Nano-lipid injection with high eicosapentaenoic acid content, and preparation method and application thereof

Abstract

The present application relates to the technical field of pharmaceutical preparation, in particular to a nano-lipid injection with high EPA content, which provides high-concentration EPA through omega-3 fatty acid glyceride, and adopts the preparation form of nano-lipid injection, so that EPA exists in the form of nanoization, and the solubility is greatly improved; further, auxiliary materials for long circulation of EPA and optional auxiliary materials for enrichment of EPA in the liver are introduced, which can assist the enrichment of EPA in the liver during the circulation in the body, so as to maintain the effective blood drug concentration and liver tissue concentration of EPA for a long time, and more efficiently play the role of the preparation in treating and / or preventing liver damage related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a nano-lipid injection with high EPA content, and a preparation method and application thereof. BACKGROUND

[0002] Liver injury, especially cholestatic liver injury, is a pathological state caused by various reasons, which leads to the disorder of bile formation, secretion and excretion, so that bile cannot be actively discharged into the intestinal cavity through bile canaliculi, and cannot normally flow into the duodenum and enter the blood. Due to various reasons, liver diseases that cause cholestasis as the main manifestation are collectively referred to as cholestatic liver disease, and cholestasis itself can further aggravate liver damage. Cholestatic liver injury usually leads to a continuous increase in the concentration of triglycerides in the liver, which cannot be metabolized and eliminated, resulting in severe vacuolization of hepatocytes, and the occurrence of diseases such as hepatitis and cirrhosis.

[0003] For various reasons, cholestatic liver disease in the liver should be intervened individually according to its cause. Clinical surgery causes great pain to patients by using partial external biliary shunt and ileal bypass. However, there are currently fewer specific drugs approved for long-term treatment of cholestatic liver disease. Studies have shown that increasing the supplementation of polyunsaturated fatty acids and monounsaturated fatty acids can be used for the treatment of cholestatic liver disease, and this class of substances is more easily accepted by people and has no obvious adverse reactions.

[0004] Omega-3 fatty acids are competitive substrates for lipoxygenase and cyclooxygenase in the body. When reaching the effective concentration, they can generate prostaglandins, thromboxanes and leukotrienes, inhibit the expression of transcription factors related to inflammation, and play an anti-inflammatory role. In addition, omega-3 fatty acids can inhibit the esterification of phospholipid acid phosphatase and acyltransferase to other fatty acids, reduce the synthesis of triglycerides in the liver, and regulate lipid metabolism.

[0005] Currently reported omega-3 fatty acid preparations have many limitations when used for the treatment of liver injury, such as low active fatty acid content, limited administration dosage form to soft capsules, easy to be metabolized due to first-pass effect, low utilization amount by liver cells, and difficult to maintain effective concentration in the liver for a long time. In 2018, the FDA approved The prescription of fish oil used for the treatment of parenteral nutrition associated cholestasis in premature infants or children has only 20% of EPA content, and the active substance accumulates in the liver, which leads to insufficient anti-inflammatory ability and insufficient regulation of lipid metabolism, and the treatment has the problems of slow effect and long medication cycle, and the medication group is limited to premature infants or children. Patent document CN106692973A provides a combination of berberine, EPA and DHA, and the dosage form adopts a traditional soft capsule. The traditional soft capsule often has the problems of swallowing difficulty and low bioavailability. Patent document CN110312509A provides an EPA and DHA combination for treating and / or preventing liver disease. The EPA and DHA in the preparation can only play a curative effect when the ratio is specific, and the preparation requirements are relatively high, and it may be difficult to adapt to the complexity of biological bodies and individual differences in the clinic.

[0006] Therefore, it is necessary to develop a new preparation for the treatment of liver damage. SUMMARY

[0007] Based on this, one object of the present application is to provide a nano-lipid injection with high EPA content, which can be used for the prevention and / or treatment of liver damage related diseases.

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

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

[0010]

[0011] Among them, the omega-3 fatty acid glyceride contains EPA ester; in all fatty acid components of the omega-3 fatty acid glyceride, the mass percentage of EPA is ≥ 35%;

[0012] The liver targeting component contains a liver targeting factor or an unsaturated phospholipid unit in its structure; the number of unsaturated bonds in the unsaturated phospholipid unit is ≥ 2, and the mass percentage of phosphatidylethanolamine is ≥ 5%;

[0013] The emulsifier includes a phospholipid component;

[0014] The second auxiliary material is a pharmaceutically acceptable auxiliary material;

[0015] The weight percentage of water in the nano-lipid injection is at least 60%.

[0016] It should be understood that the amount of water is an appropriate amount of water, so that the total content of each component is 100%.

[0017] In some embodiments of the present application, the weight percentage of the omega-3 fatty acid glyceride in the nano-lipid injection is 2wt%-40wt%;

[0018] In some embodiments of the present application, the weight percentage of the omega-3 fatty acid glyceride in the nano-lipid injection is 2wt%-40wt%;

[0019] The weight percentage of the liver targeting component in the nano-lipid injection is 0.01wt%-10wt%, and / or the weight percentage of the polyethylene glycol lipid derivative in the nano-lipid injection is 0.001wt%-12wt%;

[0020] In some embodiments of the present application, the weight percentage of the omega-3 fatty acid glyceride in the nano-lipid injection is 2wt%-40wt%;

[0021] In some embodiments of the present application, the weight percentage of the omega-3 fatty acid glyceride in the nano-lipid injection is 2wt%-40wt%;

[0022] In some embodiments of the present application, the omega-3 fatty acid glyceride is selected from one or more of the following group: triglyceride type fish oil lipids extracted from one or more of the following fish: Engraulis japonicus, Psettodes eromaculatus, Gadus macrocephalus, Clupanodon pellegrini, Scomber japonicus, Thunnus thynnus, Sardinella zunasi, etc., and artificially synthesized glyceride type fish oil; and / or,

[0023] In some embodiments of the present application, the weight percentage of the omega-3 fatty acid glyceride in the nano-lipid injection is 2wt%-40wt%;

[0024] In some embodiments of the present application, the weight percentage of the omega-3 fatty acid glyceride in the nano-lipid injection is 2wt%-40wt%;

[0025] The polyethylene glycol lipid derivative contains a phospholipid unit or a fatty acid ester unit in its structure; and / or,

[0026] The nano-lipid injection is an oil-in-water dispersion system, and the average particle size of the droplets therein is ≤500nm.

[0027] In some embodiments of the present application, the liver targeting factor is selected from one or more of the following group: glycyrrhetinic acid and its derivatives, mannose and its derivatives, galactose and its derivatives; and / or,

[0028] The liver targeting component contains an unsaturated phospholipid unit in its structure, and the unsaturated phospholipid unit has a C 12-20fatty acyl; wherein, in the unsaturated phospholipid unit, the number of unsaturated bonds is ≥ 2, and the mass fraction of phosphatidylethanolamine is ≥ 5%; and / or,

[0029] The structure of the liver targeting component contains or does not contain a polyethylene glycol unit; wherein, when containing a polyethylene glycol unit, the molecular weight of the polyethylene glycol unit in the structure of the liver targeting component is 1000 Da to 5000 Da; and / or,

[0030] The polyethylene glycol lipid derivative is selected from phosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol-C 12-20 fatty acid ester and polyethylene glycol-bis C 12-20 fatty acid ester; and / or,

[0031] The molecular weight of the PEG unit in the polyethylene glycol lipid derivative is 1000 Da to 6000 Da; and / or,

[0032] The iodine value of the phospholipid component in the emulsifier is greater than 80; and / or,

[0033] The emulsifier is selected from one or more of egg yolk lecithin, soybean phospholipid, synthetic phospholipid, polyene phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, etc.; and / or,

[0034] The second adjuvant is selected from one or more of an antioxidant, an interfacial film stabilizer, an osmotic pressure regulator, a pH regulator, a linolenic acid-rich lipid, etc., wherein the mass fraction of linolenic acid in the linolenic acid-rich lipid is 30wt% to 70wt%, and the linolenic acid-rich lipid is not an omega-3 fatty acid glyceride; wherein, in the nano-lipid injection, the content of the antioxidant is 0 to 5wt%, the content of the interfacial film stabilizer is 0 to 3wt%, the content of the osmotic pressure regulator is 0 to 10wt%, and the content of the linolenic acid-rich lipid is 0 to 10wt%; and / or,

[0035] In the nano-lipid injection, the average particle size of the droplets is ≤ 300 nm.

[0036] In some embodiments of the present application, the liver targeting component is selected from any one of the following (A) to (E): (A) the unsaturated phospholipid unit of the liver targeting component is polyene phosphatidylcholine or polyene phosphatidylethanolamine, and the unsaturated phospholipid unit is selected from one or more of dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, and dimyristoyl phosphatidylcholine; (B) the unsaturated phospholipid unit in the liver targeting component has at least one of lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, etc.; (C) the liver targeting component is phosphatidylethanolamine-polyethylene glycol-liver targeting factor, or phosphatidylethanolamine-liver targeting factor, or polyene phospholipid; wherein “-” represents a chemical bond or a linker; (D) the liver targeting component includes a liver targeting component containing a polyethylene glycol unit, and the liver targeting component containing a polyethylene glycol unit is selected from one or more of phosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol-liver targeting factor, and phosphatidylethanolamine-polyethylene glycol monomethyl ether; (E) the liver targeting factor is mannose or glycyrrhetinic acid or galactose; and / or,

[0037] The polyethylene glycol lipid derivative is selected from one or more of distearoyl phosphatidylethanolamine-polyethylene glycol 2000, distearoyl phosphatidylethanolamine-polyethylene glycol 5000, soybean phosphatidylethanolamine-polyethylene glycol monomethyl ether 2000, polyethylene glycol 4000 oleate, polyethylene glycol 6000 oleate, polyethylene glycol 6000 dioleate, polyethylene glycol 200 laurate, etc., wherein “-” represents a chemical bond or a linker; and / or,

[0038] The synthetic phospholipid is selected from one or more of distearoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, and dimyristoyl phosphatidylcholine; and / or,

[0039] The iodine value of the egg yolk lecithin is ≥ 60; the egg yolk lecithin contains ≥ 68 wt% of phosphatidylcholine and ≤ 20 wt% of phosphatidylethanolamine, wherein the total content of phosphatidylcholine and phosphatidylethanolamine is ≥ 80 wt%; and / or,

[0040] The soybean phospholipid is polyene phosphatidylcholine or polyene phosphatidylethanolamine; and / or,

[0041] The iodine value of the soybean lecithin is ≥ 80, the soybean lecithin contains ≥ 45 wt% of phosphatidylcholine and ≤ 30 wt% of phosphatidylethanolamine, wherein the total content of phosphatidylcholine and phosphatidylethanolamine is ≥ 70 wt%; and / or,

[0042] the antioxidant is selected from one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, ascorbic acid and esters thereof, alpha-tocopherol and esters thereof, beta-tocopherol and esters thereof, gamma-tocopherol and esters thereof, delta-tocopherol and esters thereof, and the like; and / or,

[0043] the osmotic pressure regulator is selected from one or more of glycerol, propylene glycol, mannitol, and the like; and / or,

[0044] the pH regulator is a buffer salt system; and / or,

[0045] in the linolenic acid-enriched lipid, the ratio of alpha-linolenic acid: gamma-linolenic acid is ≥ 5:1; and / or,

[0046] the average particle size of the nano-lipid injection is 50-300 nm.

[0047] In some embodiments of the present application, part or all of the liver targeting component is a polyethylene glycol lipid derivative. That is, part or all of the liver targeting component is also a polyethylene glycol lipid derivative.

[0048] In some embodiments of the present application, the liver targeting component is selected from one or more of phosphatidylethanolamine-polyethylene glycol-mannose, phosphatidylethanolamine-glycyrrhetinic acid-mannose, phosphatidylethanolamine-glycyrrhetinic acid, and the like; wherein each of the phosphatidylethanolamine units is independently selected from any one of distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidyl, dioleoylphosphatidylethanolamine, and the like, wherein "-" represents a chemical bond or a linker.

[0049] According to a second aspect of the present application, a method for preparing the nano-lipid injection of the first aspect of the present application is provided, comprising the following steps:

[0050] mixing the oil phase components including the omega-3 fatty acid glyceride under heating conditions to prepare an oil phase;

[0051] dissolving the water phase components in an aqueous solvent to prepare a water phase, or using water as the water phase;

[0052] mixing and dispersing the oil phase and the water phase to prepare an oil-in-water dispersion system;

[0053] after preparing the oil-in-water dispersion system, optionally filtering, optionally sterilizing, and optionally packaging are further performed.

[0054] According to a third aspect of the present application, the nano-lipid injection of the first aspect of the present application, or the nano-lipid injection obtained by the method of the second aspect of the present application, is used in the preparation of a medicament for preventing and / or treating a liver injury-related disease.

[0055] In some embodiments of the present application, the liver injury related disease is cholestatic liver disease.

