sterilized at high temperature (preferably 100°C to 121°C) to obtain a sterilized EPA-EE nano-lipid preparation.

[0246] In some specific embodiments of the present application, the preparation method comprises the following steps:

[0247] S100: mixing the oil phase components under the protection of inert gas until a uniform oil solution is formed, heating in a water bath to 50°C to 70°C to obtain a preheated oil phase; preferably, the inert gas is nitrogen;

[0248] S200: mixing the water phase components in the formula under the protection of inert gas, stirring and dissolving until a uniform aqueous solution is formed, heating in a water bath to 50°C to 70°C to obtain a preheated water phase; preferably, the inert gas is nitrogen;

[0249] S300: mixing the obtained preheated water phase and oil phase to form an oil-in-water sub-micro emulsion (a non-sterilized EPA-EE nano-lipid preparation) by shearing or high-pressure homogenization;

[0250] Further, the obtained sub-micro emulsion water bath can be heated to 50-70℃, and then filtered, sterilized and packaged, in which inert gas protection is adopted. The sterilized EPA-EE nano-lipid preparation is obtained.

[0251] In the case of specifying the kind and amount of raw materials, the skilled in the art can implement the above preparation method according to the above instructions to obtain the EPA-EE nano-lipid preparation of the present application.

[0252] Fourth aspect of the invention

[0253] According to the fourth aspect of the present application, the use of the EPA-EE nano-lipid composition of the first aspect of the present application, or the EPA-EE nano-lipid preparation of the second aspect of the present application, or the EPA-EE nano-lipid preparation obtained by the preparation method of the third aspect of the present application is provided, and further, the use includes the use in the preparation of prophylactic and / or therapeutic drugs (especially drugs for cardiovascular diseases), and also includes the use in medical foods and health foods.

[0254] In some embodiments of the present application, the use of the EPA-EE nano-lipid composition of the first aspect of the present application, or the EPA-EE nano-lipid preparation of the second aspect of the present application, or the EPA-EE nano-lipid preparation obtained by the preparation method of the third aspect of the present application in the preparation of drugs is provided, preferably, the drugs are used for preventing and / or treating diseases related to fat accumulation.

[0255] In some preferred embodiments of the present application, the cardiovascular disease is atherosclerosis.

[0256] Through the inventor's experimental verification, the EPA-EE nano-lipid preparation prepared by the present application contains a high concentration of eicosapentaenoic acid-EE, which can promote the oral absorption of EPA, and the peak time can be advanced by 1h compared with directly taking fish oil, and also improves the bioavailability of EPA. With the stabilizer for maintaining the plasma concentration of EPA and the first adjuvant for promoting the combination of EPA with lipoprotein, the EPA-EE nano-lipid preparation can reduce or delay the rapid clearance of EPA in the plasma, increase the interaction time of EPA with the lipid components in the plasma, and increase the content of eicosapentaenoic acid in the lipoprotein through active combination with the lipoprotein. Compared with ordinary fish oil and fish oil capsules, the EPA-EE nano-lipid preparation prepared by the present application has higher bioavailability, can fully exert the effects of EPA in reducing blood lipids and treating atherosclerosis, and promote the body to restore normal physiological state.

[0257] Fifth aspect of the invention

[0258] According to a fifth aspect of the present application, there is provided a method for preventing and / or treating cardiovascular diseases, comprising administering to a subject in need thereof a therapeutically effective amount of the EPA-EE nanolipid composition (first aspect) or the EPA-EE nanolipid formulation (second aspect or third aspect) of the present application. Further, the cardiovascular diseases can be atherosclerosis.

[0259] As used herein, "therapeutically effective amount" means an amount of the EPA-EE nanolipid of the present application (or an amount of EPA) that will elicit the biological or medical response of a subject, for example, an amount of the EPA-EE nanolipid of the present application (or an amount of EPA) that will have a physiologically and / or pharmacologically positive effect on the subject, including, but not limited to, reducing or inhibiting an enzyme or protein activity or ameliorating symptoms, alleviating conditions, slowing or delaying disease progression, or preventing a disease, etc.

[0260] As used herein, "pharmaceutically acceptable" means those agents, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for administration to a patient and are commensurate with a reasonable benefit / risk ratio.

[0261] As used herein, "patient" means an animal, preferably a mammal, and more preferably a human. The term "mammal" primarily refers to warm-blooded vertebrate classes of mammals, including, but not limited to, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs, cows, sheep, horses, and humans.

[0262] As used herein, "administering" means, unless otherwise specified, administering the EPA-EE nanolipid formulation of the present application.

[0263] In some embodiments of the present application, the subject is a rat, and the administration is at a dose of 400 mg / kg, either single or multiple administrations.

[0264] In some embodiments of the present application, the EPA-EE nanolipid formulation of the present application is administered to a rat orally, and the maximum blood concentration is achieved within 2 hours after the rat is orally administered 400 mg / kg of the formulation, and is higher than 550 μg / mL, and in some embodiments, higher than 700 μg / mL.

