Zinc liposome formulations for liver delivery and treatment of liver diseases

Abstract

This application relates to zinc liposome formulations for liver delivery and treatment of liver diseases. The liposome composition comprises: a) a lipid bilayer containing phospholipids (e.g., electrically neutral phospholipids, such as EPC or HSPC) and cholesterol, and b) zinc encapsulated within the bilayer. The method involves administering the composition containing zinc-containing liposomes to an individual.

Description

Technical Field

[0001] This invention relates to zinc liposome formulations for liver delivery and treatment of liver diseases. Background Technology

[0002] Liver fibrosis is a leading cause of death because it alters the structure of certain organs and disrupts normal function. Liver fibrosis is a histological consequence of the wound healing process caused by chronic liver diseases such as viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, and other liver disorders. Excessive deposition of extracellular matrix (ECM) rich in collagen, which forms fibers, is a hallmark of liver fibrosis. This excessive ECM deposition alters the normal structure of the liver, leading to pathophysiological damage. Effective treatments for liver fibrosis are still needed.

[0003] All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety. Summary of the Invention

[0004] This application provides methods for increasing and / or maintaining liver zinc levels (e.g., liver zinc concentration), methods for delivering zinc to the liver of an individual, methods for treating, reversing, or reducing liver fibrosis, and methods for promoting liver regeneration in an individual by administering a composition comprising liposomes (i.e., a "liposomal composition"), wherein the liposomes comprise a lipid bilayer and zinc, wherein the zinc is encapsulated by the lipid bilayer. This application also provides compositions comprising liposomes, said liposomes comprising a) a lipid bilayer comprising phospholipids and cholesterol and b) a zinc compound.

[0005] In one aspect of this application, a method for increasing the half-life of zinc in the liver of an individual is provided, comprising administering to the individual a composition comprising: a) a lipid bilayer; and b) a zinc compound encapsulated by the lipid bilayer.

[0006] In one aspect of this application, a method for treating liver fibrosis in an individual is provided, comprising injecting the individual with a composition containing liposomes, the composition comprising a) a lipid bilayer; and b) zinc encapsulated by the lipid bilayer. In some embodiments, liver fibrosis is associated with alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), or viral infection. In some cases, liver fibrosis is associated with drug-induced liver injury (DILI), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), or other liver diseases.

[0007] In some embodiments of any of the above methods, the lipid bilayer comprises phospholipids and cholesterol, wherein the phospholipids comprise electrically neutral phospholipids. In some embodiments, the electrically neutral phospholipids comprise phosphatidylcholine (PC). In some embodiments, PC is lecithin (EPC) or hydrogenated soybean lecithin (HSPC). In some embodiments, the molar ratio of phospholipids to cholesterol is about 1:1 to about 4:1. In some embodiments of any of the above methods, the phospholipids have a phase transition temperature of at least 30°C. In some embodiments of any of the above methods, the phospholipids further comprise negatively charged phospholipids. In some embodiments, the negatively charged phospholipids comprise distearate phosphoglycerol (DSPG). In some embodiments, the molar ratio of negatively charged phospholipids to electrically neutral phospholipids is at least about 1:10.

[0008] In some embodiments according to any of the methods described above, the average particle size of the liposomes is from about 50 nm to about 200 nm. In some embodiments, the average particle size of the liposomes is from about 90 nm to about 140 nm.

[0009] In some embodiments according to any of the above methods, the absolute value of the zeta potential of the liposomes is about or greater than about 25.

[0010] In some embodiments according to any of the methods described above, the zinc is selected from organic zinc salts, inorganic zinc salts, and zinc chelate compounds bound to zinc.

[0011] In some embodiments according to any of the above methods, zinc is in an aqueous solution in a readily ionizable or ionized form.

[0012] In some embodiments according to any of the methods described above, the zinc comprises zinc sulfate.

[0013] In some embodiments according to any of the above methods, less than about 10% of zinc is released from the liposomes in vitro over 0.5 hours.

[0014] In some embodiments according to any of the above methods, less than about 20% of zinc is released from the liposomes in vitro over 48 hours.

[0015] In some embodiments according to any of the above methods, the liver zinc concentration of an individual increased by at least about 20% approximately 48 hours after administration of the liposomes, compared with the liver zinc concentration before administration of the liposomes.

[0016] In some embodiments according to any of the methods described above, the composition is administered parenterally. In some embodiments, the composition is administered intravenously.

[0017] In some embodiments of any of the methods described above, the individual is a person.

[0018] In some embodiments of any of the methods described above, the individual suffers from liver disease. In some embodiments, the liver disease is selected from alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

[0019] In some embodiments according to any of the methods described above, the composition is applied approximately once a week.

[0020] In some embodiments according to any of the methods described above, the zinc content of each applied zinc liposome is less than about 1.6 mg / kg (e.g., for humans). In some embodiments, the zinc content of each applied zinc liposome is less than about 0.8 mg / kg (e.g., for humans).

[0021] In some embodiments according to any of the methods described above, the zinc content of each applied zinc liposome is at least about 0.08 mg / kg (e.g., for humans). In some embodiments, the zinc dose per application is at least about 0.4 mg / kg (e.g., for humans).

[0022] In some embodiments according to any of the methods described above, the composition is applied for approximately two weeks.

[0023] In some embodiments according to any of the above methods, the composition is applied for approximately four months.

[0024] In one aspect of this application, a composition comprising liposomes is provided, the liposomes comprising a) a lipid bilayer comprising phospholipids and cholesterol, wherein the phospholipids comprise electrically neutral phospholipids; and b) zinc, wherein the zinc is encapsulated by the lipid bilayer. In some embodiments, the phospholipids further comprise negatively charged phospholipids. In some embodiments, the electrically neutral phospholipids comprise phosphatidylcholine (PC). In some embodiments, PC is selected from egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the molar ratio of phospholipids to cholesterol is from about 1:1 to about 4:1. In some embodiments, the average particle size of the liposomes is from about 50 nm to about 200 nm. In some embodiments, the average particle size of the liposomes is from about 90 to 140 nm. In some embodiments, the phospholipids further comprise negatively charged phospholipids comprising distearate phosphatidylglycerol (DSPG). In some embodiments, the molar ratio of negatively charged phospholipids to electrically neutral phospholipids is at least about 1:10. In some embodiments, the absolute value of the zeta potential of the liposomes is about or greater than about 25. In some embodiments, the zinc is selected from organic zinc salts, inorganic zinc salts, and zinc chelates bound to zinc. In some embodiments, the zinc is in an easily ionized or ionized form in aqueous solution. In some embodiments, the zinc comprises zinc sulfate. In some embodiments, the average particle size of the liposomes is about 50 nm to about 200 nm. In some embodiments, the average particle size of the liposomes is about 90 nm to about 140 nm. In some embodiments, less than about 10% of the zinc is released from the liposomes in vitro after 0.5 hours. In some embodiments, less than about 20% of the zinc is released from the liposomes in vitro after 48 hours.

[0025] In one aspect of this application, a composition comprising liposomes is provided, the liposomes comprising: a) a lipid bilayer comprising phospholipids and cholesterol, and b) zinc, wherein the zinc is encapsulated by the lipid bilayer, wherein the phospholipid is hydrogenated soybean lecithin (HSPC), wherein the molar ratio of HSPC to cholesterol is about 4:1, and wherein the particle size of the liposomes is about 50 nm to about 200 nm.

[0026] In one aspect of this application, a composition comprising liposomes is provided, the liposomes comprising a) a lipid bilayer comprising phospholipids and cholesterol, and b) zinc, wherein the zinc is encapsulated by the lipid bilayer, wherein the phospholipids comprise hydrogenated soybean lecithin (HSPC) and negatively charged phospholipids, wherein the molar ratio of HSPC to cholesterol is about 4:1, and wherein the average particle size of the liposomes is about 50 nm to about 200 nm.

[0027] In one aspect of this application, a method for preparing any of the compositions described herein is provided, the method comprising mixing a formulation containing phospholipids and cholesterol with zinc. In some embodiments, the encapsulation efficiency of the liposomes is at least about 80%. Attached Figure Description

[0028] Figure 1 The survival of the test animals was shown during the 14-day observation period following a single application of zinc liposomes.

[0029] Figure 2 This shows the changes in body weight of the experimental animals during the 14-day observation period following a single application of zinc liposomes.

[0030] Figure 3 This shows the concentration of zinc in the liver within 168 hours after a single administration of zinc liposomes and zinc sulfate.

[0031] Figure 4 The diagram shows the experimental progress. Kunming mice underwent bile duct ligation (BDL surgery) to induce liver fibrosis. Four weeks post-surgery, they were treated with zinc for two weeks, followed by endpoint sampling.

[0032] Figure 5 The survival rates of mice in different treatment groups during treatment are shown. Mortality was observed only in the group receiving standard zinc treatment.

[0033] Figure 6 The changes in body weight in different treatment groups after treatment are shown. BDL surgery resulted in weight loss in the animals, but with the use of zinc liposomes, BDL did not affect the animals' body weight.

[0034] Figure 7 This shows serum zinc levels after treatment.