[0056] The nano-lipid injection provided by the present application can provide a high concentration of active eicosapentaenoic acid (EPA) by ω-3 fatty acid glyceride, can significantly increase the effective amount of EPA, and can effectively prevent and / or treat liver injury related diseases, including but not limited to cholestatic liver disease. The nano-lipid injection has the advantages of low viscosity and good dispersibility, and the EPA exists in the form of nano-particles, the solubility is greatly improved, and the liquid droplets with a particle size of less than 500 nm help the absorption of EPA and the effective arrival of EPA at the liver site.

[0057] The introduction of auxiliary materials (preferably liver targeting components) that can assist in the enrichment of EPA in the liver in the nano-lipid injection can promote the effective enrichment of EPA in the liver and increase the uptake of EPA by liver cells. The introduction of polyethylene glycol lipid derivatives in the nano-lipid preparation can provide a protective layer for the nano-carrier and reduce the hydrolysis of EPA by lipoprotein lipase when it enters the blood circulation. The introduction of liver targeting components and polyethylene glycol lipid derivatives can reduce the hydrolysis of EPA by lipoprotein lipase when it enters the blood circulation, target the delivery of EPA to the liver, assist in the effective enrichment of EPA in the liver during in vivo circulation, and maintain a long-term effective blood drug concentration and liver tissue concentration of EPA, thereby more effectively exerting the therapeutic and / or preventive effect of the preparation on liver injury related diseases (including but not limited to cholestatic liver injury). The enrichment of EPA in the liver and the uptake of EPA by liver cells can be increased while the blood action time of EPA is prolonged, and the preventive and / or therapeutic effect on liver injury related diseases is significantly improved.

[0058] Optimizing the types and amounts of emulsifiers and phospholipids and further adding auxiliary materials with long circulation and liver enrichment effects can achieve better liver injury treatment effects.

[0059] The introduction of EPA-rich lipids and the mass fraction of EPA in the lipids being 40% or more can reduce the intake of saturated fatty acids and prevent the aggravation of fat metabolism burden in the body. BRIEF DESCRIPTION OF DRAWINGS

[0060] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed in the embodiment description will be briefly introduced as follows.

[0061] Figure 1 HE staining results for investigating the effects of different preparations in an embodiment of the present application on the liver tissue morphology of rats. DETAILED DESCRIPTION

[0062] The present application will be further described below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not intended to limit the scope of the present application, and the purpose of providing these embodiments and examples is to make the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms obtained thereby also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0063] 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 application belongs. The terminology used in the description of the application herein is for the purpose of describing embodiments and examples only and is not intended to be limiting of the application.

[0064] Terminology

[0065] Unless otherwise indicated or unless contradicted by context, terms or phrases used herein have the following meanings:

[0066] The selection scope of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in this 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").

[0067] 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.

[0068] As used herein, "combinations thereof", "any combinations thereof", "any combination thereof", and the like include all suitable combinations of any two or more of the listed items.

[0069] As used herein, "suitable", "suitable", "any suitable", and the like in "suitable combination", "suitable manner", "any suitable manner" and the like are subject 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.

[0070] As used herein, "preferably", "better", "better", "preferably" are only to describe the better effect of the embodiments or examples, and it should be understood that they do not constitute a limitation on the scope of protection of the present application.

[0071] In the present application, "further", "further", "in particular" and the like are used to describe the purpose, indicating the difference in content, but should not be understood as a limitation on the scope of protection of the present application.

[0072] In the present application, "optionally", "optional", "optional" means optional, that is, selected from "yes" 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. In the present application, "optionally contains", "optionally contains" and the like indicate "contains or does not contain". "Optional component X" means that component X exists or does not exist.

[0073] In the present application, "first aspect", "second aspect", "third aspect", "fourth aspect" and the like, the terms "first", "second", "third", "fourth" and the like are only for the purpose of description, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0074] In the present application, the technical features described in an open manner include both the closed technical solutions consisting of the listed features and the open technical solutions containing the listed features.

[0075] In the present application, when a numerical interval (i.e. a numerical range) is involved, unless otherwise specified, the numerical values within the numerical interval are considered to be continuous and include both numerical endpoints (i.e. the minimum and maximum values) of the numerical range and every numerical value between the two numerical endpoints. When a numerical interval refers to integers within the numerical interval, unless otherwise specified, both numerical endpoints and every integer between the two numerical endpoints are included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, ranges disclosed herein are to be understood to include any and all sub-ranges subsumed therein.

[0076] In the present application, when an approximate number is involved, unless otherwise specified, the fluctuation range is generally ±10%, and can further be ±8%, ±5%, ±3%, etc. In the present application, an approximate number provides both the listed numerical value and the numerical interval represented by the approximate number. For example, approximately 200 nm provides both a technical solution of “200 nm” and a technical solution of a numerical interval of “200 nm ± fluctuation range”.

[0077] In the present application, a temperature parameter, unless otherwise specified, allows for constant temperature treatment and allows for fluctuations within a certain temperature interval. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0078] In the present application, the term “room temperature” generally refers to 4°C to 35°C, and preferably refers to 20°C ± 5°C. In some embodiments of the present application, room temperature refers to 20°C to 30°C.

[0079] In the present application, % (w / w) and wt% both refer to weight percentage.

[0080] All documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. Unless and to the extent that the cited documents conflict with the purpose and / or technical solutions of the present application, the cited documents are incorporated by reference in their entirety. When the present application refers to cited documents, the definitions of relevant technical features, terms, names, phrases, etc. in the cited documents are also incorporated by reference. When the present application refers to cited documents, examples and preferred modes of the cited relevant technical features can also be incorporated by reference into the present application, subject to the implementation of the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is given priority or is modified according to the description in the present application.

[0081] The abbreviations in the present disclosure, if not specifically defined, 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, PC refers to phosphatidylcholine, and PE refers to phosphatidylethanolamine.

[0082] In the present disclosure, the unsaturation degree of phospholipids is mainly characterized by iodine value, if not specifically defined, which refers to average iodine value.

[0083] In the present disclosure, the molecular weight refers to average molecular weight, which can be number average molecular weight or weight average molecular weight, if not specifically defined, which refers to weight average molecular weight.

[0084] In the present disclosure, "linker" refers to a structure connecting two groups, which can be connected by atoms or atom groups.

[0085] In the present disclosure, the molecular weight of PEG refers to average molecular weight, which can be number average molecular weight or weight average molecular weight, if not specifically defined, which refers to weight average molecular weight.

[0086] In the present disclosure, "above" and "below" each independently contain the number, if not specifically defined.

[0087] In the present disclosure, "≥" and "greater than or equal to" have the same meaning and can be used interchangeably, both of which mean greater than or equal to. "≤" and "less than or equal to" have the same meaning and can be used interchangeably, both of which mean less than or equal to.

[0088] According to the first aspect of the present disclosure, a nano-lipid injection is provided, which provides active fatty acids in the form of ω-3 fatty acid glyceride with high EPA content, and the mass percentage of EPA in the total fatty acid composition of the ω-3 fatty acid glyceride is ≥ 35%, which can significantly increase the effective amount of EPA and effectively prevent and / or treat liver injury related diseases, including but not limited to cholestatic liver disease.

[0089] In some embodiments of the present disclosure, a nano-lipid injection is provided, which comprises the following components in the weight percentage of 0.1-10% of the total weight of the nano-lipid injection:

[0090]

[0091] The ω-3 fatty acid glyceride comprises EPA ester, and the mass percentage of EPA in the total fatty acid composition of the ω-3 fatty acid glyceride is ≥ 35%.

[0092] The structure of the liver targeting component contains a liver targeting factor, or contains a phospholipid unit with an unsaturation bond number ≥ 2 and a phosphatidylethanolamine proportion ≥ 5%.

[0093] The emulsifier comprises a phospholipid;

[0094] The second adjuvant is a pharmaceutically acceptable other adjuvant. It should be understood that the second adjuvant is different from the liver targeting component, the polyethylene glycol lipid derivative, and the emulsifier.

[0095] It should be understood that the water in the nano-lipid injection is an appropriate amount of water, so that the sum of the weight percentages of the components does not exceed 100%. Preferably, the weight percentage of water in the nano-lipid injection is ≥ 60% (w / w), but the sum of the weight percentages of the components does not exceed 100%.

[0096] It should be understood that the sum of the weight percentages of the omega-3 fatty acid glyceride, the liver targeting component, the polyethylene glycol lipid derivative, the emulsifier, the pharmaceutically acceptable other adjuvant, and the water does not exceed 100%, and one of them is preferably 100%.

[0097] In some embodiments of the present application, the nano-lipid injection comprises 0.2wt% to 40wt% of omega-3 fatty acid glyceride, 0 to 10wt% of liver targeting component, 0.001wt% to 12wt% of polyethylene glycol lipid derivative, 0.05wt% to 10wt% of emulsifier, 0 to 30wt% of pharmaceutically acceptable other adjuvant (denoted as second adjuvant), and an appropriate amount of water (the minimum content is 60wt%), and various components cooperate with each other to promote the effective arrival of high content of EPA at the liver site.

[0098] The nano-lipid injection described above comprises omega-3 fatty acid glyceride, emulsifier, water, optional liver targeting component, polyethylene glycol lipid derivative, and optional pharmaceutically acceptable other adjuvant.

[0099] The nano-lipid injection of the present application can provide a large amount of active EPA. The inventors have found through extensive exploration and research that EPA is a key active fatty acid for the treatment of liver injury diseases, and high-purity EPA can inhibit the expression of transcription factors related to inflammation and effectively exert anti-inflammatory effects. The present application loads EPA in the form of nano-lipid preparation, which has the advantages of low viscosity, good dispersibility, etc., and can greatly improve the solubility and stability of EPA. Special functional adjuvants can also be further added to further improve the performance of the preparation.

[0100] In the nano-lipid injection, the content of the liver targeting component can be 0 (i.e., no liver targeting component). When the content of the liver targeting component is not 0, by introducing the liver targeting component into the nano-lipid injection, the effective enrichment of EPA at the liver site can be promoted, and the uptake of EPA by liver cells can be increased.

[0101] By introducing polyethylene glycol lipid derivatives into the nano-lipid injection, a protective layer can be provided for the nano-carrier, reducing the hydrolysis of EPA by lipoprotein lipase when entering the blood circulation, prolonging the blood action time of EPA, and achieving the long circulation effect of EPA.

[0102] When the contents of the liver targeting component and the polyethylene glycol lipid derivative in the nano-lipid injection are both not 0, i.e., both components are contained, the dual effects of long circulation and liver targeting can be achieved, the hydrolysis of EPA by lipoprotein lipase when entering the blood circulation can be reduced, EPA can be targeted to the liver, and EPA can be effectively enriched in the liver during circulation in the body. By maintaining a long-term effective blood drug concentration and liver tissue concentration of EPA, the preparation can more efficiently play a role in treating and / or preventing liver damage-related diseases (including but not limited to cholestatic liver damage).

[0103] The inventors have found that increasing the enrichment of EPA in the liver is beneficial for fully exerting the preventive and relieving effects of EPA on diseases such as cholestatic liver disease.

[0104] In some embodiments of the present application, the nano-lipid injection comprises a polyethylene glycol lipid derivative, and can optionally comprise a liver targeting component.

[0105] In some embodiments of the present application, the nano-lipid injection comprises omega-3 fatty acid glyceride, emulsifier, water, optional liver targeting component, polyethylene glycol lipid derivative, and optional second excipient.

[0106] In some embodiments of the present application, the nano-lipid injection comprises omega-3 fatty acid glyceride, emulsifier, water, liver targeting component, polyethylene glycol lipid derivative, and optional second excipient.

[0107] In some embodiments of the present application, the nano-lipid injection comprises omega-3 fatty acid glyceride, emulsifier, water, liver targeting component, polyethylene glycol lipid derivative, and second excipient.

[0108] omega-3 fatty acid glycerides

[0109] In the present application, the nano-lipid injection contains omega-3 fatty acid glyceride.

[0110] In some embodiments of the present application, the content of omega-3 fatty acid glyceride in the nano-lipid injection is 0.2% to 40% by weight, and can be further 2% to 40%, and specifically, any one of the following percentages or a percentage interval formed by any two of the following percentages: 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, and the like, and a percentage interval such as 5% to 30%, 5% to 25%.

[0111] The omega-3 fatty acid glyceride in the present application can provide a high content of EPA. The nano-lipid preparation prepared by using the omega-3 fatty acid glyceride with a high EPA content can reduce the intake of useless fat and prevent the aggravation of fat metabolism burden in the body. While effectively treating liver damage, the adverse effects on the body are reduced.

[0112] In some preferred embodiments of the present application, the mass percentage of EPA in the total fatty acid components of the omega-3 fatty acid glyceride is preferably ≥ 35%, and can be further ≥ 40%, and can be further ≥ 50%. In some embodiments, the mass percentage of EPA is any one of the following percentages or a percentage interval formed by any two of the following percentages: 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, and the like. If the content of EPA is too low (such as < 20%), the blood drug concentration required for treatment cannot be provided after administration. The nano-lipid injection of the present application encapsulates a high concentration of eicosapentaenoic acid, which can meet the required dose for treatment.