[0265] In some embodiments of the present application, the EPA-EE nanolipid formulation of the present application is administered to a rat orally, and the total EPA concentration in the blood (including serum or plasma) of the rat is maintained at a range higher than 200 μg / mL for a time period of 2.5 h or more, and 100 μg / mL for a time period of 9 h or more, after the rat is orally administered 400 mg / kg of the formulation. Further, the total EPA concentration is maintained at a range higher than 200 μg / mL for a time period of 3 h or more, and 100 μg / mL for a time period of 10 h or more. Specific embodiments

[0267] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. These examples are only used to illustrate the present application, and are intended to illustrate the specific formula composition, preparation method, and functions and effects thereof, and should not be understood as any form of limitation on the scope of the present application. Without departing from the technical principles of the present application, several improvements and adjustments can also be made, which should also be considered as the protection scope of the present application. The experimental methods in the following examples without specific conditions are preferred to refer to the guidance given in the present application, and can also be carried out according to the experimental manual or conventional conditions in the art, or according to the conditions suggested by the manufacturer, or according to the experimental methods known in the art.

[0268] In the following specific examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range if not otherwise specified. The temperature and time parameters allow for acceptable deviations caused by instrument testing accuracy or operation accuracy.

[0269] In the following examples, "room temperature" refers to 20-30°C.

[0270] The raw material information of EPA-EE, phospholipids and the like used in the following examples and comparative examples is shown in Table 1.

[0271] Table 1. Raw material information of EPA-EE, phospholipids and the like used in the examples and comparative examples of the present application.

[0272]

[0273]

[0274] In the following examples, EPA-EE represents eicosapentaenoic acid-EE (also denoted as EPA-EE), and PC represents phosphatidylcholine. In the following examples, the particle size was tested by Zetasizer Nano ZS 90 (Malvern) laser particle size instrument.

[0275] 1. Formulation Examples

[0276] Example 1.1. Preparation of eicosapentaenoic acid-EE nano-lipid preparation containing eicosapentaenoic acid-EE and emulsifier

[0277] 1.1.1. Raw materials

[0278] The eicosapentaenoic acid-EE nano-lipid preparation (also recorded as EPA-EE nano-lipid preparation) was prepared by using the raw material composition and amount shown in Table 2. Among them, the EPA-EE 60 was selected from KinOmega Company, model 6015 EE EPA 60%+DHA 12%, containing EPA-EE 60 g per 100 g. The EPA-EE 80 was selected from BASF Company K85EE Omega-3-acid-EE (EPAEE 86227-47-6), containing EPA-EE 80 g per 100 g. The EPA-EE 97 was selected from BASF Company Maxomega EPA 97EE, containing about 97 g of EPA-EE per 100 g. The information of each raw material also refers to Table 1.

[0279] Table 2. Composition and particle size of different eicosapentaenoic acid-EE nano-lipid preparations

[0280]

[0281]

[0282] Among them,

[0283] The first emulsifier used in preparation 1-1, 1-6, 1-11, preparation 2-1, 2-6, 2-11, preparation 3-1, 3-6, 3-11 is soybean phospholipid Lipoid S75; the second emulsifier used is egg yolk lecithin E80; the antioxidant is α-tocopherol; and the base oil is an equal mixture of corn oil and olive oil.

[0284] The first emulsifier used in preparation 1-2, 1-7, 1-12, preparation 2-2, 2-7, 2-12, preparation 3-2, 3-7, 3-12 is soybean phospholipid Lipoid S100; the second emulsifier used is polysorbate 80; the antioxidant is α-tocopherol; and the base oil is corn oil.

[0285] The first emulsifier used in preparation 1-3, 1-8, 1-13, preparation 2-3, 2-8, 2-13, preparation 3-3, 3-8, 3-13 is sunflower seed phospholipid Lipoid H100; the second emulsifier used is polysorbate 80; the antioxidant is α-tocopherol; and the base oil is corn oil.

[0286] The first emulsifier used in preparation 1-4, 1-9, 1-14, preparation 2-4, 2-9, 2-14, preparation 3-4, 3-9, 3-14 is polyenyl phosphatidylcholine; the second emulsifier used is sorbitan oleate 80; the antioxidant is α-tocopherol; and the base oil is olive oil.

[0287] The first emulsifier used in Formulations 1-5, 1-10, 1-15 is an equal mixture of soybean phospholipids Lipoid S75; the second emulsifier used is sorbitan oleate 80; the antioxidant is alpha-tocopherol; and the base oil is olive oil.

[0288] Wherein, "Particle size" refers to the average particle size of the emulsion droplets in different batches under the same prescription and preparation process. The same meaning is indicated in the following examples.

[0289] 1.1.2. Preparation

[0290] The liposoluble components such as eicosapentaenoic acid-EE (EPA-EE), emulsifiers, antioxidants, etc. are added to the same container according to the amounts in Table 2, preheated to about 50-70°C, and stirred vigorously until evenly dispersed, as the oil phase matrix. The water-soluble components such as pH adjusters, flavorings, etc. are added to the same container according to the amounts in Table 2, preheated to about 50-70°C, and stirred vigorously until evenly dispersed, as the aqueous matrix. Under the action of a stator-rotor type shear stirring at 7000-10000 rpm, the oily matrix is added to the aqueous matrix, stirred for 3-5 min until evenly dispersed, forming a milky white primary emulsion, and then water is added to make up to 1000 mL; then the primary emulsion is subjected to homogenization emulsification, with a homogenization pressure range of 200-800 bar, 6-10 times, until the average particle size is below 300 nm as determined by sampling. Then filtered through a 0.65 μm microporous filter. The emulsion particle size is tested (the results are shown in Table 2). Nitrogen charging, filling, high-temperature sterilization at 115°C for 30 min, and storage of the obtained nano-lipid preparation at room temperature are carried out.