[0035] Figure 8 This shows the level of liver zinc after treatment.

[0036] Figure 9A This shows representative images of histological staining from different groups of liver fibrosis. Figure 9B The results of quantitative analysis of histological staining are shown. Figure 9C The amount of hydroxyproline measured in different groups is shown. The unit is micrograms of hydroxyproline per milligram of liver tissue.

[0037] Figure 10 A schematic diagram showing the experimental timeline. Rhesus monkeys underwent liver fibrosis induction surgery (BDL). Two months after BDL, rhesus monkeys with confirmed liver fibrosis were treated with zinc liposomes. Treatment lasted for four months, with liver biopsies performed monthly.

[0038] Figure 11 The changes in body weight under different dosage regimens are shown. Weight gain is generally related to the dosage of zinc liposomes.

[0039] Figure 12Representative images from histological examination are shown. Sirius red staining highlights collagen deposition, while Fast Green co-staining provides background staining for the liver tissue. An image of normal liver tissue without fibrosis is shown on the right side of the figure for comparison. Every two images in the same column are examination results from the same animal before and after 4 months of treatment.

[0040] Figure 13 The results of histological examination are presented as a quantitative analysis. The percentage area stained with Sirius red was obtained using computer software. The level of normal tissue is represented by dashed lines. Error bars represent the mean and standard deviation of results from more than one animal in a single treatment group.

[0041] Figure 14 The survival rates of mice in different treatment groups during the treatment period are shown as described in Example 9.

[0042] Figure 15 Quantitative analysis of Sirius red histological staining is shown, as described in Example 10. Detailed Implementation

[0043] This application provides a zinc-containing liposome composition and a method for preparing the same. In some embodiments, the liposome composition comprises liposomes including a lipid bilayer and zinc encapsulated within the lipid bilayer, wherein the lipid bilayer is composed of phospholipids and cholesterol, and the zinc is encapsulated within the lipid bilayer. In some embodiments, the phospholipids are composed of electrically neutral phospholipids (e.g., EPC or HSPC). The methods described herein include administering the zinc-containing liposome composition to an individual (e.g., an individual with liver fibrosis). In some embodiments, the methods treat, reverse, or reduce liver fibrosis. In some embodiments, the methods promote liver regeneration. In some embodiments, the methods improve the individual's quality of life. In some embodiments, the methods deliver zinc liposomes to the liver and / or increase, regulate, or maintain liver levels in the individual. In some embodiments, the methods promote zinc retention in the individual's liver.

[0044] Zinc deficiency in the liver is an important comorbidity with chronic liver disease. Currently, zinc is mainly supplemented orally. However, previous studies have shown that oral zinc supplements have failed to restore liver zinc levels.

[0045] Zinc deficiency in the liver has been observed to be a concomitant phenomenon with chronic liver disease, especially in patients with liver fibrosis or cirrhosis. See Bartholomay, AF, et al., N Engl J Med, 1956. 255(9): p. 403-8; Vallee, BL, et al., N Engl J Med, 1957. 257(22): p. 1055-65; and Grungreiff, K. et al., Ann Hepatol, 2016. 15(1): p. 7-16. Several studies have examined the effects of zinc supplementation on liver fibrosis, see Gimenez, A., et al., J Hepatol, 1994. 21(3): p. 292-8 and Cabre, M., et al., Int J VitamNutrRes, 2001. 71(4): p. 229-36. Zinc supplementation has been shown to restore serum zinc levels and sometimes alleviate associated zinc deficiency. Yanagisawa, H., Yakugaku Zasshi, 2008, 128(3): p.333-9. Mohammad, MK, et al., Nutr Clin Pract, 2012, 27(1): p.8-20. However, oral zinc supplementation has been reported not to significantly alter the zinc deficiency status in the liver of patients with cirrhosis. Zarski, JP, et al., Gastroenterol ClinBiol, 1987, 11(12): p.856-60. Similarly, most findings are attributed primarily to the hepatoprotective effect of zinc supplementation rather than its therapeutic effect on existing liver fibrosis. To date, no human clinical studies have demonstrated that zinc can treat liver fibrosis / cirrhosis. See Gimenez, A., et al., J Hepatol, 1994.21(3):p.292-8; Anttinen, H., et al., Gastroenterology, 1984.86(3): p.532-9 and Atsushi Hosui, NH, Hepatology, 2016.64(S1).

[0046] On the other hand, zinc is essential for the function of more than 300 enzymes and 1,000 transcription factors, and is stored and transported in metallothionein. Insufficient or excessive zinc intake can be harmful. Excessive zinc, in particular, impairs copper absorption, as metallothionein absorbs both metals. Nausea, vomiting, pain, cramps, and diarrhea may occur after oral administration of very high doses of zinc (300 mg zinc per day), and there is evidence that it induces copper deficiency, changes in blood lipoprotein levels, elevated LDL levels, and decreased HDL levels with long-term intake of 100 mg zinc / day. See Fosmire GJ (February 1990). Am.J.Clin.Nutr.51(2):225–7. Therefore, the subtle regulation of zinc is crucial for the individual.

[0047] Unbound by theory, it is assumed that patients with liver fibrosis may lack the ability to retain zinc in a lasting manner, and thus still suffer from liver zinc deficiency despite zinc supplementation.

[0048] Liposomes are spherical artificial vesicles composed primarily of phospholipids, and come in various sizes and structures. Common liposomal vesicles consist of a hydrophilic core and a surrounding lipid bilayer. The lipid bilayer separates the hydrophilic core from the external environment, thus enabling liposomes to function as drug delivery systems. It can protect the active pharmaceutical ingredient from degradation, thereby increasing circulation time, reduce unnecessary contact with non-target cells, thereby reducing toxicity, and / or enhance target specificity through liposome modification, thereby improving drug efficacy. Currently, many liposomal drugs have been approved by numerous institutions worldwide, and the number of drug candidates in clinical trials is also increasing. [19, 20] Depending on their properties, different liposomes may have different organ distributions. Liposomes must cross the epithelial layer to accumulate in organs, and their physicochemical properties greatly influence this process.

[0049] A major challenge facing liposomes as a drug delivery system is the need to identify, neutralize, and eliminate multiple defense systems of invading substances, including the reticuloendothelial system (RES), opsonization mechanisms, and the immune system. The RES is a major site of lipid accumulation after systemic administration. Key organs associated with the RES include the liver, spleen, kidneys, lungs, bone marrow, and lymph nodes. Resident macrophages clear liposomes from the RES through direct interaction with phagocytes. Large charged liposomes are cleared by the liver within minutes, and by the spleen in less than an hour. See Sercombe et al., FrontPharmacol. 2015; 6: 286. The liver has been shown to rapidly clear liposomes smaller than 200 nm specifically delivered to it. For example, administration of liposomes containing glycyrrhetinic acid (GA) resulted in a rapid increase in GA concentration in the liver within minutes after administration, but the hepatic GA concentration decreased sharply within hours, only returning to approximately zero after 12 hours post-administration. See Chen et al, Oncotarget, 2017, Vol.8 (No. 60), pp:102046-102066.

[0050] This application is based, at least in part, on the various surprising advantages demonstrated by liposomes and the methods of using the liposomal compositions described herein. First, zinc liposomes, once introduced into the liver, remain there (e.g., for at least 48 hours). This contrasts sharply with patients or individuals receiving the same dose of conventional zinc intravenously, where liver zinc levels show a slight increase within minutes of administration but disappear within 30 minutes. Furthermore, patients or individuals receiving zinc liposomes exhibit 100% survival and appropriate weight gain with few adverse reactions. In contrast, patients or individuals receiving the same dose of conventional zinc show lower survival and weight loss. The favorable results are likely due to the zinc protection provided by the zinc liposomes. Moreover, patients or individuals with liver fibrosis who receive zinc liposome treatment show a significant reduction in liver fibrosis, indicating that zinc liposome treatment is effective for liver fibrosis. Remarkably, at certain doses (e.g., approximately 1-2 mg zinc / kg in monkeys every other day), the recovery of fibrosis in patients or individuals receiving zinc liposome treatment is unexpected, reaching almost complete recovery.

[0051] Furthermore, the exemplary liposomes and methods demonstrated in the examples involve liposomes using much lower doses of zinc than previously studied. For example, in a clinical study, patients with alcoholic cirrhosis were given oral zinc supplements to determine whether zinc therapy could reduce liver damage (inflammation, oxidative stress, cell death) and scarring. In this study, the zinc administration regimen was 50 mg of elemental zinc (220 mg of zinc sulfate) orally daily. [8, 13] In another animal study, rats were given zinc via drinking water at a daily supplementation of approximately 20–30 mg / kg. See Dashti et al., Nutrition Vol. 13, March 1997. Conversely, as described in this application, administering zinc liposomes at much lower doses (e.g., administering about 0.35-5 mg / kg of zinc liposomes to mice or monkeys every two days, equivalent to about 6-22 mg / kg per day) can a) effectively and persistently increase hepatic zinc levels, b) reduce fibrosis (by up to 80-90% compared to the control group), and 3) protect patients or individuals from adverse events and weight loss.