[0113] In some preferred embodiments of the present application, the mass percentage of DHA in the total fatty acid components of the omega-3 fatty acid glyceride is < 35%, and the mass percentage of other omega-3 fatty acids (referring to fatty acids other than EPA and DHA) is < 20%. Further, the mass percentage of DHA is < 30%, and the mass percentage of other omega-3 fatty acids is < 16%.

[0114] In some embodiments of the present application, the mass percentage of EPA in the total fatty acid components of the omega-3 fatty acid glyceride is preferably ≥ 35%, the mass percentage of DHA is < 35%, and the mass percentage of other omega-3 fatty acids is < 20%.

[0115] In some embodiments of the present application, the mass percentage of EPA in the total fatty acid component of the omega-3 fatty acid glyceride is preferably ≥ 40%, the mass percentage of DHA is < 30%, and the mass percentage of other omega-3 fatty acids is < 16%.

[0116] In the present application, the omega-3 fatty acid glyceride contains EPA esters. In some embodiments of the present application, the mass percentage of EPA glycerol triesters in the EPA esters is ≥ 50% (preferably > 50%), for example, any one of the following percentages or a percentage range 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%, etc. Further, in the EPA esters, the mass percentage of EPA glycerol monoesters is < 30%, and the mass percentage of EPA glycerol diesters is < 30%.

[0117] In some embodiments of the present application, the omega-3 fatty acid glyceride is selected from one or more of the following: triglyceride-type fish oil lipids extracted from one or more of the following fish: anchovy, finfish, cod, herring, mackerel, tuna, sardine, etc., and artificially synthesized glyceride-type fish oil. The artificially synthesized glyceride-type fish oil can be prepared by the following method: ethanol substitution, separation and purification of natural fish oil or glycerides extracted therefrom, hydrolysis to release free fatty acids, and then esterification synthesis by connecting specific types of fatty acids to the glycerol backbone as needed. The artificially synthesized glyceride-type fish oil can also be obtained by other processes.

[0118] emulsifiers

[0119] In the present application, the nano-lipid injection contains an emulsifying agent, and the composition can be prepared into an emulsion for injection.

[0120] In some embodiments of the present application, the content of the emulsifying agent in the nano-lipid injection is 0.1% to 10% by weight, and further can be 0.5% to 5%, for example, 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., for example, 0.5% to 10%.

[0121] The emulsifier used in the present application preferably comprises a phospholipid component. The phospholipid component can be an independent phospholipid molecule or a modified phospholipid molecule. Examples of the modified phospholipid molecule include a high-molecular derivative of phospholipid (e.g. a polyethylene glycol phospholipid derivative), a conjugate of a phospholipid molecule and a liver targeting factor, a conjugate of a "liver targeting factor-high-molecular block-phospholipid molecule" (e.g. a liver targeting factor-PEG-phospholipid), etc. In some embodiments, the phospholipid unit in the phospholipid component accounts for 10wt% to 100wt% of the emulsifier, for example 11%, 12%, 28%, 60%, 80%, 100%.

[0122] Further, the iodine value of the phospholipid component in the emulsifier is preferably greater than 80, more preferably ≥ 85.

[0123] In some embodiments of the present application, the emulsifier is selected from one or more of egg yolk lecithin, soybean lecithin, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine, sphingomyelin, polyene phosphatidylcholine, polyene phosphatidylethanolamine, etc.

[0124] In some embodiments of the present application, the aforementioned synthetic phospholipid is selected from one or more of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), etc. In this case, the synergistic effect between the liver targeting component is better, preferably a polyethylene glycol derivative of the liver targeting component, further preferably a polyethylene glycol lipid derivative of the liver targeting component (e.g. DSPE-PEG-glycyrrhetinic acid), which can be referred to in Example 2.

[0125] In some preferred embodiments of the present application, the iodine value of the egg yolk lecithin is ≥ 60, for example 60, 65, 70, etc. In some embodiments, the egg yolk lecithin contains ≥ 68wt% of phosphatidylcholine and ≤ 20wt% of phosphatidylethanolamine, further, the total content of phosphatidylcholine and phosphatidylethanolamine is ≥ 80wt%.

[0126] In some preferred embodiments of the present application, the soybean phospholipid is polyene phosphatidylcholine or polyene phosphatidylethanolamine.

[0127] In some preferred embodiments of the present application, the iodine value of the soybean lecithin is ≥ 80, for example 80, 85, 90, etc. In some embodiments, the soybean lecithin contains ≥ 45wt% of phosphatidylcholine and ≤ 30wt% of phosphatidylethanolamine, further, the total content of phosphatidylcholine and phosphatidylethanolamine is ≥ 70wt%.

[0128] water

[0129] In the present application, the nano-lipid injection necessarily contains water so as to be able to make an injection preparation which is easy to apply to a patient.

[0130] In the present application, the water in the nano-lipid injection can be deionized water, distilled water, sterile water, etc., as long as it is suitable for the preparation of a pharmaceutical preparation. The amount of water used in the nano-lipid injection is an appropriate amount of water, as long as it can be prepared into a preparation with the required particle size of the present application.

[0131] It should be understood that the amount of water is an appropriate amount of water, so that the sum of the contents of each component in the nano-lipid injection does not exceed 100%. The appropriate amount of water allows the nano-lipid injection to form a suitable ratio of oil phase and water phase, and can form an injection suitable for clinical use.

[0132] In some embodiments of the present application, the minimum mass content of water in the nano-lipid injection is 59%. That is, the minimum weight percentage of water in the nano-lipid injection is 59%.

[0133] In some embodiments of the present application, the weight percentage of water in the nano-lipid injection is ≥59%. For example, 60%, 65%, 70%, etc.

[0134] In some embodiments of the present application, on the basis of ω-3 fatty acid glyceride, liver targeting component (optional), polyethylene glycol lipid derivative, emulsifier, second auxiliary material (optional), water is added to a total weight percentage of 100%.

[0135] liver targeting component

[0136] In the present application, the nano-lipid injection optionally contains a liver targeting component, which can target the delivery of EPA to the liver site, significantly improving the accumulation of EPA at the liver site.

[0137] The liver targeting component contains a liver targeting factor in its structure, or contains an unsaturated phospholipid unit; further, in the unsaturated phospholipid unit, the number of unsaturated bonds is ≥2, and the mass percentage of phosphatidylethanolamine is ≥5%.

[0138] In some embodiments of the present application, the liver targeting component contains a liver targeting factor, wherein the liver targeting factor can be an independent molecule or a part of a modified liver targeting factor. Examples of the modified liver targeting factor include a high molecular weight modified liver targeting factor (e.g., a polyethylene glycol modified liver targeting factor), a phospholipid modified liver targeting factor, a polyethylene glycol phospholipid modified liver targeting factor, etc. In some embodiments of the present application, the liver targeting component is present in the nano-lipid injection at a content of 0% to 10% by weight, further at a content of 0.001% to 10% by weight, further at a content of 0% to 6% by weight, and more specifically at a content of any one of the following percentages or a content of any two of the following percentages or a content of a percentage range formed 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%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc., and more specifically at a content of any one of the following percentage ranges or a content of any two of the following percentage ranges: 0.5% to 10%, 1% to 10%, 2% to 10%, 0.5% to 8%, 1% to 8%, 2% to 8%, 0.5% to 6%, 1% to 6%, 2% to 6%, etc.

[0139] In some embodiments of the present application, the liver targeting factor is selected from one or more of glycyrrhetinic acid and its derivatives, mannose and its derivatives, galactose and its derivatives, etc.

[0140] In some embodiments of the present application, the liver targeting factor is mannose or glycyrrhetinic acid.

[0141] In some embodiments of the present application, the liver targeting component contains an unsaturated phospholipid unit, and further preferably, the unsaturated phospholipid unit contains a C 12-20Fatty acyl group. In some embodiments, the number of unsaturated bonds in the unsaturated phospholipid unit is ≥ 2, and the mass fraction of phosphatidyl ethanolamine in the unsaturated phospholipid unit is ≥ 5%. The number of unsaturated bonds in the unsaturated phospholipid unit can be, for example, 2, 3, 4, etc. The mass fraction of phosphatidyl ethanolamine in the unsaturated phospholipid unit can be, for example, 5%, 6%, 7%, etc. In some embodiments of the present application, the unsaturated phospholipid unit in the liver targeting component has at least one of lauroyl group, myristoyl group, palmitoyl group, stearoyl group, oleoyl group, etc. In some embodiments, the unsaturated phospholipid unit in the liver targeting component is polyenyl phosphatidyl choline or polyenyl phosphatidyl ethanolamine, and further, the unsaturated phospholipid unit can be selected from one or more of dioleoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, distearoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine, dimyristoyl phosphatidyl choline, etc.

[0142] In some embodiments of the present application, the liver targeting component does not contain a polyethylene glycol unit in its structure.

[0143] In some embodiments of the present application, the liver targeting component contains a polyethylene glycol unit (also referred to as PEG unit) in its structure, and in this case, the liver targeting component is a PEG derivative of the liver targeting component. When the chain length of the PEG unit is appropriate, the PEG derivative of the liver targeting component can simultaneously produce the dual effects of liver targeting and long circulation.

[0144] In some embodiments of the present application, part or all of the liver targeting component is a PEG derivative of the liver targeting component.

[0145] In some embodiments of the present application, part or all of the liver targeting component is a polyethylene glycol lipid derivative. That is, part or all of the liver targeting component simultaneously functions as a polyethylene glycol lipid derivative.

[0146] In some embodiments of the present application, the liver targeting component includes a liver targeting component containing a polyethylene glycol unit, and further, the liver targeting component containing a polyethylene glycol unit can be selected from one or more of phosphatidyl ethanolamine-polyethylene glycol, phosphatidyl ethanolamine-polyethylene glycol-liver targeting factor, and phosphatidyl ethanolamine-polyethylene glycol monomethyl ether.

[0147] In some embodiments of the present application, the liver targeting component contains both a phospholipid unit and a polyethylene glycol unit in its structure, and in this case, it is a polyethylene glycol phospholipid derivative of the liver targeting component. The definition, preferences, and examples of the phospholipid unit are consistent with the above.

[0148] When the liver targeting component contains a PEG unit in its structure, the definition, the preference and the example of the molecular weight of the PEG unit in the structure of the liver targeting component can refer to the polyethylene glycol lipid derivative section. Further, the molecular weight is preferably not more than 5000 Da, further preferably not more than 4000. In some embodiments, the molecular weight of the polyethylene glycol unit in the structure of the liver targeting component is 1000 Da to 5000 Da, further exemplified by 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.

[0149] In some embodiments of the present application,

[0150] In some embodiments of the present application, the structure of the liver targeting component is any one of phosphatidylethanolamine-polyethylene glycol-liver targeting factor, phosphatidylethanolamine-liver targeting factor, polyene phosphatide, etc., wherein the “-” represents a chemical bond or a linker. Examples of the linker herein include, but are not limited to, -OCH2CH2NH-PEG-, -OCH2CH2NH-C(=O)-PEG-, -OCH2CH2NHC(=O)O-PEG-, -PEG-O-C(=O)-TF (wherein TF represents a liver targeting factor), -PEG-O-C(=O)NH-TF, etc. Further, TF is mannose or glycyrrhetinic acid.

[0151] In some embodiments of the present application, the liver targeting component is selected from one or more of phosphatidylethanolamine-polyethylene glycol-mannose, phosphatidylethanolamine-glycyrrhetinic acid-mannose, phosphatidylethanolamine-glycyrrhetinic acid, etc. Further, the phosphatidylethanolamine unit therein can each independently be selected from any one of distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidyl, dioleoylphosphatidylethanolamine, etc., wherein the “-” represents a chemical bond or a linker. Examples of the linker herein include, but are not limited to, those described above.

[0152] In some embodiments of the present application, the nano-lipid injection comprises omega-3 fatty acid glyceride (e.g., 0.2wt%~40wt%), liver targeting component (e.g., 0.01wt%~10wt%), emulsifier (e.g., 0.05wt%~10wt%), and water, further comprising polyethylene glycol lipid derivative (0.001wt%~12wt%), and optionally other pharmaceutically acceptable excipients. In the liver targeting component, part or all of the liver targeting component is a PEG derivative. In some preferred embodiments, in the liver targeting component, part or all of the liver targeting component is a polyethylene glycol phospholipid derivative, i.e., the formulation contains polyethylene glycol lipid derivative, and it is understood that other types of polyethylene glycol lipid derivative can also be present in the formulation.

[0153] polyethylene glycol lipid derivative

[0154] In the present application, the nano-lipid injection preferably comprises polyethylene glycol lipid derivative. When containing polyethylene glycol lipid derivative, the hydrolysis of EPA by lipoprotein lipase when entering the blood circulation can be reduced, and EPA can be efficiently targeted to the liver, maintaining the effective concentration of EPA in the liver during circulation for a long time, and achieving better liver injury treatment effect.