[0291] Example 1.2. Preparation of eicosapentaenoic acid-EE nano-lipid preparations containing different emulsifiers, stabilizers, and auxiliary materials for promoting lipoprotein binding

[0292] According to the types and amounts of raw materials in Table 3, each ingredient is weighed, and the nano-lipid preparation is prepared according to the method of 1.1.2. in Example 1.1. The particle size is measured. After preparation is completed, it is stored at room temperature.

[0293] Table 3. Composition and particle size of different eicosapentaenoic acid-EE nano-lipid preparations

[0294]

[0295] Wherein, all the formulations in Table 3 contain, in addition to the above amounts, antioxidant alpha-tocopherol 1 g and olive oil 30 g.

[0296] The first emulsifier used in Formulation 4-1, 4-6, 4-11, Formulation 5-1, 5-6, 5-11, Formulation 6-1, 6-6, 6-11 is soybean phospholipid Lipoid S75; the stabilizer used is TPGS; the first adjuvant is phosphatidylserine;

[0297] The first emulsifier used in Formulation 4-2, 4-7, 4-12, Formulation 5-2, 5-7, 5-12, Formulation 6-2, 6-7, 6-12 is soybean phospholipid Lipoid S75; the stabilizer used is DSPE-PEG; the first adjuvant is sodium glutamate;

[0298] The first emulsifier used in Formulation 4-3, 4-8, 4-13, Formulation 5-3, 5-8, 5-13, Formulation 6-3, 6-8, 6-13 is soybean phospholipid Lipoid S75; the stabilizer used is S40; the first adjuvant is phosphatidylserine;

[0299] The first emulsifier used in Formulation 4-4, 4-9, 4-14, Formulation 5-4, 5-9, 5-14, Formulation 6-4, 6-9, 6-14 is soybean phospholipid Lipoid S75; the stabilizer used is S40; the first adjuvant is taurine;

[0300] The first emulsifier used in Formulation 4-5, 4-10, 4-15, Formulation 5-5, 5-10, 5-15, Formulation 6-5, 6-10, 6-15 is soybean phospholipid Lipoid S75; the second emulsifier used is an equal mixture of TPGS and S40; the first adjuvant is an equal mixture of taurine and sodium glutamate.

[0301] Comparative Example

[0302] Comparative Example 1 (denoted as D1): 100 g of ordinary fish oil (EPA content 18.2%) was mixed with 10 g of soybean phospholipid, water was added to 1 kg, and a nano-lipid preparation was prepared according to the method of Reference Example 1, which was stored at room temperature until the implementation evaluation.

[0303] Comparative Example 2 (denoted as D2): Vascepa, a product on the market developed by Amarin Corporation (USA) Ltd., is an EPA-EE capsule with a purity of more than 97%.

[0304] 2. Effect Evaluation

[0305] In this example, "drug" refers to a component that provides EPA.

[0306] Example 2.1. In vitro release of pentacosanoid acid-EE-rich lipid preparation

[0307] Simulated fasting state intestinal fluid: 100 mM Tris, 300 mM NaCl, 10 mM CaCl2, 10 mM sodium cholate, 2.5 mM phospholipid (soybean phospholipid S100, Germany Lipoid), corresponding proportion of purified water, stirring and dissolving and mixing, adjusting pH to 7.5±0.05 with 0.5 g / mL aqueous maleic acid solution. 100 mg of porcine pancreatic lipase was added to 1 mL of the above solution to obtain an in vitro fasting state digestion simulation buffer.

[0308] An equal amount of experimental group preparations (preparation 4-1, preparation 1-6, preparation 1-11, preparation 2-6, preparation 3-6, preparation 4-6), control group preparations (control example 1 uses preparation D1, control example 2 uses preparation D2) were taken into dialysis bags, the two ends were tightly tied, placed in a beaker, and 200 mL of artificial intestinal fluid was added, the temperature was maintained at 37°C, the rotation speed was 100 rpm, and 10 mL (V 取出体积 ) was taken out at regular time intervals (0.25 h, 0.5 h, 1 h, 2 h, 4 h), and the corresponding amount of dissolution medium at the same temperature was promptly supplemented. After methylation, the in vitro release was determined by gas chromatography.

[0309] The experimental parameters for methylation are as follows:

[0310] Step one: Take a clean glass test tube with a stopper, add 100 μL of 300 μg / mL methyl heptadecanoate (internal standard), dry under N2, then add 100 μL of the sample to be tested, 2 mL of 0.5 mol / L KOH-MeOH solution, 0.5 mL of BHT in isooctane solution, seal with a cap, vortex for 60 s, mix well, and then stand for 10-15 min. Collect the isooctane layer in a clean sample vial with a small amount of anhydrous sodium sulfate added.