[0052] definition

[0053] The terms “zinc,” “zinc ion,” “elemental zinc,” and “zinc element” are used interchangeably in this document.

[0054] The term "negatively charged phospholipid" refers to phospholipids that carry one or more negative charges at physiological pH. Examples of negatively charged phospholipids include, but are not limited to, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol (PI), and phosphatidylserine.

[0055] "Positively charged phospholipids" refer to phospholipids that carry one or more positive charges at physiological pH values.

[0056] "Electrically neutral phospholipids" are phospholipids that do not carry a positive or negative charge at physiological pH. Examples of electrically neutral phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin.

[0057] For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by the disease, reduction of the severity of the disease, stabilization of the disease (e.g., prevention (or delay of disease progression), delay or slowing of disease progression, improvement of disease status, improvement of quality of life, and / or prolongation of survival. In some embodiments, the treatment reduces the severity of one or more symptoms associated with liver fibrosis by at least one of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to corresponding symptoms in the same patient or individual before treatment, or compared to corresponding symptoms in other patients or individuals who have not received treatment, at 95% or 100%. "Treatment" also includes a reduction in the pathological outcome of liver fibrosis. The methods of this application cover any one or more of these aspects of treatment.

[0058] As used herein, “delay” means to postpone, hinder, slow, delay, stabilize, and / or postpone the development of a disease. This delay can have varying durations, depending on the history of the disease and / or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay can indeed include prevention, as the individual will not develop the disease.

[0059] As used herein, the term "effective amount" refers to an amount of compound or composition sufficient to treat a particular condition, symptom, or disease, such as alleviating, reducing, and / or delaying one or more of its symptoms. In the context of liver fibrosis, an effective amount includes amounts sufficient to reduce the severity of liver fibrosis or to prevent or delay its progression.

[0060] As understood in the art, an "effective dose" can refer to one or more doses, i.e., a single or multiple doses may be required to achieve the desired therapeutic endpoint. An effective dose may be considered when liposomes are administered in combination with another drug if a desirable or beneficial result can be achieved or obtained in combination with one or more other drugs. Liposomes and another drug may be administered sequentially, simultaneously, or co-administered, and the components may be administered via the same or different routes. Therefore, a combination therapy regimen of effective doses comprises an effective dose of a first treatment method and an effective dose of a second treatment method, which, when administered sequentially, simultaneously, or co-administered, can produce the desired therapeutic effect.

[0061] As used herein, "composition" includes and applies to the compositions of this application. This application also provides pharmaceutical compositions comprising the components described herein.

[0062] Unless otherwise expressly stated, “individual” as used herein refers to mammals, including but not limited to primates, humans, cattle, horses, cats, dogs or rodents.

[0063] As used herein, the term "patient" encompasses all mammalian species, including but not limited to primates, humans, cattle, horses, cats, dogs, or rodents.

[0064] As used herein, the term “treatment” encompasses the treatment of disease conditions in mammals, particularly humans, and includes: (a) suppressing disease conditions, i.e., preventing their development; and / or (b) alleviating disease conditions, i.e. causing disease conditions to subside.

[0065] As used herein, the term "prevention" refers to protective treatment of a disease condition to reduce and / or minimize the risk of a disease condition and / or reduce the risk of recurrence of a disease condition by administering a therapeutically effective amount of a substance to the patient. Patients may be selected for preventive treatment based on factors known to increase the risk of developing a clinical disease condition compared to the general population. For preventive treatment, the clinical disease condition may or may not be present. "Preventive" treatment can be categorized into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment aimed at reducing or minimizing the risk of a disease condition in a patient who has not yet developed a clinical disease condition, while secondary prevention is defined as minimizing or reducing the risk of recurrence or a second occurrence of the same or similar clinical disease condition.

[0066] It should be understood that the aspects and embodiments of the invention described herein include aspects and embodiments that are “composed of” and / or “substantially composed of”.

[0067] As used herein, “combination therapy” refers to a first therapy that includes a second therapy (e.g., surgery or a therapeutic agent) for treating, stabilizing, preventing, and / or treating liver fibrosis, comprising a liposomal composition containing zinc-containing liposomes. Delaying liver fibrosis. Administration in “combination” with another compound includes administration in sequence, simultaneously, or sequentially, with the same or different compositions. In some embodiments, the combination therapy optionally includes one or more pharmaceutically acceptable carriers or excipients, non-pharmaceutical active compounds, and / or inert substances.

[0068] As used herein, the term “simultaneous administration” means administering the first and second treatments in a combination therapy at time intervals not exceeding about 15 minutes (e.g., not exceeding any one of about 10 minutes, 5 minutes, or 1 minute). When the first and second treatments are administered simultaneously, the first and second treatments may be contained in the same composition (e.g., a composition containing the first and second treatments) or in separate compositions (e.g., the first treatment is contained in one composition and the second treatment is contained in another composition).

[0069] As used herein, the term "sequential administration" means administering the first and second treatments in a combination therapy at time intervals greater than about 15 minutes (e.g., greater than any of about 20, 30, 40, 50, 60, or more minutes). The first or second treatment may be administered first. The first and second treatments are contained in separate compositions, which may be contained in the same or different packages or kits.

[0070] As used in this article, the term "simultaneous administration" means that in combination therapy, the administration of the first therapy and the second therapy overlap.

[0071] As used herein, "pharmaceutical acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable; for example, the material can be incorporated into a pharmaceutical composition administered to a patient without causing any apparent undesirable biological effects or interacting with any other component of the composition in a harmful manner. Pharmaceutically acceptable carriers or excipients preferably meet the requirements of toxicological and manufacturing testing standards and / or are included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0072] References to “about” values ​​or parameters in this document include (and describe) variations of that value or parameter itself. For example, a description of “about X” includes a description of “X”.

[0073] The term “about XY” as used herein has the same meaning as “about X to about Y”. The expression “about X, Y or Z” as used herein has the same meaning as “about X, Y or Z”.

[0074] As used in this article, references to "not" values ​​or parameters typically refer to and describe values ​​or parameters that are "different from" .

[0075] As used herein and in the appended claims, the singular forms “a” or “an” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “liposome” includes a plurality of such liposomes, reference to “pharmaceutical” includes reference to one or more pharmaceuticals known to those skilled in the art, and so on.

[0076] It will be apparent to those skilled in the art that individuals who need to be assessed, selected, and / or treated are those who require such activities.

[0077] How to use

[0078] This application provides methods of using the liposome compositions described herein. In some embodiments, a method of delivering zinc to the liver of an individual is provided, comprising delivering the liposome composition to the individual. In some embodiments, a method of promoting or regulating hepatic zinc (e.g., promoting the half-life of hepatic zinc) in an individual is provided, comprising administering the liposome composition to the individual. In some embodiments, a method of maintaining hepatic zinc is provided, comprising applying the liposome composition to the individual. In some embodiments, a method for promoting hepatic zinc retention in an individual (e.g., promoting zinc half-life, promoting the area under the liver curve (i.e., AUC) of zinc) is provided, the method comprising administering a liposome composition to the individual. In some embodiments, a method for treating liver fibrosis in an individual is provided, comprising administering a liposome composition to the individual. In some embodiments, a method for reversing liver fibrosis in an individual is provided, comprising administering a liposome composition to the individual. In some embodiments, a method for reducing liver fibrosis in an individual is provided, comprising administering the liposome composition to the individual. In some embodiments, a method for promoting liver regeneration in an individual is provided, comprising administering the liposome composition to the individual. In some embodiments, a method for improving the quality of life of an individual suffering from liver fibrosis is provided, the method comprising administering a liposome composition to the individual. The liposome composition in some embodiments comprises a) a lipid bilayer consisting of phospholipids and cholesterol and b) zinc, wherein the zinc is encapsulated in the lipid bilayer.

[0079] Methods of zinc delivery

[0080] In some embodiments, a method of delivering zinc to the liver of an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; in some embodiments, the individual is a human, and the amount of zinc administered each time does not exceed about 120 mg / m². 2 (for example, not exceeding approximately 100 mg / m²) 2 80mg / m 2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some implementations, the individual is a human, and the amount of zinc applied each time is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 60 mg / m2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m 2 Approximately 15 mg / m 2 or about 12mg / m 2In some embodiments, liposomes are administered at least once a week (e.g., at least once every six, five, four, three, or two days). In some embodiments, liposomes are administered at least once every two days. In some embodiments, liposomes are administered at a frequency of about or at least about one, two, three, four, five, or six weeks, or at least about one, two, three, or four months. In some embodiments, the individual has liver fibrosis that has not progressed to cirrhosis prior to liposome injection. In some embodiments, the patient's liver fibrosis has progressed to cirrhosis prior to administration. In some cases, the individual's liver zinc concentration after administration is greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. In some cases, the increase in hepatic zinc concentration was sustained for at least 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least 1, 2, 3, 4, 5, 6, or 7 days after a single dose. In some embodiments, an individual's collagen content decreased after liposome administration compared to pre-liposome collagen content (e.g., a decrease in collagen content level of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%). In some embodiments, patients experienced weight gain after taking liposomes. For example, within 1, 2, 3, or 4 months after liposome administration, patients experienced a weight gain of at least 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30%. In some cases, patients did not experience serious adverse reactions after using liposomes. In some cases, patients do not experience more than 5, 4, 3, 2, or 1 non-serious adverse events after using liposomes. In some embodiments, individuals do not develop more than 5, 4, 3, 2, or 1 non-serious adverse events. In some embodiments, the liposomes have an average particle size of about 50-200 nm (e.g., about 90-140 nm). In some embodiments, the lipid bilayer consists of electrically neutral phospholipids having a phase transition temperature of at least about 10 °C (e.g., at least about 20 °C, 30 °C, 40 °C, or 50 °C). In some embodiments, the electrically neutral phospholipids consist of egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the phospholipid bilayer consists of negatively charged phospholipids (e.g., DSPG). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc. In some embodiments, this composition is not suitable for oral administration.