[0155] In some embodiments of the present application, the content of polyethylene glycol lipid derivative in the nano-lipid injection is 0.001%~12% by weight, further 0.01%~12%, further 0.01%~10%, and specifically any one of the following percentages or the percentage interval formed 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%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, and the like, and the percentage interval is, for example, 0.5%~12%, 1%~12%, 2%~12%, 0.5%~10%, 1%~10%, 2%~10%, 0.5%~8%, 1%~8%, 2%~8%, 0.5%~6%, 1%~6%, 2%~6%, and the like.

[0156] In some embodiments of the present application, the polyethylene glycol lipid derivative contains a phospholipid unit in its structure, or contains a fatty acid ester unit. Among them, the phospholipid unit independently preferably includes a phosphatidyl ethanolamine unit. The fatty acid ester unit independently preferably C 12-20 A fatty acid ester unit (with 12 to 20 carbon atoms, for example 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, related to factors such as the type of ester.

[0157] In some embodiments of the present application, the polyethylene glycol lipid derivative is selected from phosphatidyl ethanolamine-polyethylene glycol, phosphatidyl ethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol-C 12-20 A fatty acid ester and polyethylene glycol-double C 12-20 One or more of the fatty acid ester and the like.

[0158] In some embodiments of the present application, the polyethylene glycol lipid derivative is selected from phosphatidyl ethanolamine-polyethylene glycol (PE-PEG, preferably containing C 12-20 A fatty acyl group (such as stearoyl), further preferably C 12-20 A fatty acyl phosphatidyl ethanolamine-polyethylene glycol), phosphatidyl ethanolamine-polyethylene glycol monomethyl ether (PE-mPEG, preferably containing C 12-20 A fatty acyl group (such as stearoyl), further preferably C 12-20 A fatty acyl phosphatidyl ethanolamine-polyethylene glycol monomethyl ether), polyethylene glycol-C 12-20 A fatty acid ester and polyethylene glycol-double C 12-20 One or more of the fatty acid ester and the like. The molecular weight of the PEG unit in the polyethylene glycol lipid derivative mainly considers the combination of various factors such as stabilization, preparation particle size, drug release, etc.

[0159] In some embodiments of the present application, the molecular weight of the PEG unit in the polyethylene glycol lipid derivative is 1000 Da to 6000 Da, and specific examples include an average molecular weight of about 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. "About" means that it can vary within a certain range, such as ± 10%, for example, about 1000 can be 1000 ± 10% (numerically equivalent to 9000-1100).

[0160] In some embodiments of the present application, the polyethylene glycol lipid derivative is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), distearoylphosphatidylethanolamine-polyethylene glycol 5000 (DSPE-PEG5000), soybean phosphatidylethanolamine-polyethylene glycol monomethyl ether 2000 (MPEG2000-SPE), polyethylene glycol 4000 oleate (PEG4000MO), polyethylene glycol 6000 oleate (PEG6000MO), polyethylene glycol 6000 dioleate, polyethylene glycol 200 laurate, etc.; wherein the numbers 2000, 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. The "-" represents a chemical bond or a linker. Examples of the linker here include -OCH2CH2NH-PEG-, -OCH2CH2NH-C(=O)-PEG-, -OCH2CH2NHC(=O)O-PEG-, etc.

[0161] Second adjuvant

[0162] In the present application, the nano-lipid injection optionally further comprises other pharmaceutically acceptable adjuvants (denoted as second adjuvant). It should be understood that the components of the second adjuvant are different from the liver targeting component, the polyethylene glycol lipid derivative, and the emulsifying agent.

[0163] In some embodiments of the present application, the second adjuvant is selected from one or more of an antioxidant, an interfacial film stabilizer, an osmotic pressure regulator, a pH regulator, a linolenic acid-rich lipid (ALA-rich lipid), etc., wherein the mass fraction of linolenic acid in the linolenic acid-rich lipid is preferably 30wt%-70wt%, and the linolenic acid-rich lipid is not an omega-3 fatty acid glyceride. In some embodiments of the present application, in the linolenic acid-rich lipid, the ratio of α-linolenic acid to γ-linolenic acid is ≥5:1.

[0164] In some embodiments of the present application, the second adjuvant is present in the nano-lipid injection in a weight percentage of 0-30%, further can be 0.01%-30%, for example, any one of the following percentages or the 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%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, etc., for example, 0.1%-30%, 0.5%-30%, 1%-30%, 0.1%-25%, 0.5%-25%, 1%-25%, 0.1%-20%, 0.5%-20%, 1%-20%, 0.1%-15%, 0.5%-15%, 1%-15%, etc.

[0165] In some embodiments of the present application, the nano-lipid injection optionally comprises an antioxidant. The antioxidant is selected from one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, ascorbic acid and its esters, a-tocopherol and its esters, β-tocopherol and its esters, γ-tocopherol and its esters, δ-tocopherol and its esters, etc. In some embodiments of the present application, the antioxidant is present in the nano-lipid injection in a mass content of 0-5wt%, for example, any one of the following percentages or the 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%, etc.

[0166] In some embodiments of the present application, the nano-lipid injection 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, etc. In some embodiments of the present application, the mass content of the interfacial film stabilizer in the nano-lipid injection is 0-3wt%, for example, any one of the following percentages or a percentage interval formed 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%, etc.

[0167] In some embodiments of the present application, the nano-lipid injection optionally comprises an osmotic pressure regulator. In some embodiments of the present application, the osmotic pressure regulator is selected from one or more of glycerol, propylene glycol, mannitol, etc. In some embodiments of the present application, the mass content of the osmotic pressure regulator in the nano-lipid injection is 0-10wt%, for example, any one of the following percentages or a percentage interval formed 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%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc.

[0168] In some embodiments of the present application, the nano-lipid injection optionally comprises a pH regulator. In some embodiments of the present application, the pH regulator is a buffer salt system. In some embodiments of the present application, the pH regulator is selected from one or more of the following: citric acid-sodium citrate, acetic acid-sodium acetate, phosphate agent, etc., and alkali (such as NaOH, etc.), acid (such as HC1, etc.), etc.

[0169] Examples

[0170] In some embodiments of the present application, the liver targeting component is present in the nano-lipid injection at a weight percentage of 0.01wt% to 10wt%, and / or the polyethylene glycol lipid derivative is present in the nano-lipid injection at a weight percentage of 0.001wt% to 12wt%.

[0171] In some embodiments of the present application, the nano-lipid injection comprises the following components at the weight percentages, based on the total weight of the nano-lipid injection: omega-3 fatty acid glyceride 0.2wt% to 40wt%, liver targeting component 0 to 10wt%, polyethylene glycol lipid derivative 0.001wt% to 12wt%, emulsifier 0.05wt% to 10wt%, second excipient 0 to 30wt%, and water (added to 100wt%). Further, the second excipient can include antioxidant 0 to 5wt%, interfacial film stabilizer 0 to 3wt%, osmotic pressure regulator 0 to 10wt%, linolenic acid-rich lipid 0 to 10wt%, and appropriate amount of PH regulator, based on the total weight of the nano-lipid injection.

[0172] In some embodiments of the present application, the nano-lipid injection comprises the following components at the weight percentages, based on the total weight of the nano-lipid injection: omega-3 fatty acid glyceride 0.2wt% to 40wt%, liver targeting component 0 to 10wt%, polyethylene glycol lipid derivative 0.001wt% to 12wt%, emulsifier 0.05wt% to 10wt%, second excipient 0 to 30wt%, and water (added to 100wt%). Further, the second excipient can include antioxidant 0 to 5wt%, interfacial film stabilizer 0 to 3wt%, osmotic pressure regulator 0 to 10wt%, linolenic acid-rich lipid 0 to 10wt%, and appropriate amount of PH regulator, based on the total weight of the nano-lipid injection.

[0173] Examples of the nano-lipid injection include, but are not limited to, those listed in Example 1 and Example 2, 1g of which can be regarded as 1 mass part.

[0174] In some embodiments of the present application, the nano-lipid injection comprises: fish oil of triglyceride type (5-250 parts by mass, further 10-200 parts by mass), flaxseed oil (0-10 parts by mass, further 0.1-10 parts by mass), egg yolk lecithin (e.g., E80, 0-25 parts by mass, further 1.2-24 parts by mass), soybean lecithin (e.g., S75, 0-15 parts by mass, further 1.2-12 parts by mass), soybean lecithin (e.g., S100, 0-15 parts by mass, further 3-12 parts by mass), polyethylene glycol (e.g., PEG4000, 0-20 parts by mass, further 1-20 parts by mass), α-tocopherol (0-0.2 parts by mass, further 0.01-0.2 parts by mass), ascorbic acid palmitate (0-0.2 parts by mass, further 0.01-0.2 parts by mass), sodium oleate (0-0.2 parts by mass, further 0-0.15 parts by mass, further 0.001-0.15 parts by mass), oleic acid (0-3 parts by mass, further 0.01-3 parts by mass, further 0.01-1.5 parts by mass), glycerol (2-50 parts by mass, further 2.25-45 parts by mass), sodium hydroxide (for adjusting pH, e.g., to pH 8-9), and water (an appropriate amount of water); further, the total weight of the nano-lipid injection can be 900-1100 parts by mass (preferably 1000 parts by mass).

[0175] In some embodiments of the present application, the nano-lipid injection comprises: a triglyceride type fish oil (5-250 parts by mass, further can be 10-200 parts by mass), a flaxseed oil (0-10 parts by mass, further can be 2-10 parts by mass), an egg yolk lecithin (such as E80, can be 0-25 parts by mass, further can be 1.2-24 parts by mass), a soybean lecithin (such as S100, can be 0-15 parts by mass, further can be 1.2-12 parts by mass), a polyethylene glycol (such as PEG4000, can be 0-20 parts by mass, further can be 1-20 parts by mass), an α-tocopherol (0-0.2 parts by mass, further can be 0.01-0.2 parts by mass), an ascorbic palmitate (0-0.2 parts by mass, further can be 0.01-0.2 parts by mass), a sodium oleate (0-0.2 parts by mass, further can be 0-0.15 parts by mass, further can be 0.001-0.15 parts by mass), an oleic acid (0-3 parts by mass, further can be 0.01-3 parts by mass, further can be 0.01-1.5 parts by mass), a glycerol (2-50 parts by mass, further can be 2.25-45 parts by mass), a sodium hydroxide (for adjusting pH, such as adjusting to pH 8-9), and water (an appropriate amount of water); further, the total weight of the nano-lipid injection can be 900-1100 parts by mass (preferably 1000 parts by mass).

[0176] In some embodiments of the present application, the nano-lipid injection comprises: fish oil of triglyceride type (5-250 mass parts, further can be 10-200 mass parts), flaxseed oil (0-10 mass parts, further can be 0.1-10 mass parts), egg yolk lecithin (such as E80, can be 0-25 mass parts, further can be 1.2-24 mass parts), soybean lecithin (such as S100, can be 0-15 mass parts, further can be 0.6-12 mass parts), polyethylene glycol (such as PEG4000, can be 0-15 mass parts, further can be 5-15 mass parts, such as 10 mass parts), α-tocopherol (0-0.2 mass parts, further can be 0.01-0.2 mass parts), ascorbic acid palmitate (0-0.2 mass parts, further can be 0.01-0.2 mass parts), DSPE-PEG-glycyrrhetinic acid (such as PEG molecular weight 1000, 2000, 4000, 5000, etc., can be 0-10 mass parts, further can be 0.1-8 mass parts), DSPE-PEG-galactose (such as PEG molecular weight 1000, 2000, 4000, 5000, etc., can be 0-10 mass parts, further can be 0.1-8 mass parts), glycerol (1.2-50 mass parts, further can be 1.2-45 mass parts), sodium hydroxide (for adjusting pH, such as adjusting to pH 8-9), and water (appropriate amount of water); further, the total weight of the nano-lipid injection can be 900-1100 mass parts (preferably 1000 mass parts).

[0177] In some embodiments of the present application, the nano-lipid injection comprises: fish oil of triglyceride type (5-250 parts by mass, further 10-200 parts by mass), flaxseed oil (0-10 parts by mass, further 0.1-10 parts by mass), egg yolk lecithin (e.g., E80, 0-15 parts by mass, further 1.2-12 parts by mass), soybean lecithin (e.g., S100, 0-12 parts by mass, further 0.6-12 parts by mass), soybean lecithin (e.g., S75, 0-15 parts by mass, further 0-12 parts by mass, such as 0 or 12 parts by mass), polyethylene glycol (e.g., PEG4000, 0-15 parts by mass, further 5-15 parts by mass, such as 10 parts by mass), α-tocopherol (0-0.2 parts by mass, further 0.01-0.2 parts by mass), ascorbic acid palmitate (0-0.2 parts by mass, further 0.01-0.1 parts by mass), DSPE-PEG-glycyrrhetinic acid (e.g., PEG molecular weight 1000, 2000, 4000, 5000, etc., 0-12 parts by mass, further 0.4-10 parts by mass), DSPE-PEG-galactose (e.g., PEG molecular weight 1000, 2000, 4000, 5000, etc., 0-6 parts by mass, further 0.1-5 parts by mass), PEG-PE (e.g., PEG molecular weight 1000, 2000, 4000, 5000, etc., 0-6 parts by mass, further 0.1-5 parts by mass), glycerol (2-50 parts by mass, further 2.25-45 parts by mass), sodium hydroxide (for adjusting pH, such as to pH 8-9), and water (an appropriate amount of water); further, the total weight of the nano-lipid injection can be 900-1100 parts by mass (preferably 1000 parts by mass). Each of the above components can be used in the form of a commercially available product, or can be prepared by a known method.