[0311] Step two: Add 2 mL of 5% H2SO4-MeOH solution to the lower layer solution of step one, seal after slightly charging N2 on the surface of the solution, mix well by vortexing, and react at 70°C for 30 min. After cooling to about 40°C, add 0.5 mL of isooctane, vortex for 30 s, add 0.5 mL of saturated sodium chloride solution, vortex for 15 s, collect the isooctane layer, and combine with the organic layer of step one. After drying with anhydrous sodium sulfate, transfer to a sample vial with a sample sleeve as the test sample solution.

[0312] The instrument for gas phase test is Agilent 7890A gas chromatograph, and the test parameters are as follows: gas chromatography conditions are (88%-cyanopropyl) aryl-polysiloxane capillary column (60 m x 0.25 mm x 0.2 μm), programmed temperature, 0 min 170°C, 3.5°C / min speed to 240°C, holding for 10 min, injector temperature is 250°C, detector temperature is 270°C. The carrier gas is helium, and the flow rate is 1.0 mL / min. Split ratio: 10:1. Injection volume 1 μL.

[0313] The method for calculating the percentage of drug release at time point (t) is as follows:

[0314]

[0315] Wherein, V 体系总体积 Refers to 200 mL; V 取出体积 Refers to 10 mL; C t检测浓度 Refers to the drug detection concentration at time t; C t-1检测浓度 Refers to the drug detection concentration at the previous time point; M 总量 Refers to the total amount of eicosapentaenoic acid-EE contained in the preparation.

[0316] The percentage of drug release at different time points is shown in Table 4. In the simulated fasting state, the intestinal juice contains less digestive enzymes. After EPA-EE is encapsulated in the capsule (preparation D2), the release rate in the fasting intestinal juice is slow, and only 49.5% is reached after 2 h, and it cannot be completely released after 4 h. In contrast, after EPA-EE is administered in the form of emulsion (each experimental group), the release rate in the fasting intestinal juice is fast, and more than 80% is reached in 1 h. This is related to the fact that nanoemulsion provides a larger specific surface area. The rapid release of EPA-EE in the intestinal environment helps to be rapidly absorbed into the blood after oral administration, and fully exert the efficacy. In addition, the addition of different emulsifiers and other excipients in Table 3 can achieve good release of nano-lipid preparation in the intestinal environment.

[0317] Table 4. In vitro release of EPA in simulated intestinal digestive juice of each group of preparations (n=3)

[0318]

[0319] Example 2.2. Pharmacokinetic study of different eicosapentaenoic acid-EE nano-lipid preparations

[0320] Male Sprague-Dawley (SD) rats were taken 54, weighing 200 ± 20 g, 6 in each group, respectively, the experimental group preparation (preparation 4-1, preparation 5-1, preparation 6-1, preparation 6-2, preparation 6-3, preparation 6-4, preparation 6-5, preparation 6-6), control group preparation (control example 1 uses preparation D1, control example 2 uses preparation D2). The dose of eicosapentaenoic acid-EE is 400 mg / kg. 0.5 mL of blood was taken at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h after oral administration, and placed in a centrifuge tube containing 1% (w / v) heparin sodium. After centrifugation at 3000 rpm for 10 min at 4°C, the supernatant was stored at -20°C for detection of drug concentration. The content of EPA in the plasma was detected by gas chromatography, and the data were statistically analyzed by Graphpad Prism software. T test was used for comparison between two groups, and variance analysis and multiple comparison were used for multiple groups. The experimental data are expressed as mean ± SD, and SD represents standard deviation.

[0321] The instrument and parameters of gas chromatography are the same as in Example 2.1.

[0322] Table 5. Evaluation of the pharmacodynamic level of each group of preparations

[0323]

[0324] After the SD rats were orally administered with the nine preparations, the content of EPA in the blood was detected at the sampling time points, and the results are shown in Table 5. According to the results, the peak time of the experimental group and control group 1 (preparation D1) is 2 h earlier than that of control group 2 (preparation D2, capsule group), and it can be seen that the nano-lipid preparation provided by the application has significant advantages in promoting absorption. In addition, the peak concentration and bioavailability of the drug between preparation D1, preparation 4-1, 5-1 and 6-1 increase with the content of EPA-EE, and it can be seen that the eicosapentaenoic acid-EE nano-lipid preparation (each experimental group) of high-purity EPA has more advantages. In the experimental group, the maintenance time of 200 μg / mL EPA concentration is more than 2.0 h, and the maintenance time of 100 μg / mL EPA concentration is more than 9 h.

[0325] Different types of stabilizers were selected in preparation 6-1, 6-2, 6-3 and 6-4. The emulsion of TPGS, DSPE-PEG and S40 as stabilizers, the maintenance time of 200 μg / mL EPA concentration can reach more than 3 h, and the maintenance time of 100 μg / mL EPA concentration is more than 10 h. However, the preparation group 6-4 with F68 as stabilizer does not show obvious stabilizing effect. It can be seen that the addition of PEG segment with lipophilic end has better effect on maintaining blood drug concentration and shows higher bioavailability (see AUC0-24h In addition, the preparation group 6-6 further added taurine to promote the binding of lipoprotein, promote the insertion of EPA into lipoprotein to maintain the concentration, and has the best effect on the basis of 6-1.