[0081] In some embodiments, a method is provided for delivering and maintaining adequate levels of zinc in the liver of an individual, comprising administering a composition comprising liposomes, wherein the liposomes comprise a) a lipid bilayer; and b) an effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, the zinc content in the liver is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% (as shortly before delivery) compared to before zinc delivery. In some embodiments, the desired level of zinc in the liver is not less than about 98%, 95%, 90%, 85%, 80%, 75%, or 70% compared to the liver zinc concentration of an individual without liver fibrosis (or the average liver zinc concentration of a group of individuals without liver fibrosis). In some embodiments, the zinc concentration in the liver is maintained for at least 30 minutes (e.g., at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days) after delivery. In some embodiments, the individual is human, and the amount of zinc used for each administration does not exceed about 120 mg / m². 2 (for example, not exceeding approximately 100 mg / m²) 2 80mg / m 2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some embodiments, the individual is a human, and the amount of zinc used for each administration is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 60 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m³ 2 Approximately 15 mg / m 2 or approximately 12 mg / m³ 2In some embodiments, liposomes are administered at least once a week (e.g., at least once every six, five, four, three, or two days). In some embodiments, liposomes are administered at least once every two days. In some embodiments, liposomes are administered at a certain frequency for about one, two, three, four, five, or six weeks or at least one, two, three, three, or four months, and in some embodiments, the individual's liver fibrosis had not progressed to cirrhosis prior to liposome administration. In some embodiments, the individual's liver fibrosis had progressed to cirrhosis prior to liposome administration. In some embodiments, the liver zinc concentration in the individual after administration is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. In some embodiments, the increased liver zinc concentration persists for at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days after a single liposome administration. In some embodiments, after liposome administration, an individual's collagen content decreases (e.g., a decrease in collagen content level of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to collagen content prior to liposome administration. In some embodiments, an individual gains weight after liposome administration. For example, within approximately 1, 2, 3, or 4 months after the start of liposome administration, the individual's weight increases by at least about 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30%). In some embodiments, the individual does not experience serious adverse events after liposome injection. In some embodiments, the individual does not develop more than 5, 4, 3, 2, or 1 non-serious adverse events. In some embodiments, the liposomes have an average particle size of about 50-200 nm (e.g., about 90-140 nm). In some embodiments, the lipid bilayer is composed of electrically neutral phospholipids having a phase transition temperature of at least about 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C). In some embodiments, the electrically neutral phospholipids are composed of egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the phospholipid bilayer is composed of negatively charged phospholipids (e.g., DSPG). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc.

[0082] In some embodiments, a method of delivering zinc to an individual's liver is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise a) a lipid bilayer; and b) an effective amount of zinc encapsulated by the lipid bilayer, wherein the zinc concentration in the liver is maintained for at least 30 minutes (e.g., at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days). In some embodiments, the desired level of zinc in the liver is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% higher than a reference liver zinc concentration prior to delivery. In some embodiments, the desired level of zinc in the liver is not less than about 98%, 95%, 90%, 85%, 80%, 75%, or 70% higher than the liver zinc concentration of an individual without liver fibrosis (or the average liver zinc concentration of a group of individuals without liver fibrosis). In some embodiments, the individual is a human, and the amount of zinc used for each administration does not exceed approximately 120 mg / m². 2 (for example, not exceeding approximately 100 mg / m²) 2 80mg / m 2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some embodiments, the individual is a human, and the amount of zinc used for each administration is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 60 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m 2 Approximately 15 mg / m 2 or about 12mg / m 2In some embodiments, liposomes are administered at least once a week (e.g., at least once every six, five, four, three, or two days). In some embodiments, liposomes are administered at least once every two days. In some embodiments, liposomes are administered at a certain frequency for about one, two, three, four, five, or six weeks or at least one, two, three, three, or four months, and in some embodiments, the individual's liver fibrosis has not progressed to cirrhosis prior to liposome administration. In some embodiments, the individual's liver fibrosis has progressed to cirrhosis prior to liposome injection. In some embodiments, the liver zinc concentration in the individual after administration is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. In some embodiments, the increased liver zinc concentration persists for at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days after a single liposome administration. In some embodiments, after liposome administration, an individual's collagen content decreases (e.g., a decrease in collagen content level of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to collagen content prior to liposome administration. In some embodiments, within 1, 2, 3, or 4 months after the start of liposome injection, the individual's body weight increases by at least about 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30%). In some embodiments, the individual does not experience serious adverse events after administration, and in some embodiments, the individual does not experience more than 5, 4, 3, 2, or 1 non-serious adverse events after liposome administration. In some embodiments, the liposomes have an average particle size of about 50-200 nm (e.g., about 90-140 nm). In some embodiments, the lipid bilayer is composed of electrically neutral phospholipids having a phase transition temperature of at least about 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C). In some embodiments, the electrically neutral phospholipids are composed of egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the phospholipid bilayer is composed of negatively charged phospholipids (e.g., DSPG). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc.

[0083] In some embodiments, a method for increasing, maintaining, or regulating liver zinc concentration in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise a) a lipid bilayer; and b) an effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, the liver zinc concentration is increased by at least about 20% (e.g., at least about 30%, 40%, 50%, or 60%) compared to a control liver zinc concentration (e.g., liver zinc concentration before administration (e.g., immediately before administration)). In some embodiments, the individual is a human, and the amount of zinc administered per dose does not exceed about 120 mg / m². 2 (If not exceeding approximately 100 mg / m 2 80mg / m 2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some embodiments, the individual is a human, and the amount of zinc used for each administration is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 60 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m 2 Approximately 15 mg / m 2 or about 12mg / m 2In some embodiments, liposomes are administered at least once a week (e.g., at least once every six, five, four, three, or two days). In some embodiments, liposomes are administered at least once every two days. In some embodiments, liposomes are administered at a certain frequency for about one, two, three, four, five, or six weeks or at least one, two, three, three, or four months, and in some embodiments, the individual's liver fibrosis has not progressed to cirrhosis prior to liposome administration. In some embodiments, the individual's liver fibrosis has progressed to cirrhosis prior to liposome injection. In some embodiments, the liver zinc concentration in the individual after administration is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. In some embodiments, the increased liver zinc concentration persists for at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days after a single liposome administration. In some embodiments, after liposome administration, an individual's collagen content decreases (e.g., a decrease in collagen content level of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to collagen content prior to liposome administration. In some embodiments, within 1, 2, 3, or 4 months after the start of liposome injection, the individual's body weight increases by at least about 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30%). In some embodiments, the individual does not experience serious adverse events after administration, and in some embodiments, the individual does not experience more than 5, 4, 3, 2, or 1 non-serious adverse events after liposome administration. In some embodiments, the liposomes have an average particle size of about 50-200 nm (e.g., about 90-140 nm). In some embodiments, the lipid bilayer is composed of electrically neutral phospholipids having a phase transition temperature of at least about 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C). In some embodiments, the electrically neutral phospholipids are composed of egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the phospholipid bilayer is composed of negatively charged phospholipids (e.g., DSPG). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc.

[0084] In some embodiments, a method is provided to promote the retention of zinc in the liver of an individual (e.g., to promote the half-life of zinc, to promote an increase in the area under the curve (AUC) of zinc in the liver), comprising administering a composition comprising liposomes, wherein the liposomes comprise a) a lipid bilayer; and b) an effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, the liver zinc concentration is increased by at least about 20% (e.g., at least about 30%, 40%, 50%, or 60%) compared to a control liver zinc concentration (e.g., the liver zinc concentration before administration). In some embodiments, the individual is a human, and the amount of zinc administered each time does not exceed about 120 mg / m². 2 (If not exceeding approximately 100 mg / m 2 80mg / m 2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some embodiments, the individual is a human, and the amount of zinc used for each administration is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 60 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m 2 Approximately 15 mg / m 2 or about 12mg / m 2In some embodiments, liposomes are administered at least once a week (e.g., at least once every six, five, four, three, or two days). In some embodiments, liposomes are administered at least once every two days. In some embodiments, liposomes are administered at a certain frequency for about one, two, three, four, five, or six weeks, or at least for about one, two, three, three, or four months. In some embodiments, the individual's liver fibrosis had not progressed to cirrhosis prior to liposome administration. In some embodiments, the individual's liver fibrosis had progressed to cirrhosis prior to liposome injection. In some embodiments, the individual's liver zinc concentration after administration is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. In some embodiments, the increased liver zinc concentration persists for at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days after a single liposome administration. In some embodiments, after liposome administration, an individual's collagen content decreases (e.g., a decrease in collagen content level of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to collagen content prior to liposome administration. In some embodiments, within 1, 2, 3, or 4 months after the start of liposome injections, the individual's body weight increases by at least about 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30%).