[0178] In some embodiments of the present application, the nano-lipid injection comprises: fish oil of triglyceride type (150-250 mass parts, further can be 200 mass parts), flaxseed oil (8-12 mass parts, further can be 10 mass parts), egg yolk lecithin (such as E80, can be 10-25 mass parts, further can be 12 or 24 mass parts), soybean lecithin (such as S100, can be 10-15 mass parts, further can be 0 or 12 mass parts), soybean lecithin (such as S75, can be 10-15 mass parts, further can be 0 or 12 mass parts), polyethylene glycol (such as PEG4000, can be 0-15 mass parts, such as 0 or 10 mass parts), α-tocopherol (0.15-0.25 mass parts, further can be 0.2 mass parts), ascorbic acid palmitate (0-0.2 mass parts, further can be 0 mass parts), DSPE-PEG-glycyrrhetic acid (such as PEG molecular weight 1000, 2000, 4000, 5000, etc., can be 0-12 mass parts, further can be 0 or 10 mass parts), DSPE-PEG-galactose (such as PEG molecular weight 1000, 2000, 4000, 5000, etc., can be 0-6 mass parts, further can be 0 or 5 mass parts), PEG-PE (such as PEG molecular weight 1000, 2000, 4000, 5000, etc., can be 0-6 mass parts, further can be 0 or 5 mass parts), glycerol (40-50 mass parts, further can be 45 mass parts), sodium hydroxide (for adjusting pH, such as adjusting to pH 8-9), and water (an appropriate amount of water); further, the total weight of the nano-lipid injection can be 900-1100 mass parts (preferably 1000 mass parts).

[0179] It should be understood that in each of the above examples, the specific examples of the types and amounts of each component can be referred to the foregoing, and can be independent of each other.

[0180] It should be understood that in each of the above embodiments, the specific examples of each component can be independent of each other. The "an appropriate amount of water" should satisfy the emulsification and be able to control the appropriate particle size.

[0181] In each of the above embodiments, the total weight of the EPA ethyl ester nano-lipid composition can be about 1000 mass parts (also refer to the formulations in Example 1 and Example 2).

[0182] nanocarrier size

[0183] In some embodiments of the present application, the nano-lipid injection is an oil-in-water dispersion system (emulsion), and further, the average droplet size is ≤500 nm. It has the advantages of low viscosity and good dispersibility, etc., can enhance the solubility of EPA and improve the stability of EPA.

[0184] In some embodiments of the present application, the drug carrier is highly dispersed to less than 1 micron (nanolipid carrier). In some embodiments of the present application, the average droplet size is less than 500 nm (e.g., 50 nm to 500 nm), further, the average droplet size can be ≤ 300 nm (e.g., 50 nm to 300 nm), further, the average droplet size can be ≤ 250 nm (e.g., 50 nm to 250 nm), further, the average droplet size can be about 200 nm. In some specific embodiments of the present application, the average droplet size is about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, 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.

[0185] In some embodiments of the present application, the droplet size of the nanolipid injection is 50 nm to 300 nm.

[0186] In some embodiments of the present application, the average droplet size is less than 500 nm, and 95% of the droplet size is not greater than 0.5 μm.

[0187] According to a second aspect of the present application, a preparation method of a nanolipid injection is provided, which can be used to prepare the nanolipid injection of the first aspect of the present application, and is suitable for industrial mass production.

[0188] In the present application, the preparation method of the nanolipid injection includes, but is not limited to, high-pressure homogenization method, high-speed shearing method, ultrasonic emulsification method, microfluidic method, and high-pressure microjet method.

[0189] In some embodiments of the present application, the preparation method includes the following steps:

[0190] S100, preparing an oil phase (preferably under inert gas protection): mixing the oil phase components including ω-3 fatty acid glyceride under heating conditions (preferably the heating temperature is 50°C to 70°C, e.g., 50°C, 55°C, 60°C, 65°C, 70°C) to form a uniform oily solution, thereby preparing an oil phase;

[0191] S200, preparing the aqueous phase (preferably under inert gas protection): dissolving the aqueous phase components in the aqueous solvent to form a uniform aqueous solution, preparing the aqueous phase, or, using water as the aqueous phase, preferably preheating the aqueous phase to a certain temperature (preferably the same or similar temperature as the oil phase, such as 50-70°C, for example 50°C, 55°C, 60°C, 65°C, 70°C);

[0192] S300, preparing the oil-in-water dispersion system: mixing the oil phase and the aqueous phase (the mixing can be carried out under heating, further can be 50-70°C), dispersing treatment, preparing the oil-in-water dispersion system.

[0193] In some embodiments of the present application, in step S300, the oil phase and the aqueous phase are mixed in a corresponding ratio.

[0194] In some embodiments of the present application, the oil-in-water dispersion system is an emulsion, and the average particle size of the droplets is ≤500nm (further preferably ≤300nm, further can be 50-300nm).

[0195] The oil phase component refers to a fat-soluble component. The aqueous phase component refers to a water-soluble component. It should be understood that some components have amphiphilic properties and can be used as both oil phase components and aqueous phase components.

[0196] As used herein, "aqueous solvent" refers to a solvent that provides a pharmaceutically acceptable aqueous phase, which can be water or a mixed solvent of water and other solvents.

[0197] In some embodiments, the pH adjuster is mixed with water (to adjust the pH of the aqueous solution with the pH adjuster), obtaining an aqueous solvent for subsequent preparation. The pH of the aqueous solvent is adjusted according to factors such as injection site, drug characteristics, etc., and then a suitable pH adjuster is selected. In some embodiments, the pH is 8-9.

[0198] In some embodiments of the present application, the nano-lipid injection can be dispersed by an in-line stator-rotor shear machine or a high-pressure homogenizer. The parameters of the dispersion process can be adjusted according to the desired particle size. In some embodiments, the parameters of the in-line stator-rotor shear are 1000-8000 bar (for example, 1000 bar, 2000 bar, 3000 bar, 4000 bar, 5000 bar, 6000 bar, 7000 bar, 8000 bar). In some embodiments, the parameters of the high-pressure homogenization are 10-600 bar (for example, 10 bar, 20 bar, 30 bar, 40 bar, 50 bar, 60 bar, 80 bar, 100 bar, 150 bar, 200 bar, 250 bar, 300 bar, 350 bar, 400 bar, 450 bar, 500 bar, 550 bar, 600 bar). The homogenization can be performed once or multiple times, preferably multiple times, for example, 3-10 times, and also for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times.

[0199] When the pH adjusting agent is included in the raw material, the pH is preferably adjusted in step S300. The pH of the system is adjusted to obtain a stable suitable particle size, not to affect the activity of the drug, and to be suitable for injection.

[0200] In some embodiments of the present application, the oil-in-water dispersion system is further filtered after being prepared.

[0201] In some embodiments of the present application, the oil-in-water dispersion system is further sterilized after being prepared.

[0202] In some embodiments of the present application, the oil-in-water dispersion system is further packaged after being prepared.

[0203] The above filtration, sterilization, and packaging steps can each be independently selected.

[0204] The inert gas used in the above preparation process can be nitrogen.

[0205] In some embodiments of the present application, the oil-in-water dispersion system is further filtered, sterilized, and packaged after being prepared, and further, the inert gas used in the process can be nitrogen.

[0206] It should be understood that the above steps of the preparation method, unless otherwise specified, are not limited in order. For example, the steps of S100 and S200 are not limited in order, but both are before S300. For another example, the order of packaging and packaging is not limited.

[0207] In some embodiments of the present application, the preparation method comprises the following steps:

[0208] S100: mixing the oil phase components (including omega-3 fatty acid glyceride) under inert gas protection until a uniform oily solution is formed, and heating the solution in a water bath to 50-70°C to obtain a preheated oil phase; preferably, the inert gas is nitrogen;

[0209] S200: preparing the water phase components in the formula into a water phase under inert gas protection, and stirring and dissolving until a uniform aqueous solution is formed, and heating the solution in a water bath to 50-70°C to obtain a preheated water phase; preferably, the inert gas is nitrogen;

[0210] S300: mixing the obtained preheated water phase and oil phase to form an oil-in-water dispersion system by an online stator-rotor shear machine or high-pressure homogenization (this is one of the modes of the nano-lipid injection of the present application);

[0211] Preferably, the method further comprises step S400: heating the obtained oil-in-water dispersion system in a water bath to 50-70°C, and then filtering, sterilizing and packaging the system under inert gas protection. A sterilized nano-lipid injection can be obtained.

[0212] In the presence of the types and amounts of the specified raw materials, those skilled in the art can implement the above-mentioned preparation method according to the above-mentioned instructions to obtain the nano-lipid injection of the present application.

[0213] According to a third aspect of the present application, the nano-lipid injection of the first aspect of the present application or the nano-lipid injection obtained by the preparation method of the second aspect of the present application is provided for use, preferably for use in the preparation of a medicament for preventing and / or treating a liver injury-related disease.

[0214] In some embodiments of the present application, the liver injury-related disease is cholestatic liver disease, liver cirrhosis, liver necrosis, and liver coma, etc.

[0215] In some embodiments of the present application, the nano-lipid injection contains both a PEG lipid derivative that assists the long circulation of EPA and a liver-targeting component that assists the enrichment of EPA in the liver, which can increase the enrichment amount of EPA in the liver and thus achieve a better therapeutic effect on liver injury.

[0216] In some embodiments of the present application, compared with the use of egg yolk lecithin alone, the use of a mixed phospholipid containing one or more of egg yolk lecithin, soybean lecithin and PEG-DSPE can reduce the hydrolysis of EPA by lipoprotein lipase when EPA enters the blood circulation, and can target the delivery of EPA to the liver.

[0217] According to a fourth aspect of the present invention, a method for preventing and / or treating liver injury-related diseases is provided, comprising administering a therapeutically effective amount of the nanolipid injection of the present invention (first aspect or second aspect) to a patient in need. Further, the liver injury-related disease may be cholestatic liver disease.

[0218] As used herein, "therapeutic effective amount" means the amount of the oral microemulsion of the present invention (or the amount of EPA) that will elicit a biological or medical response in an individual, such as the amount of the oral microemulsion of the present invention (or the amount of EPA) that will bring about positive physiological and / or pharmacological effects in an individual, including but not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression or preventing disease.

[0219] As used herein, “pharmaceutically acceptable” means those reagents, materials, compositions, and / or dosage forms that are appropriate for administration to patients within the bounds of reasonable medical judgment and that are commensurate with a reasonable benefit / risk ratio.

[0220] As used in this article, "patient" refers to an animal, preferably a mammal, and more preferably a human. The term "mammal" primarily refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats, pigs, cattle, sheep, horses, and humans.

[0221] As used herein, "administration" unless otherwise specified refers to administering the nanolipid injection of the present invention.

[0222] In some embodiments of the present invention, the drug is administered to rats, and the drug is administered once or multiple times.

[0223] In some embodiments of the present invention, the target of administration is rats, and the dosage is 0.7 mL / kg to 3 mL / kg of the nanolipid injection per day based on body weight.

[0224] In some embodiments of the present invention, the administration method is injection, such as intravenous injection.

[0225] In some embodiments of the present invention, the target organism is a rat; further, the nanolipid injection is administered daily at a rate of 0.7 mL / kg to 3 mL / kg of body weight for 7 consecutive days. Within one hour of the seventh administration, the peak plasma concentration is reached, exceeding 5500 μg / mL, and the concentration reaching the liver exceeds 1000 μg / g. The nanolipid injection of the present invention is preferably a formulation capable of achieving the above-mentioned effects.

[0226] In some embodiments of the present application, the subject is a rat; further, the nano-lipid injection is infused at 0.7 mL / kg to 3 mL / kg per day based on body weight, and after 7 days of continuous administration, the maximum blood drug concentration is higher than 6000 μg / mL within 1 hour after the seventh administration, and the concentration reaching the liver site is higher than 2000 μg / g. The nano-lipid injection of the present application is preferably a preparation capable of achieving the above effects.