[0326] Example 2.3. Investigation of EPA content in lipoprotein in rats in vivo by different eicosapentaenoic acid-EE nano-lipid preparations

[0327] Take 54 male Sprague-Dawley (SD) rats, weighing 200±20g, free to eat basic feed for one week, and adapt to the environment. After the animals are randomly divided into blank control group and model group, the blank group is fed with ordinary feed, and the model group is fed with high-fat feed for two weeks to complete the modeling. The experimental groups are given preparations (preparation 4-1, preparation 5-1, preparation 6-1, preparation 6-2, preparation 6-3, preparation 6-4, preparation 6-5, preparation 6-6), and the control group is given preparations (control example 1 uses preparation D1, and control example 2 uses preparation D2). The dose of eicosapentaenoic acid oil phase is 400mg / kg. Intragastrically administered once a day, free to eat and drink water, and the intervention ends at the end of the 11th week (the intervention time is 8 weeks).

[0328] The mice in each group were anesthetized after administration, and blood was taken from the heart and placed in a centrifuge tube containing 1% (w / v) heparin sodium. Low temperature 3000rpm, centrifugal 10min to separate plasma. The lipoprotein-containing fraction was separated from the plasma by iodixanol density gradient centrifugation. The lipids were extracted and separated by acid / methanol / chloroform, and after centrifugation, purified by isohexane and solid phase extraction, and confirmed complete hydrolysis and methylation of lipids (acid / methanol, 50℃ overnight). Full titration of EPA was performed, and the verified liquid chromatography / tandem mass spectrometry method was used to measure the EPA concentration. The test results were statistically analyzed by Graphpad Prism software, and the T test was used for comparison between two groups, and the variance analysis and multiple comparisons were performed between multiple groups. The experimental data is expressed as mean±SD, P<0.05 is statistically significant, and SD represents the standard deviation.

[0329] Method for testing EPA content: The total titration of EPA is based on the EPA methyl ester formed during the transmethylation process. For non-esterified EPA, a loran inhibitor solution (0.5 g sodium fluoride, 1.0 g L-ascorbic acid and 0.25 g 5-methylisoxazole-3-carboxylic acid per 10 mL water) is added to each 1 mL of plasma sample to prevent degradation. The lipids are extracted with methanol / chloroform (without hydrolysis or methylation), centrifuged, and then purified by protein precipitation and solid phase extraction. The EPA concentration is measured using a validated liquid chromatography / tandem mass spectrometry (Charles River Laboratories Ltd, Elphinstone Research Center, Tranent, Scotland, UK) method. The analyte is separated by a Perkin Elmer liquid chromatography system (Perkin Elmer, Beaconsfield, Cheshire, UK) using an ascesis R Express C18 column: 2.7 mm (Sigma-Aldrich Co. Ltd, Poole, UK) at a flow rate of 1 mL / min and a column temperature of 60°C with a mobile phase of 60% / 40% (A / B) to 100% A. The mobile phase A is acetonitrile / acetic acid (100 / 0.5, v / v) and the mobile phase B is water / acetic acid (100 / 0.5, v / v).

[0330] The results of the experiment are shown in Table 1. Figure 1 The EPA content in the low density lipoprotein varied greatly after oral gavage of the seven formulations in SD rats. The EPA content in the low density lipoprotein was low in the control group 1 (formulation D1) with low purity of eicosapentaenoic acid (about 20%) despite the promotion of absorption by the lipid formulation. The EPA content in the low density lipoprotein was low in the control group 2 (formulation D2) which was a capsule formulation with slow absorption and metabolism. Formulations 4-1, 5-1 and 6-1 were nanoemulsions containing more than 60% EPA-EE and the use of different emulsifiers did not affect the binding of EPA to the low density lipoprotein. Effective stabilizers, such as formulations 6-1, 6-2 and 6-3, allowed the EPA to stay in the blood for a long time by maintaining the blood concentration, which created conditions for the accumulation of EPA in the low density lipoprotein. In addition, the inclusion of auxiliary materials that competed with lipoproteins in formulation 6-6 promoted the active enrichment of EPA in lipoproteins.

[0331] Example 2.4. Investigation of different eicosapentaenoic acid-EE nano-lipid formulations improving blood lipid levels

[0332] SD male rats (Shanghai Experimental Animal Research Center) weighing 200 ± 20 g. The animals were randomly divided into a blank control group and a model group after being fed with basic feed for one week and adapting to the environment. The blank group was fed with ordinary feed and the model group was fed with high-fat feed for two weeks to complete modeling.

[0333] After the molding was completed, the blank control group and the model control group rats were given deionized water by gavage every day, and the rest of the groups were given the experimental group preparations (preparation 3-1, preparation 3-2, preparation 3-6, preparation 6-1, preparation 6-2, preparation 6-6, preparation 6-7), control group preparations (control example 1 uses preparation D1, control example 2 uses preparation D2), for 28 days, and the amount of administration is equivalent to the dose of EPA-EE of 400 mg / kg. After 14 days and 28 days of administration, the animals were fasted for 4 h, anesthetized, and whole blood samples (not less than 0.5 mL) were collected, centrifuged at 4°C and 4000 rpm for 15 min, and the supernatant serum was taken. The wavelength was measured at 510 nm by ultraviolet-visible spectrophotometer. The serum total cholesterol (TC) was determined by CHOD-PAP method, and the serum triglyceride (TG) was determined by GPO-PAP method.