[0085] In some embodiments, no serious adverse events occur in the individual after administration. In some embodiments, no more than 5, 4, 3, 2, or 1 non-serious adverse events occur in the individual after administration of the liposomes. In some embodiments, the liposomes have an average particle size of about 50-200 nm (e.g., about 90-140 nm). In some embodiments, the lipid bilayer is composed of electrically neutral phospholipids having a phase transition temperature of at least about 10 °C (e.g., at least about 20 °C, 30 °C, 40 °C, or 50 °C). In some embodiments, the electrically neutral phospholipids are composed of egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the phospholipid bilayer is composed of negatively charged phospholipids (e.g., DSPG). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc.

[0086] In some embodiments, a method is provided for delivering zinc to the liver of an individual or increasing the liver zinc concentration of an individual, comprising administering a composition comprising liposomes, wherein the liposomes comprise a) a lipid bilayer; and b) an effective amount of zinc encapsulated in the lipid bilayer. The individual is a human, and the amount of zinc administered each time does not exceed approximately 15 mg / m² per administration. 2(e.g., approximately 1 mg / m³) 2 Approximately 15 mg / m 2 For example, approximately 12 mg / m³ 2 In some embodiments, liposomes are administered at a frequency of about once or at least once every 2, 3, 4, 5, 6, or 7 days. In some embodiments, a method for promoting zinc retention in the liver of an individual is provided, comprising administering a composition comprising: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the individual is a human and the amount of zinc administered each time does not exceed about 15 mg / m². 2 (e.g., approximately 1 mg / m³) 2 Approximately 15 mg / m 2 For example, approximately 12 mg / m³ 2 In some embodiments, liposomes are administered approximately or at least once every 2, 3, 4, 5, 6, or 7 days. In some embodiments, liposomes are administered approximately every 1-3 days (e.g., approximately every two days) for approximately or at least 1, 2, 3, 4, 5, or 6 weeks. In some embodiments, liposomes are administered approximately every 1-3 days (e.g., approximately every two days) for approximately or at least 1, 2, 3, or 4 months. In some embodiments, the individual has liver fibrosis.

[0087] In some embodiments, a method of delivering zinc to the liver or increasing the zinc concentration in an individual's liver is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer consisting of electrically neutral phospholipids with a phase transition temperature of at least 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C); b) an effective amount of zinc encapsulated in the lipid bilayer, the zinc being sufficiently encapsulated within the lipid bilayer, wherein the average particle size of the liposomes is about 50-200 nm (e.g., about 90-130 nm). In some embodiments, a method of delivering zinc to an individual's liver is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer consisting of HSPCs; b) an effective amount of zinc encapsulated in the lipid bilayer, the zinc being sufficiently encapsulated within the lipid bilayer, wherein the average particle size of the liposomes is about 50-200 nm (e.g., about 90-130 nm). In some embodiments, the phospholipids further comprise negatively charged phospholipids. In some embodiments, the negatively charged phospholipids comprise or are DSPG. In some embodiments, the molar ratio of neutral phospholipids to negatively charged phospholipids is approximately 50:1 to 10:1 (e.g., approximately 50:1 to 20:1, or 20:1 to 10:1). In some embodiments, the molar ratio of neutral phospholipids to negatively charged phospholipids does not exceed 20:1 or 10:1. In some embodiments, the phospholipids do not contain negatively charged phospholipids. In some embodiments, the molar ratio of phospholipids (e.g., neutral phospholipids, such as PC) to cholesterol is not greater than 8:1 (e.g., not greater than 7:1, 6:1, 5:1, or 4:1). In some embodiments, the phospholipids do not contain positively charged phospholipids. In some embodiments, the phospholipids are not polyethylene glycol-derived phospholipids. In some embodiments, the zinc may be composed of organic zinc salts, inorganic zinc salts, or zinc chelates of conjugated zinc. In some embodiments, the zinc is readily ionized or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the PDI (polydispersity index) of the liposomes is not greater than 1 (e.g., not greater than 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05). In some embodiments, the absolute value of the zeta potential of the liposomes is at least or 5 mV (e.g., at least or 10 mV, 15 mV, 20 mV, 25 mV, or 30 mV). In some embodiments, the in vitro zinc release rate from the liposomes over 0.5 hours is not greater than or less than 20% (e.g., not greater than or less than 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, the in vitro release rate of zinc from liposomes over 48 hours is no greater than or less than 50% (e.g., no greater than or less than 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%).In some embodiments, at least about 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) of the liposomes have a lipid bilayer. In some embodiments, the individual has liver fibrosis. In some embodiments, a method of delivering zinc to the liver or increasing the zinc concentration in the liver of an individual is provided, comprising administering a composition comprising: a) a lipid bilayer consisting of phospholipids and cholesterol; b) zinc, wherein the zinc is encapsulated in the liposome bilayer, wherein the phospholipid is hydrogenated soybean lecithin (HSPC), wherein the molar ratio of HSPC to cholesterol is 4:1, and wherein the average particle size of the liposomes is 50 nm to 200 nm (e.g., 90-140 nm). In some embodiments, a method is provided for delivering zinc to the liver or increasing the zinc concentration in an individual's liver, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) zinc, wherein the zinc is encapsulated within the liposome bilayer, wherein the phospholipids are hydrogenated soybean lecithin (HSPC) and negatively charged phospholipids, wherein the molar ratio of HSPC to cholesterol is 4:1, and wherein the average particle size of the liposomes is 50 nm to 200 nm (e.g., 90-140 nm). The individual is a human, and the amount of zinc administered each time does not exceed about 15 mg / m². 2 (e.g., approximately 1 mg / m³) 2 Approximately 15 mg / m 2 For example, approximately 12 mg / m³ 2 In some embodiments, the liposomes are administered approximately once every 2, 3, 4, 5, 6, or 7 days. In some embodiments, the individual is a human, and the amount of zinc administered each time is approximately 1-15 mg / m². 2 Furthermore, liposomes should be applied at least every two days.

[0088] In some embodiments, a method is provided to promote zinc retention in an individual liver (e.g., increase zinc half-life, promote zinc bioavailability in the liver), comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) zinc, wherein the zinc is encapsulated in the liposome bilayer, wherein the phospholipid is hydrogenated soybean lecithin (HSPC), wherein the molar ratio of HSPC to cholesterol is 4:1, and wherein the average particle size of the liposomes is 50 nm to 200 nm (e.g., 90-140 nm). In some embodiments, a method is provided to promote zinc retention in an individual liver (e.g., increase zinc half-life, promote zinc bioavailability in the liver), comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) zinc, wherein the zinc is encapsulated in the liposome bilayer, wherein the phospholipids are hydrogenated soybean lecithin (HSPC) and negatively charged phospholipids, wherein the molar ratio of HSPC to cholesterol is 4:1, and wherein the average particle size of the liposomes is 50 nm to 200 nm (e.g., 90-140 nm).

[0089] In some implementations, the individual is a human, and the amount of zinc administered each time does not exceed approximately 15 mg / m². 2 (e.g., approximately 1 mg / m³) 2 Approximately 15 mg / m 2 For example, approximately 12 mg / m³ 2 In some embodiments, the liposomes are administered approximately once every 2, 3, 4, 5, 6, or 7 days. In some embodiments, the individual is a human, and the amount of zinc administered each time is approximately 1-15 mg / m². 2 Furthermore, liposomes should be applied at least every two days.

[0090] Treatment and / Or methods to prevent liver fibrosis

[0091] In some embodiments, a method for treating and / or preventing liver fibrosis in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, a method for reducing liver fibrosis in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, a method for reversing liver fibrosis in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, the individual is a human, and the amount of zinc administered each time does not exceed about 120 mg / m². 2(for example, not exceeding approximately 100 mg / m²) 2 80mg / m 2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some implementations, the individual is a human, and the amount of zinc applied each time is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 60 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m 2 Approximately 15 mg / m 2 or about 12mg / m 2In some embodiments, liposomes are administered at least once a week (e.g., at least once every six, five, four, three, or two days). In some embodiments, liposomes are administered at least once every two days. In some embodiments, liposomes are administered at a certain frequency for about one, two, three, four, five, or six weeks, or at least for about one, two, three, three, or four months. In some embodiments, the individual's liver fibrosis had not progressed to cirrhosis prior to liposome administration. In some embodiments, the individual's liver fibrosis had progressed to cirrhosis prior to liposome injection. In some embodiments, the individual's liver zinc concentration after administration is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. In some embodiments, the increased liver zinc concentration persists for at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days after a single liposome administration. In some embodiments, the individual's collagen content is reduced after intervention (e.g., a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to pre-liposome collagen content. In some embodiments, the individual's body weight increases by at least about 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% within 1, 2, 3, or 4 months after the start of liposome injection. In some embodiments, the individual does not experience serious adverse events after administration; in some embodiments, the individual does not experience more than 5, 4, 3, 2, or 1 non-serious adverse events after liposome administration. In some embodiments, the liposomes have an average particle size of about 50-200 nm (e.g., about 90-140 nm). In some embodiments, the lipid bilayer is composed of electrically neutral phospholipids having a phase transition temperature of at least about 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C). In some embodiments, the electrically neutral phospholipids are composed of egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the phospholipid bilayer is composed of negatively charged phospholipids (e.g., DSPG). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc.