[0227] In some embodiments of the present application, the subject is a rat; the nano-lipid injection is infused at 0.7 mL / kg to 3 mL / kg per day based on body weight, which is equivalent to 0.2 g / kg to 0.6 g / kg of EPA-enriched lipids. Taking a rat with a body weight of 200 g as an example, the daily administration amount is 0.14 mL to 0.57 mL. The rat is administered once a day and continuously for 7 days, and after the seventh administration, the total EPA concentration in the blood (including serum or plasma) is maintained at a range higher than 700 μg / mL for more than 6 hours, the total EPA concentration at the liver site is maintained at a range higher than 500 μg / g for more than 12 hours, further, the total EPA concentration in the blood (including serum or plasma) is maintained at a range higher than 1000 μg / mL for more than 6 hours, and the total EPA concentration at the liver site is maintained at a range higher than 500 μg / g for more than 24 hours. Specific embodiments

[0229] 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 specific formula composition, preparation method, function and effect of the present application, 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 not specified in the following examples are preferred to refer to the guidelines 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 known experimental methods in the art.

[0230] In the following specific examples, the amount of raw material components is measured, and slight deviations within the weighing accuracy range are allowed unless otherwise specified. Acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed for temperature and time parameters.

[0231] In the following examples, "room temperature" refers to 20℃ to 30℃.

[0232] In the following examples, "particle size" refers to average particle size unless otherwise specified.

[0233] The following examples 1 formulation involving the amount ratio, such as no special limit, refers to the mass ratio.

[0234] In the following examples, PEG-PE represents polyethylene glycol-phosphatidyl ethanolamine, PEG-DSPE represents polyethylene glycol-distearylphosphatidyl ethanolamine; ALT represents glutamic-pyruvic transaminase, AST represents glutamic-oxaloacetic transaminase, ALP represents serum alkaline phosphatase, γ-GT represents γ-glutamyl transpeptidase, T-BIL represents total bilirubin, D-BIL represents direct bilirubin, I-BIL represents indirect bilirubin, and TBA represents total bile acid.

[0235] The raw material information of fish oil, phospholipids and the like used in the following examples and comparative examples is shown in Table 1.

[0236] Table 1. Raw material information of fish oil, phospholipids and the like used in the examples and comparative examples of the present application.

[0237]

[0238] In the following examples, a Malvern particle size analyzer (model ZetaSizer Nano ZS) was used to test the particle size.

[0239] Example 1. Formulation example

[0240] Formulation example 1.1. Preparation of nano-lipid injection containing different proportions of high-purity EPA in glyceride form and different phospholipids

[0241] 1.1.1. Raw materials

[0242] The fish oil in formulations 1-3, 1-5 and 1-7 is from EPAX 4030 TGN of Epax Company, and the fish oil raw material contains 40 g of EPA per 100 g, and the proportion of monoglyceride and diglyceride in total glyceride is about 21%; the fish oil in formulations 1-4 and 1-9 is from KinOmega EPA7010TG, and the fish oil raw material contains 70% of EPA, and the proportion of monoglyceride and diglyceride in total glyceride is 8%. The fish oil in other formulations is from EPAX 6015 TGN of Epax Company, and the fish oil raw material contains about 55 g of EPA per 100 g, and the proportion of monoglyceride and diglyceride in total glyceride is 10%. In the formulation comparative examples D1-1 and D1-2, the fish oil is from EPAX 6015 TGN.

[0243] Egg yolk lecithin E80 (iodine value 60-73), soybean lecithin S75 (iodine value 85-95), S100 (iodine value 103) from Lipoid, Germany. The emulsifier used in preparation D1-1, D1-2, 1-4, 1-6, 1-10 is egg yolk lecithin Lipoid E80; the emulsifier used in preparation 1-3 is egg yolk lecithin Lipoid S75, the emulsifier used in preparation 1-8 is egg yolk lecithin Lipoid S100, the emulsifier used in preparation 1-7, 1-11 is a mixture of the above three kinds of lecithin with mass ratio S75:E80:S100 = 1:1:0; the emulsifier used in preparation 1-5 is a mixture of the above three kinds of lecithin with mass ratio S75:E80:S100 = 1:0:1; the emulsifier used in preparation 1-9 is a mixture of the above three kinds of lecithin with mass ratio S75:E80:S100 = 0:1:1. The lipid rich in linolenic acid used in preparation D1-1, 1-3, 1-5 is from linolenic acid oil; the lipid rich in linolenic acid used in preparation D1-2, 1-4, 1-6 is from perilla oil; the lipid rich in linolenic acid used in preparation 1-7, 1-8, 1-9, 1-10, 1-11 is from a mixture of the above two kinds of lipids with ratio perilla oil:linolenic acid oil = 1:1. The antioxidant used in preparation D1-1, 1-3, 1-5 is from α-tocopherol; the antioxidant used in preparation D1-2, 1-4, 1-6 is from ascorbyl palmitate; the antioxidant used in preparation 1-7, 1-8, 1-9, 1-10, 1-11 is from a mixture of the above two kinds of antioxidants with ratio α-tocopherol:ascorbyl palmitate = 200:1. The interfacial film stabilizer used in preparation D1-1, 1-3, 1-5, 1-7, 1-9, 1-11 is from oleic acid; the interfacial film stabilizer used in preparation D1-2, 1-4, 1-6, 1-8, 1-10 is from sodium oleate. The pH adjuster used in all preparations is sodium hydroxide.

[0244] 1.1.2. Preparation of the formulations

[0245] According to the types and amounts of each component in each group of Table 2, the ω-3 fatty acid-rich nano-lipid preparation (formulation 1-1 to formulation 1-11) was prepared as follows:

[0246] Step a (preparation of oil phase): TG type fish oil, linseed oil, α-tocopherol, ascorbyl palmitate, sodium oleate, oleic acid (components not in the prescription table are not added) were weighed and preheated to 60-70°C under nitrogen protection as the oil phase.

[0247] Step b (preparation of water phase): egg yolk lecithin, PEG4000, glycerol, galactosylceramide, phosphocholine, galactosylceramide, phosphoethanolamine (not added if not in the prescription table), add water to 1 L (including oil phase), mix well, preheat to 60-70°C, as water phase.

[0248] Step c (mixing of oil phase and water phase): the oil phase and water phase are sheared by a pipeline customized rotor shearing machine at a mass ratio of 1:100 to 1:3, parameters are 7000 rpm, 5 min, to obtain an oil-in-water dispersion system, and the pH value of the colostrum is adjusted to 8.0-9.0 by using sodium hydroxide aqueous solution or sodium oleate.

[0249] The mass ratio of the oil phase and the water phase in the preparation D1-1, D1-2 and the preparation 1-3 to the preparation 1-11 is 1:3.8, 1:8.8, 1:97.9, 1:98.8, 1:18, 1:17.9, 1:8.5, 1:8.5, 1:8.4, 1:3.7, 1:3.9, respectively.

[0250] Step d (homogeneous emulsification): then homogeneous emulsification is carried out at a pressure of 10-600 bar, and the emulsification times are 6-8 times.

[0251] Step e (dispensing and sterilization): dispensing into glass infusion bottles or infusion soft bags, and sterilization is carried out at 121°C for 8-15 min or at 115°C for 30 min.

[0252] The sterilization temperature in the preparation D1-1, D1-2 and the preparation 1-3 to the preparation 1-11 is 121°C for 15 min, respectively.

[0253] 1.1.3. Preparation of comparative examples

[0254] The preparation comparative examples D1-1, D1-2 are prepared according to the prescription in Table 2, and the method of the preparation experimental examples in 1.1.2. above is referred to.

[0255] Table 2. Nanolipid preparation prescription table

[0256]

[0257] The "particle size" refers to the average particle size of the prepared nanolipid injection. "-" means not added.

[0258] 1.1.4. Results

[0259] The preparation 1-1 to 1-11 and the preparation D1-1, D1-2 prepared according to Table 2 can all form nanolipid injections with good stability, and the particle size of the preparation 1-1 to 1-11 and the preparation D1-1, D1-2 is 100-300 nm.

[0260] Formulation Example 1.2. Preparation of nano-lipid injection containing different proportions of high-purity glyceride-type EPA and different proportions of phospholipids

[0261] According to the types and amounts of raw materials in Table 3, each component was weighed, and the nano-lipid preparation was prepared according to the method of 1.1.2. in Example 1.1. The particle size was measured. Among them, the fish oil in formulation 2-1, formulation 2-4, formulation 2-7 comes from EPAX 4030TGN of Epax company, and the fish oil raw material contains 40g of EPA per 100g. The fish oil in formulation 2-2, formulation 2-5, formulation 2-8 comes from KinOmega EPA70TG, which contains 70% EPA. The fish oil in other formulations comes from EPAX 6015 TGN of Epax company, which contains about 55% EPA. The linolenic acid-rich lipid in formulation 2-1, 2-4, 2-7, 2-10 comes from flaxseed oil, the linolenic acid-rich lipid in formulation 2-2, 2-5, 2-8, 2-11 comes from perilla oil, and the linolenic acid-rich lipid in formulation 2-3, 2-6, 2-9, 2-12 comes from sunflower seed oil. The emulsifier in formulation 2-1, 2-4, 2-10 comes from egg yolk lecithin E80 (iodine value 60-73) of Lipoid in Germany, and the emulsifier in formulation 2-2, 2-3, 2-8, 2-9 is selected from soybean phospholipid S100 (iodine value 103) of Lipoid in Germany. The emulsifier in formulation 2-5, 2-6 is selected from the mixed phospholipid of the above two (E80:S100=1:1), the emulsifier in formulation 2-7 is selected from the mixed phospholipid of the above two (E80:S100=2:1), and the emulsifier in formulation 2-11, 2-12 is selected from the mixed phospholipid of the above two (E80:S100=3:1). The antioxidant in formulation 2-1, 2-4, 2-7, 2-10 comes from alpha tocopherol. The antioxidant in formulation 2-2, 2-5, 2-8, 2-11 comes from ascorbyl palmitate, and the antioxidant in formulation 2-3, 2-6, 2-9, 2-12 comes from a mixture of alpha tocopherol and ascorbyl palmitate, with a ratio of 4:1, 3:1, 2:1, 1:1 respectively. The interfacial membrane stabilizer in formulation 2-1, 2-3, 2-5, 2-7, 2-9, 2-11 is selected from sodium oleate, and the interfacial membrane stabilizer in other formulations is selected from oleic acid. The pH regulator in all the above formulations is selected from sodium hydroxide.

[0262] Formulations 2-1 to 2-12 prepared according to Table 3 can all form nano-lipid injections with good stability, and the particle sizes of formulations 2-1 to 2-12 are all between 100nm and 300nm.

[0263] Table 3. Formulation table of nano-lipid preparation

[0264]

[0265] wherein "-" means no addition.

[0266] Preparation of nano-lipid injection containing different proportions of high-purity glyceride-type EPA content and glycyrrhizic acid, galactose

[0267] According to the types and amounts of raw materials in Table 4, each component was weighed, and the nano-lipid preparation was prepared according to the method of 1.1.2. in Example 1.1. The particle size was measured. Among them, the fish oil in preparation 3-1, preparation 3-3, preparation 3-5 comes from EPAX 4030TGN of Epax Company. The fish oil in other preparations is selected from EPAX 6015 TGN of Epax Company. Egg yolk lecithin E80 (iodine value of 60-73), soybean phospholipid S100 (iodine value of 103) come from Lipoid in Germany. Among them, the PEG molecular weight in DSPE-PEG-glycyrrhizic acid, DSPE-PEG-galactose is 4000. The emulsifier used in preparation 3-1, 3-3, 3-5 is egg yolk lecithin Lipoid E80; the emulsifier used in preparation 3-2 is egg yolk lecithin Lipoid S75, the emulsifier used in preparation 3-4 is egg yolk lecithin Lipoid S100, the emulsifier used in preparation 3-6, 3-8 is a mixture of the above three phospholipids, the proportion is S75:E80:S100=1:1:0; the emulsifier used in preparation 3-7 is a mixture of the above three phospholipids, the proportion is S75:E80:S100=0:1:1; the liver targeting component used in preparation 3-1, 3-3, 3-5 comes from DSPE-PEG-glycyrrhizic acid; the liver targeting component used in preparation 3-2, 3-4, 3-6 comes from DSPE-PEG-galactose; the liver targeting component used in preparation 3-7, 3-8 comes from a mixture of the above two, the proportion is DSPE-PEG-galactose:DSPE-PEG-glycyrrhizic acid=1:1. The linolenic acid-rich lipid used in preparation 3-1, 3-3, 3-5 comes from linolenic acid oil; the linolenic acid-rich lipid used in preparation 3-2, 3-4, 3-6 comes from perilla oil; the linolenic acid-rich lipid used in preparation 3-7, 3-8 comes from a mixture of the above two, the proportion is perilla oil:linolenic acid oil=1:1. The antioxidant used in preparation 3-1, 3-3, 3-5 comes from α-tocopherol; the antioxidant used in preparation 3-2, 3-4, 3-6 comes from ascorbic acid palmitate; the antioxidant used in preparation 3-7, 3-8 comes from a mixture of the above two antioxidants, the proportion is α-tocopherol:ascorbic acid palmitate=200:1. The interfacial film stabilizer used in preparation 3-1, 3-3, 3-5, 3-7 comes from oleic acid; the interfacial film stabilizer used in preparation 3-2, 3-4, 3-6, 3-8 comes from sodium oleate. The pH adjuster used in all preparations is sodium hydroxide.