[0334] CHOD-PAP method: total cholesterol determination kit (Jiangsu Yingnuohua Medical Technology Co., Ltd.) was used. The enzyme reagent and diluent were mixed at a ratio of 1:4 to prepare the working solution. In the centrifuge tubes of the blank group, standard group and test group, reagents were added in turn: distilled water 10 μL, standard solution (different concentrations of cholesterol solution) 10 μL, serum standard solution 10 μL, and then enzyme working solution 10 μL. After mixing, 37°C water bath for 15 min. At wavelength 510 nm, blank zero, read the absorbance of each tube, and the serum total cholesterol concentration was calculated according to the standard curve.

[0335] GPO-PAP method: triglyceride detection kit (Beijing Legen Biological Technology Co., Ltd.) was used. In the centrifuge tubes of the blank group, standard group and test group, reagents were added in turn: distilled water 10 μL, standard solution (different concentrations of triglyceride solution) 10 μL, serum standard solution 10 μL, and then enzyme working solution standard solution 10 μL. After mixing, 37°C water bath for 15 min. At wavelength 510 nm, blank zero, read the absorbance of each tube, and the serum triglyceride concentration was calculated according to the formula: TG (mmol / L) = {(test tube absorbance-blank absorbance) / (standard tube absorbance-blank absorbance)} x 1.7 mmol / L.

[0336] Table 6. Lipid-lowering effect of different groups (TC and TG levels)

[0337]

[0338] The experimental results are shown in Table 6. The EPA-EE nanolipid composition exhibits good lipid-lowering effects. The phospholipid emulsifiers used in formulations 3-1, 3-2, and 3-6 have different iodine values. Group 3-2, with its higher iodine value, exhibits the best lipid-regulating effect, resulting in lower TC and TG levels in rat blood. Furthermore, the more effective stabilizer groups 6-1 and 6-2 show more significant lipid-regulating effects compared to group 3-2. Additionally, excipient group 6-7, which combines lipoprotein binding, further amplifies the lipid-lowering effect of the lipid nanocomposition.

[0339] Observation of the experimental animals during the experiment showed that, except for the model group, the rats in all groups exhibited normal activity, and their physical appearance and feces showed no abnormalities. After four weeks of administration, there was no statistically significant difference in body weight among the groups. These experimental results demonstrate the safety of the aforementioned nanoliposome formulation.

[0340] Example 2.5. Pharmacodynamic study of different eicosapentaenoic acid-EE nanolipid formulations in reducing plaque.

[0341] 120 SPF-grade male ApoEs aged 6-8 weeks - / - Mice (weighing 18-22g) were randomly housed in cages. They were acclimatized for one week with a standard diet. If no abnormalities were observed after one week, they were fed a high-fat diet with free access to food and water. This process was repeated for 12 weeks to establish an arterial plaque model. At the end of the 12th week, mice were randomly selected, processed according to pathological sampling methods, and subjected to H&E staining. The presence and morphology of plaques were observed to confirm successful model replication. Based on confirmed model success, in the 13th week, the remaining model mice were further sorted by weight and randomly assigned to 11 groups: atorvastatin group (PD, positive control group), experimental groups (formulations 2-1, 3-1, 3-6, 6-1, 6-2, 6-6), formulation control group (formulations D1, D2), and model group (M, given an equal amount of water). Additionally, 10 healthy mice were not used for modeling and served as the control group (N).

[0342] After weighing the mice in each group, the intervention was started by gavage at 13 weeks. The dosing regimen for each group of mice was as follows: the PD group was given atorvastatin at 5 mg / kg / time; the experimental group and the formulation control group were converted to a dose containing EPA-EE at 100 mg / kg / time. The model group (M) and the model control group (N) were given an equal volume of distilled water (distilled water prepared in the same way as the drug). Gavage was performed twice daily, and the mice were allowed to eat and drink freely. The drug intervention was performed until the end of the 12th week (the drug intervention time was 8 weeks). After the start of the drug intervention, the body weight of the mice was measured once a week. After 24 h of the last intervention, the mice in each group were weighed again, and pathological sampling was performed. Subsequently, the mice were sacrificed by cervical dislocation after ether anesthesia, and the thoracic cavity was quickly opened to bluntly dissect the aorta. Macroscopic observation and photography were performed. The aorta was fixed in 4% formaldehyde solution, sectioned, stained with H&E, and observed and photographed under an optical microscope. The lumen and plaque areas were analyzed using an Image ProPlus 6.0 computer image analysis system, and the percentage of the plaque area to the lumen area was calculated.

[0343] The results are shown in Table 1. Figure 2 Compared with the model group (M), EPA-EE encapsulated by the lipid preparation had the effect of delaying the progression of arterial plaques, and the higher the purity of the raw material, the smaller the plaque area. Consistent with the blood lipid regulation, the groups containing emulsifiers with higher iodine values (formulation 3-1), non-ionic high molecular segment derivatives (formulations 6-1, 6-2, and 6-6) had better therapeutic effects. In particular, in the auxiliary material 6-6 group with the function of promoting the combination of EPA-EE and low-density lipoprotein, the therapeutic effect was close to the positive control group PD using statin drugs. Compared with the control groups (D1 and D2), each of the above experimental groups could significantly slow down the development of arterial plaques.