[0092] In some embodiments, a method for promoting liver regeneration in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, the individual is a human, and the amount of zinc administered each time does not exceed about 120 mg / m². 2 (for example, not exceeding approximately 100 mg / m²) 2 80mg / m2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some embodiments, the individual is a human, and the amount of zinc used for each administration is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 60 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m 2 Approximately 15 mg / m 2 or about 12mg / m 2In some embodiments, liposomes are administered at least once a week (e.g., at least once every six, five, four, three, or two days). In some embodiments, liposomes are administered at least once every two days. In some embodiments, liposomes are administered at a certain frequency for about one, two, three, four, five, or six weeks or at least one, two, three, three, or four months. In some embodiments, the individual's liver fibrosis had not progressed to cirrhosis prior to liposome administration. In some embodiments, the individual's liver fibrosis had progressed to cirrhosis prior to liposome injection. In some embodiments, the individual's liver fibrosis had progressed to cirrhosis prior to liposome injection. In some embodiments, the individual's liver zinc concentration after administration is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. In some embodiments, the increased hepatic zinc concentration persists for at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days after a single liposome administration. In some embodiments, the individual's collagen content decreases after intervention (e.g., a decrease of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to pre-liposome administration. In some embodiments, the individual's body weight increases by at least about 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% within 1, 2, 3, or 4 months after the start of liposome injections. In some embodiments, no serious adverse events occur in the individual after administration, and in some embodiments, no more than 5, 4, 3, 2, or 1 non-serious adverse events occur in the individual after administration of the liposomes. In some embodiments, the liposomes have an average particle size of about 50-200 nm (e.g., about 90-140 nm). In some embodiments, the lipid bilayer is composed of electrically neutral phospholipids having a phase transition temperature of at least about 10 °C (e.g., at least about 20 °C, 30 °C, 40 °C, or 50 °C). In some embodiments, the electrically neutral phospholipids are composed of egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the phospholipid bilayer is composed of negatively charged phospholipids (e.g., DSPG). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc.

[0093] For example, a liver regeneration assay can be performed on the liver of an individual whose liver has been partially damaged or removed. The liposome composition described herein is then applied to the individual, and the rate of tissue regeneration is determined. The rate of liver regeneration can be compared to the rate observed in control or untreated cases. Other parameters that can be determined during a liver regeneration assay include, but are not limited to, symptoms or outcomes such as pain or the person experiencing pain, signs or symptoms of inflammation, the final degree of regeneration, and the quality of regeneration. In some embodiments, the liver regeneration assay described herein includes assessing one or more liver function parameters. For example, liver regeneration can be assessed by measuring the regeneration of hepatocytes, liver tissue, and / or the entire liver organ. Liver regeneration can also be assessed by measuring the activation / stimulation of hepatocytes capable of repairing liver structure and / or refilling liver parenchyma. Liver regeneration can also be measured / observed by measuring postoperative albumin levels, total protein levels, cholinesterase (CHE) levels, lactate dehydrogenase (LDH) levels, aspartate aminotransferase (GOT) levels, alanine aminotransferase (GPT) levels, alkaline phosphatase (AP) levels, and / or iron levels, as described in EP2554176B1 or US14 / 237,016. Liver regeneration can also be determined by analyzing the expression of hepatocyte-specific RNAs, such as AFP (alpha-fetoprotein), CK19 (cytokeratin), CK7, Cx43 (connector protein) (typical of early differentiated hepatocytes), and / or CYP3A4 (cytochrome), PCK1 (phosphoenolpyruvate carboxykinase), CPS (pyrophosphate synthase), CK18, CX32, CD26, ALB, and / or CYP3A4 (cytochrome), PCK1 (phosphoenolpyruvate carboxykinase), CPS (pyrophosphate synthase), CK18, CX32, CD26, ALB. Alternatively or additionally, liver regeneration can be assessed histologically by examining liver specimens, which can be obtained, for example, by biopsy. Furthermore, as an option or supplement, liver regeneration can be assessed by immunofluorescence of glycogen or CK19. See EP2554176B1.

[0094] In some embodiments, a method for reversing or reducing liver fibrosis in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, a method for reducing liver fibrosis in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the individual is a human, and the amount of zinc administered each time does not exceed about 15 mg / m². 2 (e.g., approximately 1 mg / m³) 2 Approximately 15 mg / m 2 (e.g., approximately 12 mg / m²) 2In some embodiments, a method for promoting liver regeneration in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the individual is a human, and the amount of zinc administered each time does not exceed about 15 mg / m². 2 (e.g., approximately 1 mg / m³) 2 Approximately 15 mg / m 2 (e.g., approximately 12 mg / m²) 2 In some embodiments, liposomes are applied at a frequency of about every 2, 3, 4, 5, 6, or 7 days or at least once. In some embodiments, liposomes are applied at a frequency of about 1-3 days (e.g., about every two days) for about or at least about 1, 2, 3, 4, 5, or 6 weeks. In some embodiments, liposomes are applied at a frequency of about 1-3 days (e.g., about every two days) for about or at least about 1, 2, 3, or 4 months.

[0095] In some embodiments, a method for treating, reversing, or reducing individual liver fibrosis is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer composed of electrically neutral phospholipids with a phase transition temperature of at least 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C); and b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the average particle size of the liposomes is about 50-200 nm (e.g., about 90-130 nm). In some embodiments, a method for treating, reversing, or reducing individual liver fibrosis is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer composed of HSPCs; and b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the average particle size of the liposomes is about 50-200 nm (e.g., about 90-130 nm). In some embodiments, a method for promoting individual liver regeneration is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer consisting of electrically neutral phospholipids having a phase transition temperature of at least 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C); and b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the average particle size of the liposomes is about 50-200 nm (e.g., about 90-130 nm).

[0096] In some embodiments, a method for promoting individual liver regeneration is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer composed of HSPCs; and b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the average particle size of the liposomes is about 50-200 nm (e.g., about 90-130 nm). In some embodiments, the phospholipids further comprise negatively charged phospholipids. In some embodiments, the negatively charged phospholipids comprise or are DSPG. In some embodiments, the molar ratio of neutral phospholipids to negatively charged phospholipids is about 50:1 to 10:1 (e.g., about 50:1 to 20:1, or 20:1 to 10:1). In some embodiments, the molar ratio of neutral phospholipids to negatively charged phospholipids does not exceed 20:1 or 10:1.

[0097] In some embodiments, the phospholipid does not contain negatively charged phospholipids. In some embodiments, the molar ratio of the phospholipid (e.g., an electrically neutral phospholipid, such as PC) to cholesterol is not greater than 8:1 (e.g., not greater than 7:1, 6:1, 5:1, or 4:1). In some embodiments, the phospholipid does not contain positively charged phospholipids. In some embodiments, the phospholipid is not a polyethylene glycol-derived phospholipid. In some embodiments, the zinc may be composed of an organic zinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc is readily ionized or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the PDI of the liposome is not greater than 1 (e.g., not greater than 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05). In some embodiments, the absolute value of the zeta potential of the liposome is at least or is 5 mV (e.g., at least 10 mV, 15 mV, 20 mV, 25 mV, or 30 mV). In some embodiments, the 0.5-hour in vitro release rate of liposomes is no greater than or less than 20% (e.g., no greater than or less than 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, the 48-hour in vitro release rate of liposomes is no greater than or less than 50% (e.g., no greater than or less than 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, at least about 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%) of the liposomes have a lipid bilayer.

[0098] In some embodiments, a method for treating, reversing, or reducing individual liver fibrosis is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer composed of phospholipids and cholesterol; b) zinc, wherein zinc is encapsulated in the liposome bilayer, wherein the phospholipid is hydrogenated soy lecithin (HSPC) and a negatively charged phospholipid, wherein the molar ratio of HSPC to cholesterol is 4:1, and wherein the average particle size of the liposomes is 50 nm to 200 nm (e.g., 90-140 nm). In some embodiments, a method for treating, reversing, or reducing individual liver fibrosis is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer composed of phospholipids and cholesterol; b) zinc, wherein zinc is encapsulated in the liposome bilayer, wherein the phospholipid is hydrogenated soy lecithin (HSPC) and a negatively charged phospholipid, wherein the molar ratio of HSPC to cholesterol is 4:1, and wherein the average particle size of the liposomes is 50 nm to 200 nm (e.g., 90-140 nm). In some embodiments, the individual is a human, and the dosage of zinc administered per dose does not exceed 15 mg / m². 2 (approximately 1 mg / m²) 2 Up to 15 mg / m 2 For example, approximately 12 mg / m 2 In some embodiments, the liposomes are administered at a frequency of approximately or at least every 2, 3, 4, 5, 6, or 7 days. In some embodiments, the patient or individual is human, and the amount of zinc administered each time is approximately 1-15 mg / m². 2 Liposomes should be administered at least once every two days.