[0268] Formulations 3-1 to 3-8 prepared according to Table 4 can all form nanolipid injections with good stability, and the particle sizes of formulations 3-1 to 3-8 are all 100 nm to 300 nm.

[0269] Table 4. Nanolipid formulation prescription table

[0270]

[0271] Formulation Example 1.4. Preparation of nanolipid injections containing different proportions of high-purity glyceride-type EPA content and polyethylene glycol lipid derivatives (PEG-DSPE, PEG-oleate, DSPE-PEG-glycyrrhetinic acid)

[0272] According to the types and amounts of raw materials in Table 5, each component was weighed, and the nanolipid formulation was prepared according to the method of 1.1.2. in Example 1.1. The particle size was measured. In formulations 4-1, 4-3, and 4-5, the fish oil was from EPAX 4030TGN of Epax Company. In other formulations, the fish oil was selected from EPAX 6015 TGN of Epax Company. In formulations 4-1, 4-3, 4-5, and 4-7, the linolenic acid-rich lipid was selected from flaxseed oil, and in other formulations, the linolenic acid-rich lipid was selected from perilla oil. In formulation 4-1, the emulsifier was selected from German Lipoid egg lecithin E80 (iodine value of 60-73), in formulation 4-2, the emulsifier was selected from German Lipoid soybean lecithin S100 (iodine value of 103), and in formulation 4-6, the emulsifier was selected from German Lipoid soybean lecithin S75 (iodine value of 85-95). In formulations 4-3, 4-4, 4-5, 4-7, and 4-8, the emulsifier was selected from a mixture of the above three, and the proportions were E80:S100:S75 = 3:2:0, 1:4:0, 2:0:1, 1:1:0, and 1:0:1, respectively. In formulations 4-1, 4-3, and 4-5, the polyethylene glycol lipid derivative was selected from DSPE-PEG, in formulations 4-2, 4-4, and 4-6, the polyethylene glycol lipid derivative was selected from PEG-oleate, and in formulations 4-7 and 4-8, the polyethylene glycol lipid derivative was selected from a mixture of the above two, and the proportions were DSPE-PEG:PEG-oleate = 1:1 and 2:1, respectively. In the above, the molecular weight of PEG in DSPE-PEG-glycyrrhetinic acid, PEG-DSPE, and PEG-oleate was 4000. In the above formulations, the liver targeting component was selected from DSPE-PEG-glycyrrhetinic acid, the antioxidant was selected from alpha tocopherol, the interfacial membrane stabilizer was selected from sodium oleate, and the pH adjuster was selected from sodium hydroxide.

[0273] Formulations 4-1 to 4-9 prepared according to Table 5 can all form microemulsions with good stability, and the particle sizes of formulations 4-1 to 4-9 are all 100 nm to 300 nm.

[0274] Table 5. Formulation table of nano-lipid preparation

[0275]

[0276] Example 2. Effect investigation

[0277] In vivo Example 2.1. Long-circulating effect of drug

[0278] 2.1.1. Preparation of preparation

[0279] According to the kind and amount of raw materials in Table 6, each component was weighed, and the nano-lipid preparation was prepared according to the method of 1.1.2. in Example 1.1. The particle size was measured. Among them, the preparation 5-1 group used the commercially available Omegaven, and the preparation 5-2 group did not add polyethylene glycol lipid derivative and liver targeting component. The fish oil in preparation 5-5 was from EPAX 4030 TGN of Epax Company. The fish oil in other preparations was from EPAX 6015 TGN of Epax Company. The emulsifier in preparation 5-2, 5-6 was from egg yolk lecithin E80 (iodine value of 60-73) of Lipoid in Germany, and the emulsifier in preparation 5-3, 5-4, 5-5, 5-7, 5-8 was a mixture of egg yolk lecithin E80 (iodine value of 60-73), soybean phospholipid S100 (iodine value of 103), and soybean phospholipid S75 (iodine value of 85-95) from Lipoid in Germany, with a ratio of E80:S100:S75 = 1:1:0, 1:1:0, 1:1:0, 1:0:1, 1:1:0, respectively. The linolenic acid-rich lipid in the above preparation was selected from flaxseed oil, the liver targeting component was selected from DSPE-PEG-glycyrrhetic acid, and the polyethylene glycol lipid derivative was selected from a mixture of DSPE-PEG and PEG-oleate with a ratio of 1:1. The antioxidant was selected from alpha tocopherol, the interfacial membrane stabilizer was selected from sodium oleate, and the pH adjuster was selected from sodium hydroxide. Among them, the molecular weight of PEG in DSPE-PEG-glycyrrhetic acid, PEG-DSPE and PEG-oleate was 4000, respectively.

[0280] The preparations 5-2 to 5-8 prepared according to Table 6 can all form microemulsions with good stability, and the particle sizes of preparations 5-2 to 5-8 are all 100 nm to 300 nm.

[0281] Table 6. Formulation table of nano-lipid preparation

[0282]

[0283] 2.1.2. Animal experiment

[0284] SD male rats 90, body weight (200 g or so), according to the body weight randomly divided into 8 groups, corresponding to the control group preparation 5-1 (Omegaven group), control group preparation 5-2, experimental group preparation 5-3, 5-4, 5-5, 5-6, 5-7, 5-8. Before the experiment, not fasting, not water, intravenous injection of EPA nano-lipid injection, intravenous injection volume conversion dose of EPA 360mg / kg, at 0h, 0.25h, 0h, 1h, 2h, 3h, 4h, 6h, 8h, 12h after administration, orbital venous plexus blood 0.5mL, collected in heparinized tube, 4℃, 3000rpm under the condition of centrifugation 10min, separation of plasma take supernatant, at-20℃ preservation for determination of its drug concentration. The content of EPA in plasma was detected by gas chromatograph, and the results were 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 were expressed as mean.

[0285] The instrument model of the gas chromatograph is Agilent 7890B, and the detection parameters are as follows: (88%-cyanopropyl) aryl-polysiloxane capillary column (60m x 0.25mm x 0.2μm), programmed temperature, 170℃ at 0min, increased to 240℃ at a speed of 5℃ / min, maintained for 6min, injector temperature is 250℃, detector temperature is 270℃. The carrier gas is helium with a flow rate of 1.0mL / min.

[0286] 2.1.3. Results

[0287] The experimental results are shown in Tables 7 and 8. The EPA blood concentration of the experimental groups (5-3 to 5-8) reached a peak at about 0.5h, and the duration of EPA concentration higher than 1000μg / mL was more than 6 hours. Among them, the preparation 5-8 group has the best long circulation effect. Compared with the preparation 5-5 group, after adding the auxiliary material with long circulation effect, the area under the drug-time curve of the preparation 5-8 can be increased by about 2 times. The area under the drug-time curve of the preparation group (5-8) using EPAX 6015 TGN as raw material is significantly better than that of the preparation group (5-2) using EPAX 4030 TGN as raw material. In addition, the area under the drug-time curve of the preparation 5-8 is slightly better than that of the preparation 5-7.

[0288] The results show that: (1) polyethylene glycol-phosphatidylethanolamine (PEG-PE), polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) and other polyethylene glycol lipid derivatives (a kind of auxiliary material with long circulation effect) produce significant long circulation effect, because the polyethylene glycol lipid derivative can improve the stability of EPA, reduce the rapid clearance of EPA in serum, effectively maintain the blood drug concentration, and has significant long circulation effect; (2) when the TG type fish oil raw material with high EPA purity and large iodine value (such as phospholipid greater than 85) is used, the long circulation effect of the preparation is easier.

[0289] Table 7. Changes in EPA blood drug concentration of different preparation groups

[0290]

[0291] Table 8. Pharmacokinetic parameters of different preparation groups

[0292]

[0293] In vivo example 2.2. Drug enrichment in liver after multiple dosing

[0294] SD male rats 90, body weight (about 200g), according to the body weight, randomly divided into 8 groups, corresponding to the control group preparation 5-1 (Omegaven group), control group 5-2, experimental group preparation 5-3, 5-4, 5-5, 5-6, 5-7, 5-8. Before the experiment, do not fast, do not water, intravenous injection of EPA lipid injection, intravenous injection volume equivalent dose of EPA 360mg / kg, continuous intravenous injection for 14 days. Dissected at 7 days, 14 days, 28 days (2h after the last administration) to get the liver, washed with normal saline, weighed, stored in-80℃ refrigerator for testing. The enrichment of EPA in the liver was detected by gas chromatograph, as shown in Table 8. The detection method is referred to the part 2.1.2 of example 2.1.

[0295] The results are shown in Table 9. The total EPA concentration in the liver of each experimental group (formulation 5-3 to formulation 5-8) was maintained at a high level of more than 500 μg / g for more than 14 days. After 28 days, the liver enrichment concentration of the experimental group (formulation 5-8) was about 2.47 times that of the control group (formulation 5-1), which had a significant liver accumulation effect. In addition, the EPAX 6015 TGN formulation group (formulation 5-8) had a significantly higher concentration of enrichment in the liver than the formulation group using EPAX 4030 TGN as the raw material (formulation 5-3). The formulation 5-8 group added a liver-targeting auxiliary material, and the liver EPA enrichment concentration was about 2.1 times that of the formulation 5-4 group 28 days after administration. Compared with the formulation groups using a single phospholipid (formulation 5-6) and having an iodine value of less than 85 (formulation 5-7), the liver targeting effect of the formulation 5-8 group was slightly better than that of the above two groups, which indicated that the liver-targeting component DSPE-PEG-glycyrrhetic acid had a synergistic effect with the phospholipid in the emulsifier.

[0296] The results show that: (1) The auxiliary material with the function of assisting EPA to accumulate in the liver (such as DSPE-PEG-glycyrrhetic acid in this example, which can also be selected from other liver-targeting components such as DSPE-PEG-galactose, DSPE-PEG-mannose, etc.) can significantly improve the enrichment rate of EPA in the liver, so that the steady-state concentration of EPA in the liver is reached faster. (2) The use of TG type fish oil raw material with high EPA purity and phospholipid with an iodine value of more than 80 is more conducive to the accumulation of EPA in the liver. (3) Compared with the use of egg yolk lecithin alone, the use of a mixture of egg yolk lecithin and soybean lecithin as phospholipid has a synergistic effect with the liver-targeting component, which is more conducive to assisting EPA to accumulate in the liver.

[0297] Table 9. Liver site concentration of EPA in different formulation groups

[0298]

[0299] In vivo Example 2.3. (Pharmacological example) Therapeutic effect on SD rat cholestatic liver injury model

[0300] Sixty SD male rats weighing about 200 g were randomly divided into 6 groups according to the body weight, namely a blank group, a model group, a positive control group (ursodeoxycholic acid), and experimental groups (formulation 5-3, formulation 5-4, formulation 5-5, formulation 5-6, formulation 5-8).

[0301] Formulation experimental groups: The nano-lipid injection of the present application was administered, namely formulation 5-3, formulation 5-4, formulation 5-5, formulation 5-6, formulation 5-7, and formulation 5-8.

[0302] The blank group: Pure water was used instead of the nano-lipid formulation of the present application for administration.

[0303] Model group: pure water instead of the nano-lipid preparation of the application for administration.

[0304] Positive control group: use ursodeoxycholic acid instead of the nano-lipid preparation of the application for administration. Ursodeoxycholic acid capsules are purchased from Losan Pharma GmbH (Germany).

[0305] 6 in each group, caged in a barrier level animal environment, free drinking water. After 3 days of adaptive feeding, the blank group was given olive oil solution by gavage, and the rest of the groups were given 1wt% α-naphthyl isothiocyanate (ANIT) olive oil solution by gavage once. Each group was fasted for 12 hours before gavage, and 48 hours after inducing cholestatic lesions, the blank group and the model group were given 20mL / kg of normal saline by gavage, the ursodeoxycholic acid group was given 100mg / kg of ursodeoxycholic acid solution by gavage, and the preparation 5-3, preparation 5-4, preparation 5-5, preparation 5-6, and preparation 5-8 groups were given EPA lipid injection by intravenous injection, with a volume-dose conversion of EPA 360mg / kg, and continuous intravenous injection for 14 days.

[0306] Serum detection: 1 hour after the end of administration on the 14th day, each group of animals was anesthetized with isoflurane, and blood was collected from the abdominal aorta, and the animals were fasted for 12 hours before blood collection. The content of ALT, AST, ALP, γ-GT, T-BIL, D-BIL, I-BIL, and TBA in the serum was detected by automatic biochemical analyzer (model JCA-BM6010 / C).

[0307] Liver histopathology and immunohistochemistry detection: The right lobe of the liver was cut and fixed with 10% formaldehyde, embedded and sectioned, HE stained, and the liver cell condition, fibrosis degree, and inflammatory cell infiltration were observed under a microscope.