[0344] Each of the technical features of the above-described embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, however, as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

[0345] The above-mentioned embodiments only express several implementation manners of the present application, facilitate concrete and detailed understanding of the technical scheme of the present application, but cannot be understood as a limitation on the patent protection scope of the present application. It should be noted that, for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. In addition, it should be understood that, after reading the above teaching content of the present application, the skilled in the art can make various modifications or modifications to the present application, and the equivalent forms obtained are also within the protection scope of the present application. It should also be understood that, on the basis of the technical scheme provided by the present application, the skilled in the art can obtain the technical scheme through logical analysis, reasoning or limited test, which is within the protection scope of the appended claims of the present application. Therefore, the protection scope of the present application patent should be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. An ethyl eicosapentaenoate (EPA-EE) nano-lipidic composition for use as an oral formulation, characterized in that, The EPA-EE nanolipid composition comprises the following components in the following weight percentages, based on the total weight of the EPA-EE nanolipid composition: EPA-EE raw material 1%~30%(w / w); First emulsifier 0.1%~10%(w / w); Second emulsifier 0%~10%(w / w); Stabilizer 0~5%(w / w); First auxiliary material 0~5%(w / w); Second auxiliary material 0~15%(w / w); and Water; wherein, The mass content of EPA-EE in the EPA-EE raw material is ≥60%; The first emulsifier is a high-unsaturation phospholipid, the iodine value of the high-unsaturation phospholipid is ≥70; and the first emulsifier is selected from one or more of soybean phospholipid, sunflower seed phospholipid, and polyene phosphatidylcholine; In the high-unsaturation phospholipid, the mass percentage of phosphatidylcholine is ≥50%; In the EPA-EE nanolipid composition, the ratio of the mass parts of EPA-EE to the mass parts of the first emulsifier is (50~500):(40~50); The second emulsifier is composed of components different from the first emulsifier, and the second emulsifier is selected from food and / or pharmaceutically acceptable raw and auxiliary materials; The stabilizer is a non-ionic high-molecular polymer; The first auxiliary material is an auxiliary material that promotes the binding of EPA and lipoprotein; The second auxiliary material is a food and / or pharmaceutically acceptable raw and auxiliary material, and is different from the first emulsifier, the second emulsifier, the stabilizer, and the first auxiliary material; The minimum weight percentage of water in the EPA-EE nanolipid composition is 65%(w / w).

2. The EPA-EE nano-lipidic composition according to claim 1, characterized in that, The content of the stabilizer is 0.1%~5%(w / w), and / or the content of the first auxiliary material is 0.1%~5%(w / w), based on the total weight of the EPA-EE nanolipid composition.

3. The EPA-EE nano-lipid composition according to claim 1, wherein, The EPA-EE nanolipid composition comprises the following components in the following weight percentages, based on the total weight of the EPA-EE nanolipid composition: EPA-EE raw material 4%~20%(w / w); First emulsifier 0.5%~5%(w / w); Second emulsifier 0.5%~5%(w / w); Stabilizer 0.1%~3%(w / w); First auxiliary material 0.1%~3%(w / w); Second auxiliary material 0.01%~10%(w / w); and Water; In the EPA-EE nanolipid composition, the ratio of the mass parts of EPA-EE to the mass parts of the first emulsifier is (50~500):

50.

4. The EPA-EE nanolipid composition according to any one of claims 1~3, wherein, The EPA-EE raw material is selected from the ethyl esterization product of oil of one or more of deep-sea fish oil, seaweed oil, and krill oil; and / or The mass content of EPA-EE in the EPA-EE raw material is ≥70%; and / or The iodine value of the high-unsaturation phospholipid is ≥90; and / or In the high-unsaturation phospholipid, the mass percentage of phosphatidylcholine is ≥70%; and / or, The EPA-EE nanolipid composition comprises an emulsifier, the content of the emulsifier in the EPA-EE nanolipid composition is 0.5% to 10% by weight; and / or, The stabilizer is an amphiphilic non-ionic high molecular polymer, and the stabilizer is selected from one or more of vitamin lipid high molecular derivative, phospholipid high molecular derivative, fatty acid ester high molecular derivative and polyoxyethylene polyoxypropylene ether block copolymer; The vitamin lipid high molecular derivative is vitamin E polyethylene glycol succinate; The phospholipid high molecular derivative is a synthetic phospholipid modified by polyethylene glycol; The fatty acid ester high molecular derivative is a fatty acid ester modified by polyethylene glycol; The molecular weight of the PEG unit in the phospholipid high molecular derivative is 400 Da to 6000 Da; and / or, The molecular weight of the PEG unit in the fatty acid ester high molecular derivative is 200 Da to 4000 Da; and / or, The first adjuvant is selected from one or more of amino acid with negative group in side chain, amino acid derivative with negative group and small peptide with negative group in side chain, wherein the amino acid derivative with negative group in side chain in the first adjuvant is selected from one or more of phosphatidylserine, hexacosanoyl-glutamic acid-glutamine, hexacosanoyl-glutamic acid-glutamic acid and hexacosanoyl-glutamic acid-asparagine; and / or, The second adjuvant is selected from one or more of antioxidant, base oil, co-emulsifier, flavoring agent, interfacial film stabilizer, thickening agent and pH regulator; and / or, The EPA-EE nanolipid composition is sub-micro emulsion, and the average particle size is ≤500 nm; and / or, The EPA-EE nanolipid composition is used as oral preparation.