[0099] In some embodiments, a method for promoting liver regeneration in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer. The phospholipid is hydrogenated soybean phospholipid (HSPC), the molar ratio of HSPC to cholesterol is 4:1, and the average particle size of the liposomes is 50 nm–200 nm (e.g., about 90–140 nm). In some embodiments, a method for promoting liver regeneration in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer composed of phospholipids and cholesterol; b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the phospholipid is hydrogenated soybean phospholipid and a negatively charged phospholipid, the molar ratio of HSPC to cholesterol is 4:1, and the average particle size of the liposomes is 50 nm–200 nm (e.g., about 90–140 nm). The individual is a human, and the amount of zinc administered each time does not exceed about 15 mg / m². 2 (e.g., approximately 1 mg / m³) 2 Approximately 15 mg / m 2 (e.g., approximately 12 mg / m²)2 In some embodiments, liposomes are applied at a frequency of about every 2, 3, 4, 5, 6, or 7 days or at least once. In some embodiments, liposomes are applied at a frequency of about 1-3 days (e.g., about every two days) for about or at least about 1, 2, 3, 4, 5, or 6 weeks. In some embodiments, liposomes are applied at a frequency of about 1-3 days (e.g., about every two days) for about or at least about 1, 2, 3, or 4 months.

[0100] Methods to improve quality of life

[0101] The present invention also provides a method for improving quality of life in individuals with liver fibrosis, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, the individual's liver fibrosis stage is F3 (severe liver fibrosis) or F4 (early cirrhosis). In some embodiments, the individual is a human, and the amount of zinc administered each time does not exceed about 120 mg / m². 2 (for example, not exceeding approximately 100 mg / m²) 2 80mg / m 2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some embodiments, the individual is a human, and the amount of zinc used for each administration is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 60 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m 2 Approximately 15 mg / m 2 or about 12mg / m 2In some embodiments, liposomes are administered at least once a week (e.g., at least once every six, five, four, three, or two days). In some embodiments, liposomes are administered at least once every two days. In some embodiments, liposomes are administered at a certain frequency for about one, two, three, four, five, or six weeks, or at least for about one, two, three, three, or four months. In some embodiments, the individual's liver fibrosis had not progressed to cirrhosis prior to liposome administration. In some embodiments, the individual's liver fibrosis had progressed to cirrhosis prior to liposome injection. In some embodiments, the individual's liver zinc concentration after administration is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. In some embodiments, the increased liver zinc concentration persists for at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days after a single liposome administration. In some embodiments, the individual's collagen content is reduced after intervention (e.g., a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to pre-liposome collagen content. In some embodiments, the individual's body weight increases by at least about 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% within 1, 2, 3, or 4 months after the start of liposome injection. In some embodiments, the individual does not experience serious adverse events after administration; in some embodiments, the individual does not experience more than 5, 4, 3, 2, or 1 non-serious adverse events after liposome administration. In some embodiments, the liposomes have an average particle size of about 50-200 nm (e.g., about 90-140 nm). In some embodiments, the lipid bilayer is composed of electrically neutral phospholipids having a phase transition temperature of at least about 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C). In some embodiments, the electrically neutral phospholipids are composed of egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the phospholipid bilayer is composed of negatively charged phospholipids (e.g., DSPG). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc.

[0102] In some embodiments, the improvement in quality of life is characterized by an increase in individual weight following administration of liposomes. In some embodiments, the patient's weight increases by at least about 1%, 2%, 3%, 4%, or 5% within about 1, 2, 3, 4, 5, or 6 weeks after liposome administration. In some cases, the patient gains at least 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% of their body weight within 1, 2, 3, or 4 months after liposome administration. In some embodiments, liposomes are repeatedly administered at least once a week (e.g., every 1, 2, 3, 4, 5, 6, or 7 days) for at least about 1, 2, 3, 4, 5, or 6 weeks or for at least 1, 2, 3, or 4 months. In some embodiments, a method for improving the quality of life of patients with liver fibrosis is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer; b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the individual is human, and the amount of zinc administered each time does not exceed about 15 mg / m² per administration. 2 (e.g., approximately 1 mg / m³) 2 Approximately 15 mg / m 2 For example, approximately 12 mg / m³ 2 In some embodiments, the individual has reached stage F3 or F4 of liver fibrosis. In some embodiments, liposomes are administered approximately or at least once every 2, 3, 4, 5, 6, or 7 days. In some embodiments, liposomes are administered approximately every 1-3 days (e.g., approximately every two days) for approximately or at least approximately 1, 2, 3, 4, 5, or 6 weeks. In some embodiments, liposomes are administered approximately every 1-3 days (e.g., approximately every two days) for approximately or at least approximately 1, 2, 3, or 4 months. In some embodiments, the individual has liver fibrosis.

[0103] In some embodiments, a method for improving the quality of life of an individual with liver fibrosis is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer consisting of electrically neutral phospholipids with a phase transition temperature of at least 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C); and b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the average particle size of the liposomes is about 50-200 nm (e.g., about 90-130 nm). In some embodiments, the individual's liver fibrosis level is F3 or F4. In some embodiments, a method for improving the quality of life of an individual with liver fibrosis is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer consisting of HSPCs; and b) an effective amount of zinc encapsulated in the lipid bilayer, wherein the average particle size of the liposomes is about 50-200 nm (e.g., about 90-130 nm). In some embodiments, the phospholipids further comprise negatively charged phospholipids. In some embodiments, the negatively charged phospholipids comprise or are DSPG. In some embodiments, the molar ratio of neutral phospholipids to negatively charged phospholipids is approximately 50:1 to 10:1 (e.g., approximately 50:1 to 20:1 or 20:1 to 10:1). In some embodiments, the molar ratio of neutral phospholipids to negatively charged phospholipids does not exceed 20:1 or 10:1. In some embodiments, the phospholipids do not contain negatively charged phospholipids. In some embodiments, the molar ratio of phospholipids (e.g., neutral phospholipids, such as PC) to cholesterol is not greater than 8:1 (e.g., not greater than 7:1, 6:1, 5:1, or 4:1). In some embodiments, the phospholipids do not contain positively charged phospholipids. In some embodiments, the phospholipids are not polyethylene glycol-derived phospholipids. In some embodiments, the zinc may be composed of organic zinc salts, inorganic zinc salts, or zinc chelates of conjugated zinc. In some embodiments, the zinc is readily ionized or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the PDI of the liposomes is not greater than 1 (e.g., not greater than 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05). In some embodiments, the absolute value of the zeta potential of the liposomes is at least or 5 mV (e.g., at least or 10 mV, 15 mV, 20 mV, 25 mV, or 30 mV). In some embodiments, the 0.5-hour in vitro release rate of the liposomes is not greater than or less than 20% (e.g., not greater than or less than 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, the 48-hour in vitro release rate of the liposomes is not greater than or less than 50% (e.g., not greater than or less than 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%).In some embodiments, at least about 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) of the liposomes have a lipid bilayer.

[0104] In some embodiments, a method for preventing or delaying liver fibrosis in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer composed of phospholipids and cholesterol; b) an effective amount of zinc encapsulated in the lipid bilayer. The phospholipids are hydrogenated soybean phospholipids, the molar ratio of HSPCs to cholesterol is 4:1, and the average particle size of the liposomes is 50 nm-200 nm (e.g., about 90-140 nm). In some embodiments, a method for preventing or delaying liver fibrosis in an individual is provided, comprising administering a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer composed of phospholipids and cholesterol; b) an effective amount of zinc encapsulated in the lipid bilayer. The phospholipids are hydrogenated soybean phospholipids and negatively charged phospholipids, the molar ratio of HSPCs to cholesterol is 4:1, and the average particle size of the liposomes is 50 nm-200 nm (e.g., about 90-140 nm). In some embodiments, the individual's liver fibrosis stage is F3 or F4. The individuals in question are humans, and the amount of zinc administered each time does not exceed approximately 15 mg / m² per administration. 2 (e.g., approximately 1 mg / m³) 2 Approximately 15 mg / m 2 For example, approximately 12 mg / m³ 2 In some embodiments, the liposomes are administered approximately once every 2, 3, 4, 5, 6, or 7 days. In some embodiments, the individual is a human, and the amount of zinc administered each time is approximately 1-15 mg / m². 2 Furthermore, liposomes should be applied at least every two days.

[0105] In some embodiments, the method described herein further includes assessing hepatic zinc levels prior to administration of the liposomes. In some embodiments, the method further includes assessing the presence or extent of liver fibrosis.