[0308] Results analysis:

[0309] (1) The effect of milk injection on serum liver function enzymes of cholestatic hepatitis rats

[0310] The results are shown in Table 10. Compared with the blank group, the serum ALT, AST, ALP, γ-GT, T-BIL, and D-BIL contents of the model group rats were significantly increased (P<0.01). Compared with the model group, the serum ALT, AST, ALP, γ-GT, T-BIL, and D-BIL contents of the rats in each preparation experimental group (preparation 5-3 to preparation 5-8) were decreased (P<0.01). The contents of the positive control group and the preparation experimental group were significantly reduced. Among them, the content of the experimental group preparation 3-8 was reduced most significantly.

[0311] Table 10. Effect of different preparation groups on serum liver function indicators of cholestatic hepatitis rats

[0312]

[0313] 3. Effects of injected emulsion on liver histopathology in rats with cholestatic hepatitis

[0314] The results are as follows Figure 1 As shown, the liver tissue in the blank group had clear cellular structure with no abnormalities; the liver tissue in the model group showed extensive inflammatory cell infiltration and significant hepatocyte necrosis in the portal areas of the liver lobules; the number of inflammatory cells infiltrating the liver tissue in the ursodeoxycholic acid group (positive control group) and the formulation experimental group was significantly reduced, the degree of hepatocyte necrosis near the portal areas of the liver lobules was significantly reduced, and no significant fibroblast proliferation was observed. Among them, the number of inflammatory cells infiltrating the liver tissue in the formulation experimental group (5-8) was reduced most significantly, the degree of hepatocyte necrosis near the portal areas of the liver lobules was reduced most significantly, and no significant fibroblast proliferation was observed. This indicates that under the same TG-type fish oil content, the EPAX 6015 TGN formulation group (5-8) had a significantly better therapeutic effect than the formulation group (5-3) using EPAX 4030 TGN as raw material.

[0315] The results showed that: (1) Using TG-type fish oil raw materials with high EPA purity helps to improve the therapeutic effect; (2) Compared with using egg yolk lecithin alone, using a mixture of egg yolk lecithin and soybean lecithin is more conducive to synergistic effects with liver-targeting components, and more conducive to reducing the hydrolysis of EPA by lipoprotein lipase when it enters the blood circulation, thus assisting the accumulation of EPA in the liver and improving the therapeutic effect; (3) Excipients that help EPA accumulate in the liver (such as DSPE-PEG-glycyrrhetinic acid in this case, and can also be selected from other liver-targeting components such as DSPE-PEG-galactose and DSPE-PEG-mannose) can improve the therapeutic effect.

[0316] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0317] 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. A nano-lipid injection characterized in that, The nano-lipid injection comprises the following components in the following weight percentages, based on the total weight of the nano-lipid injection: ω-3 fatty acid glyceride 0.2wt%-40wt%; liver targeting component 0.001-10wt%; polyethylene glycol lipid derivative 0.001wt%-12wt%; emulsifier 0.05wt%-10wt%; second excipient 0-30wt%; and water; wherein the ω-3 fatty acid glyceride comprises EPA ester; the mass percentage of EPA in all fatty acid components of the ω-3 fatty acid glyceride is ≥35%; the ω-3 fatty acid glyceride is selected from triglyceride type fish oil lipid and artificially synthesized glyceride type fish oil; the liver targeting component contains a liver targeting factor or contains an unsaturated phospholipid unit in its structure; the number of unsaturated bonds in the unsaturated phospholipid unit is ≥2, and the mass percentage of phosphatidylethanolamine is ≥5%; the targeting factor is part of the structure of a modified liver targeting factor, and the modified liver targeting factor is selected from a polyethylene glycol modified liver targeting factor, a phospholipid modified liver targeting factor or a polyethylene glycol phospholipid modified liver targeting factor; the liver targeting factor is selected from one or more of glycyrrhetinic acid, mannose and galactose; the polyethylene glycol lipid derivative contains a phospholipid unit or a fatty acid ester unit in its structure, and the liver targeting component is partially or entirely simultaneously used as the polyethylene glycol lipid derivative; the liver targeting component as the polyethylene glycol lipid derivative is selected from a polyethylene glycol unit containing liver targeting component, and the polyethylene glycol unit containing liver targeting component is selected from one or more of phosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol-liver targeting factor and phosphatidylethanolamine-polyethylene glycol monomethyl ether; The polyethylene glycol lipid derivative containing a phospholipid unit or containing a fatty acid ester unit is selected from the group consisting of phosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol-C 12-20 fatty acid ester and polyethylene glycol-dual C 12-20 one or more of the fatty acid esters; the emulsifier comprises a phospholipid component; the second excipient is a pharmaceutically acceptable other excipient; the minimum weight percentage of water in the nano-lipid injection is 59%.

2. The nano-lipid injection according to claim 1, wherein the weight percentage of the ω-3 fatty acid glyceride in the nano-lipid injection is 2wt%-40wt%; the weight percentage of the liver targeting component in the nano-lipid injection is 0.05wt%-10wt%, and / or the weight percentage of the polyethylene glycol lipid derivative in the nano-lipid injection is 0.05wt%-10wt%; in all fatty acid components of the ω-3 fatty acid glyceride, the mass percentage of DHA is <35%, and the mass percentage of other ω-3 fatty acids is <20%; the other ω-3 fatty acids refer to fatty acids other than EPA and DHA; in the emulsifier, the mass percentage of the phospholipid unit in the phospholipid component is 10wt%-100wt%.

3. The nano-lipid injection according to claim 1, wherein the ω-3 fatty acid glyceride is selected from one or more of the following: triglyceride type fish oil lipid extracted from one or more of the following fish: anchovy, finfish, cod, herring, mackerel, tuna and sardine, and artificially synthesized glyceride type fish oil; and / or The mass percentage of EPA in the total fatty acid component of the omega-3 fatty acid glyceride is ≥40%, the mass percentage of DHA is <30%, and the mass percentage of other omega-3 fatty acids is <16%; and / or, In the EPA ester, the mass percentage of EPA triglyceride is ≥50%, the mass percentage of EPA monoglyceride is <30%, and the mass percentage of EPA diglyceride is <30%; and / or, The polyethylene glycol lipid derivative contains a phospholipid unit or a fatty acid ester unit in its structure; and / or, The nano-lipid injection is an oil-in-water dispersion system, and the average droplet size is ≤500 nm.

4. The nano-lipid injection according to any one of claims 1-3, characterized in that, The liver targeting factor is selected from one or more of glycyrrhetinic acid, mannose, and galactose; and / or, The structure of the liver targeting component contains an unsaturated phospholipid unit, the unsaturated phospholipid unit has C 12-20 fatty acyl; wherein, in the unsaturated phospholipid unit, the number of unsaturated bonds is greater than or equal to 2, and the mass percentage of phosphatidyl ethanolamine is greater than or equal to 5%; and / or, The liver targeting component contains or does not contain a polyethylene glycol unit in its structure; when it contains a polyethylene glycol unit, the molecular weight of the polyethylene glycol unit in the structure of the liver targeting component is 1000 Da-5000 Da; and / or, The polyethylene glycol lipid derivative is selected from the group consisting of phosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol-C 12-20 fatty acid ester and polyethylene glycol-bis C 12-20 one or more of a fatty acid ester and polyethylene glycol-bis C The molecular weight of the PEG unit in the polyethylene glycol lipid derivative is 1000 Da-6000 Da; and / or, The iodine value of the phospholipid component in the emulsifier is greater than 80; and / or, The emulsifier is selected from one or more of egg yolk lecithin, soybean phospholipid, synthetic phospholipid, polyene phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin; and / or; The second adjuvant is selected from one or more of an antioxidant, an interfacial film stabilizer, an osmotic pressure regulator, a pH regulator, and a lipid rich in linolenic acid, wherein the mass percentage of linolenic acid in the lipid rich in linolenic acid is 30wt%-70wt%, and the lipid rich in linolenic acid is not an omega-3 fatty acid glyceride; wherein in the nano-lipid injection, the content of the antioxidant is 0-5wt%, the content of the interfacial film stabilizer is 0-3wt%, the content of the osmotic pressure regulator is 0-10wt%, and the content of the lipid rich in linolenic acid is 0-10wt%; and / or, The average droplet size in the nano-lipid injection is ≤300 nm.

5. The nano-lipid injection according to claim 4, characterized in that, The liver targeting component is selected from any one of the following (A) to (E): (A) the unsaturated phospholipid unit of the liver targeting component is polyene phosphatidylcholine or polyene phosphatidylethanolamine, and the unsaturated phospholipid unit is selected from one or more of dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine and dimyristoyl phosphatidylcholine; (B) the unsaturated phospholipid unit in the liver targeting component has at least one of lauroyl, myristoyl, palmitoyl, stearoyl and oleoyl; (C) the liver targeting component is phosphatidylethanolamine-polyethylene glycol-liver targeting factor, or phosphatidylethanolamine-liver targeting factor, or polyene phospholipid; wherein "-" represents a chemical bond or a linker; (D) the liver targeting component includes a liver targeting component containing a polyethylene glycol unit, and the liver targeting component containing a polyethylene glycol unit is selected from one or more of phosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol-liver targeting factor and phosphatidylethanolamine-polyethylene glycol monomethyl ether; (E) the liver targeting factor is mannose, galactose or glycyrrhetinic acid; and / or, The polyethylene glycol lipid derivative is selected from one or more of distearoyl phosphatidylethanolamine-polyethylene glycol 2000, distearoyl phosphatidylethanolamine-polyethylene glycol 5000, soybean phosphatidylethanolamine-polyethylene glycol monomethyl ether 2000, polyethylene glycol 4000 oleate, polyethylene glycol 6000 oleate, polyethylene glycol 6000 dioleate and polyethylene glycol 200 laurate; wherein "-" represents a chemical bond or a linker; and / or, The synthetic phospholipid is selected from one or more of distearoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine and dimyristoyl phosphatidylcholine; and / or, The iodine value of the egg yolk lecithin is ≥60; the egg yolk lecithin contains ≥68 wt% phosphatidylcholine and ≤20 wt% phosphatidylethanolamine, and the total content of phosphatidylcholine and phosphatidylethanolamine is ≥80 wt%; and / or, The soybean phospholipid is polyene phosphatidylcholine or polyene phosphatidylethanolamine; and / or, The iodine value of the soybean lecithin is ≥80; the soybean lecithin contains ≥45 wt% phosphatidylcholine and ≤30 wt% phosphatidylethanolamine, and the total content of phosphatidylcholine and phosphatidylethanolamine is ≥70 wt%; and / or, The antioxidant is selected from one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, ascorbic acid and its esters, α-tocopherol and its esters, β-tocopherol and its esters, γ-tocopherol and its esters, δ-tocopherol and its esters; and / or, The osmotic pressure regulator is selected from one or more of glycerol, propylene glycol and mannitol; and / or, The pH regulator is a buffer salt system; and / or, The ratio of α-linolenic acid to γ-linolenic acid in the linolenic acid-rich lipid is ≥5:1; and / or, The average particle size of the nano-lipid injection is 50 nm to 300 nm.

6. Nanolipidic injectable formulation according to any one of claims 1 to 3, characterized in that, All of the liver targeting components belong to the polyethylene glycol lipid derivatives; the liver targeting components belonging to the polyethylene glycol lipid derivatives are selected from the group consisting of polyethylene glycol unit-containing liver targeting components selected from one or more of phosphatidylethanolamine-polyethylene glycol, phosphatidylethanolamine-polyethylene glycol-liver targeting factor and phosphatidylethanolamine-polyethylene glycol monomethyl ether.

7. Nanolipidic injectable formulation according to claim 6, characterized by the fact that, The liver targeting components are selected from one or more of phosphatidylethanolamine-polyethylene glycol-mannose, phosphatidylethanolamine-glycyrrhetinic acid-mannose and phosphatidylethanolamine-glycyrrhetinic acid; the phosphatidylethanolamine units are each independently selected from any one of distearoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine and dioleoylphosphatidylethanolamine, wherein "-" represents a chemical bond or a linker.

8. Nanolipidic injectable formulation according to claim 4, characterized by the fact that, The liver targeting components simultaneously contain phospholipid units and polyethylene glycol units in their structures.

9. A process for the preparation of nano-lipid injection as claimed in any one of claims 1 to 8, wherein, The method comprises the following steps: mixing the oil phase components including the omega-3 fatty acid glyceride under heating conditions to prepare an oil phase; dissolving the water phase components in an aqueous solvent to prepare a water phase, or using water as the water phase; mixing and dispersing the oil phase and the water phase to prepare an oil-in-water dispersion system.

10. A process for the preparation of nano-lipid injection as claimed in claim 9 wherein, After the oil-in-water dispersion system is prepared, at least one of the following is further performed: filtration, sterilization and packaging.

11. Use of the nano-lipid injection according to any one of claims 1-8 or prepared by the method of claim 9 or 10 in the preparation of a medicament for preventing and / or treating a liver injury-related disease. The liver injury-related disease is cholestatic liver disease.

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