5. The EPA-EE nanolipid composition according to claim 4, wherein, The iodine value of the first emulsifier is greater than 90, and the first emulsifier is selected from one or more of soybean phospholipid, sunflower seed phospholipid and polyene phosphatidylcholine; and / or, The second emulsifier is selected from one or more of phospholipid different from the first emulsifier, sucrose ester, citric acid fatty acid glyceride, fatty acid glyceride, polysorbate, fatty acid sorbitan, polyoxyethylene fatty acid ester, span, alginate and caseinate; and / or, The PEG unit in the stabilizer provides a terminal group, and the terminal group is OH or methoxy; and / or, The vitamin lipid high molecular derivative is selected from one or more of d-α-tocopherol polyethylene glycol 200 succinate, d-α-tocopherol polyethylene glycol 400 succinate, d-α-tocopherol polyethylene glycol 1000 succinate, d-α-tocopherol polyethylene glycol 1500 succinate, d-α-tocopherol polyethylene glycol 2000 succinate and d-α-tocopherol polyethylene glycol 4000 succinate; and / or, the phospholipid high molecular weight derivative is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 5000, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 2000, dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000, soybean phosphatidylethanolamine-polyethylene glycol monomethylether 2000, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, dilauroylphosphatidyl-polyethylene glycol 2000, and dioleoylphosphatidylethanolamine-polyethylene glycol; and / or, the fatty acid ester high molecular weight derivative is selected from one or more of polyethylene glycol 400 oleate, polyethylene glycol 600 oleate, polyethylene glycol 4000 oleate, polyethylene glycol 6000 oleate, polyethylene glycol 400 dioleate, polyethylene glycol 600 dioleate, polyethylene glycol 200 laurate, polyethylene glycol 200 dilaurate, polyethylene glycol 400 laurate, polyethylene glycol 400 dilaurate, polyethylene glycol 400 stearate, and polyethylene glycol 400 distearate; and / or, the polyoxyethylene polyoxypropylene ether block copolymer is selected from one or more of Pluronic L65, Pluronic F68; and / or, the amino acid in the first adjuvant having a side chain with a negatively charged group is selected from one or more of aspartic acid, glutamic acid, taurine; and / or, the small peptide in the first adjuvant having a side chain with a negatively charged group is glutathione; and / or, the antioxidant in the second adjuvant is selected from one or more of vitamin E, alpha-tocopherol, alpha-tocopherol, gamma-tocopherol, mixed tocopherols, alpha-tocopherol acetate, alpha-tocopherol acetate, gamma-tocopherol acetate, mixed tocopherols acetate, ascorbic acid, ascorbic acid palmitate, ascorbic acid stearate, ascorbic acid myristate, sodium ascorbate, butylated hydroxyanisole, dibutylated hydroxytoluene, propyl gallate, and tertiary butylhydroquinone; and / or, the base oil in the second adjuvant is from one or more of soybean oil, olive oil, jojoba oil, sweet almond oil, grape seed oil, corn oil, walnut oil, sea buckthorn oil, olive oil, coix seed oil, grape seed oil, ginger oil, coconut oil, camellia oil, rose oil, peppermint oil, lemon oil, and medium-chain triglycerides; and / or, the average particle size of the EPA-EE nano-lipid composition is 100 nm ~ 300 nm.

6. An EPA-EE nano-lipid formulation characterized in that, The EPA-EE nano-lipid composition according to any one of claims 1-5; the EPA-EE nano-lipid preparation is an oral preparation.

7. The EPA-EE nano-lipidic formulation according to claim 6, wherein, The EPA-EE nano-lipid preparation is an oral emulsion.

8. The method of claim 6 or 7, wherein the EPA-EE nano-lipid preparation is prepared by, The method comprises the following steps: mixing the oil phase components including the EPA-EE raw material under heating conditions to prepare an oil phase matrix; dissolving the water phase components in an aqueous solvent to prepare a water phase matrix, or using water as the water phase matrix; mixing the oil phase matrix and the water phase matrix, and shearing to prepare an oil-in-water primary emulsion; subjecting the oil-in-water primary emulsion to high-pressure homogenization to prepare a sub-micro emulsion.

9. The method of claim 8, wherein the EPA-EE nano-lipid formulation is prepared by, After the sub-microemulsion is made, at least one of the following is performed: filtration, packaging, sterilization.

10. Use of the EPA-EE nano-lipidic composition for use as oral formulation according to any one of claims 1 to 5, or of the EPA-EE nano-lipidic formulation according to claim 6 or 7, or of the EPA-EE nano-lipidic formulation obtained according to the preparation process of claim 8 or 9, for the preparation of a medicament for the prevention and / or treatment of cardiovascular diseases; or, Use of the EPA-EE nano-lipidic composition according to any one of claims 1 to 5, or of the EPA-EE nano-lipidic formulation according to claim 6 or 7, or of the EPA-EE nano-lipidic formulation obtained according to the preparation process of claim 8 or 9, for the preparation of a medical food, a health food.

11. Use according to claim 10, characterized in that, The cardiovascular disease is atherosclerosis.

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