[0106] Dosage and administration

[0107] In some embodiments, the amount of zinc applied per dose does not exceed about 20 mg / kg, 15 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4.5 mg / kg, 4 mg / kg, 3.5 mg / kg, 3 mg / kg, 2.5 mg / kg, 2 mg / kg, 1.5 mg / kg, or 1 mg / kg. In some embodiments, the amount of zinc applied per dose is at least about 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 0.8 mg / kg, or 1 mg / kg. In some embodiments, the amount of zinc applied each time is from about 0.01 mg / kg to about 20 mg / kg (e.g., from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 2.5 mg / kg, from about 0.8 mg / kg to about 2 mg / kg, from about 1 mg / kg to about 2 mg / kg, from about 1 mg / kg to about 1.8 mg / kg, from about 1 mg / kg to about 1.5 mg / kg, or from about 1.4 mg / kg to about 1.5 mg / kg). In some embodiments, the amount of zinc applied each time is about 1.4 mg / kg.

[0108] In some implementations, the individual is a human, and the amount of zinc administered each time does not exceed approximately 120 mg / m². 2 (for example, not exceeding approximately 100 mg / m²) 2 80mg / m 2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some embodiments, the individual is a human, and the amount of zinc administered each time is at least about 0.1 mg / m². 2 0.2 mg / m 2 0.5mg / m 2 0.8 mg / m 2 1mg / m 2 2mg / m 2 3mg / m 2 or 4mg / m 2 In some embodiments, the individual is a human, and the amount of zinc used for each administration is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³2 Approximately 60 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m 2 Approximately 15 mg / m 2 or about 12mg / m 2 In some implementations, the individual is a human, and the amount of zinc administered each time does not exceed approximately 3.5 mg / kg, 3 mg / kg, 2.5 mg / kg, 2 mg / kg, 1.8 mg / kg, 1.6 mg / kg, 1.4 mg / kg, 1.2 mg / kg, 1 mg / kg, 0.8 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, or 0.08 mg / kg. In some embodiments, the individual is a human, and the amount of zinc administered each time is at least about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, or 0.4 mg / kg. In some embodiments, the individual is a human, and the amount of zinc administered each time is at least about 0.1 mg / kg to about 1 mg / kg (e.g., about 0.2 mg / kg to about 0.8 mg / kg, about 0.3 mg / kg to about 0.6 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg).

[0109] In some embodiments, the daily (e.g., average) amount of zinc administered does not exceed about 20 mg / kg, 15 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4.5 mg / kg, 4 mg / kg, 3.5 mg / kg, 3 mg / kg, 2.5 mg / kg, 2 mg / kg, 1.5 mg / kg, or 1 mg / kg. In some embodiments, the daily (e.g., average) amount of zinc administered is at least about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, or 0.5 mg / kg. In some embodiments, the daily (e.g., average) amount of zinc used for administration is at least about 0.01 mg / kg to about 20 mg / kg (e.g., about 0.01 mg / kg to about 15 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 8 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, about 0.1 mg / kg to about 2 mg / kg, about 0.2 mg / kg to about 1 mg / kg, or about 0.5 mg / kg to 0.7 mg / kg).

[0110] In some embodiments, the daily (e.g., average) zinc dosage administered to a mouse does not exceed about 20 mg / kg, 15 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4.5 mg / kg, 4 mg / kg, 3.5 mg / kg, 3 mg / kg, 2.5 mg / kg, 2 mg / kg, 1.5 mg / kg, or 1 mg / kg. In some embodiments, the daily (e.g., average) zinc dosage administered to a monkey is at least about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, or 0.5 mg / kg. In some embodiments, the daily (e.g., average) amount of zinc used for administration is at least about 0.01 mg / kg to about 20 mg / kg (e.g., about 0.01 mg / kg to about 15 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 8 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, about 0.1 mg / kg to about 2 mg / kg, about 0.2 mg / kg to about 1 mg / kg, or about 0.5 mg / kg to 0.7 mg / kg) in mice or monkeys.

[0111] In some implementations, the individual is a human being, and the amount of zinc applied daily (e.g., on average) does not exceed about 60 mg / m². 2 50mg / m 2 40mg / m2 30mg / m 2 25mg / m 2 20mg / m 2 17.5 mg / m 2 15mg / m 2 12.5 mg / m 2 10mg / m 2 or 7.5 mg / m 2 In some implementations, the individual is a human, and the daily (e.g., average) amount of zinc to be applied is at least about 0.1 mg / m². 2 0.5mg / m 2 1.0 mg / m 2 1.2 mg / m 2 or 1.5 mg / m 2 In some implementations, the individual is a human, and the daily (e.g., average) amount of zinc applied is approximately 0.1 mg / m². 2 Approximately 60 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 40 mg / m 2 Approximately 0.1 mg / m³ 2 Approximately 30 mg / m 2 Approximately 0.1 mg / m³ 2 Approximately 20 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 15 mg / m 2 Approximately 1 mg / m 2 Approximately 10 mg / m 2 Approximately 2mg / m 2 Approximately 8 mg / m 2 or about 5mg / m 2 Approximately 6 mg / m 2In some embodiments, the individual is a human being, and the daily (e.g., average) amount of zinc applied is not more than about 2 mg / kg, 1.8 mg / kg, 1.6 mg / kg, 1.4 mg / kg, 1.2 mg / kg, 1 mg / kg, 0.8 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, 0.08 mg / kg, 0.06 mg / kg, or 0.04 mg / kg. In some embodiments, the individual is a human being, and the amount of zinc applied per dose is at least about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, or 0.2 mg / kg. In some implementations, the individual is a human being, and the amount of zinc applied each time is from about 0.05 mg / kg to about 0.5 mg / kg (e.g., from about 0.1 mg / kg to about 0.4 mg / kg, from about 0.1 mg / kg to about 0.3 mg / kg, from about 0.2 mg / kg to about 0.3 mg / kg).

[0112] In some embodiments, the amount of zinc applied every 48 hours (e.g., on average) does not exceed about 20 mg / kg, 15 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4.5 mg / kg, 4 mg / kg, 3.5 mg / kg, 3 mg / kg, 2.5 mg / kg, 2 mg / kg, 1.5 mg / kg, or 1 mg / kg. In some embodiments, the amount of zinc applied each time in a 48-hour cycle (e.g., on average) is at least about 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 0.8 mg / kg, or 1 mg / kg. In some embodiments, the amount of zinc applied each time in a 48-hour cycle (e.g., on average) is about 0.01 mg / kg to about 20 mg / kg (e.g., about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 2.5 mg / kg, about 0.8 mg / kg to about 2 mg / kg, about 1 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.8 mg / kg, about 1 mg / kg to about 1.5 mg / kg, or about 1.4 mg / kg to about 1.5 mg / kg).

[0113] In some implementations, the individual is a human, and the amount of zinc applied every 48 hours (e.g., on average) does not exceed about 120 mg / m². 2 100mg / m 280mg / m 2 60mg / m 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 or 15mg / m 2 In some implementations, the individual is a human, and the amount of zinc applied every 48 hours (e.g., on average) is at least about 0.1 mg / m². 2 0.2 mg / m 2 0.5mg / m 2 0.8 mg / m 2 1mg / m 2 2mg / m 2 3mg / m 2 or 4mg / m 2 In some embodiments, the individual is a human, and the amount of zinc applied every 48 hours (e.g., on average) is approximately 0.1 mg / m². 2 Approximately 120 mg / m 2 (e.g., approximately 0.1 mg / m²) 2 Approximately 80 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 60 mg / m 2 Approximately 0.5 mg / m³ 2 Approximately 30 mg / m 2 Approximately 1 mg / m 2 Approximately 20 mg / m 2 Approximately 5mg / m 2 Approximately 15 mg / m 2 Approximately 10 mg / m³ 2 Approximately 15 mg / m 2 or approximately 12 mg / m³ 2In some embodiments, the individual is a human being, and the amount of zinc per 48 hours (e.g., on average) does not exceed about 3.5 mg / kg, 3 mg / kg, 2.5 mg / kg, 2 mg / kg, 1.8 mg / kg, 1.6 mg / kg, 1.4 mg / kg, 1.2 mg / kg, 1 mg / kg, 0.8 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, or 0.08 mg / kg. In some embodiments, the individual is a human, and the amount of zinc applied every 48 hours (e.g., on average) is at least about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, or 0.4 mg / kg. In some embodiments, the individual is a human, and the amount of zinc applied every 48 hours (e.g., on average) is about 0.1 mg / kg to about 1 mg / kg (e.g., about 0.2 mg / kg to about 0.8 mg / kg, about 0.3 mg / kg to about 0.6 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg).

[0114] In some embodiments, liposomes are administered once. In some embodiments, liposomes are administered at a dose of less than 60 mg / m³. 2 50mg / m 2 40mg / m 2 30mg / m 2 25mg / m 2 20mg / m 2 Or 15mg / m 2 A single dose is administered. In some embodiments, liposomes are administered multiple times. In some embodiments, liposomes are administered at least once a week (e.g., at least once every six, five, four, three, or two days). In some embodiments, liposomes are administered at least once every two days. In some embodiments, liposomes are administered daily.

[0115] In some embodiments, liposomes are administered for about or at least about 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, liposomes are administered for about or at least about 1, 2, 3, 4, 5, or 6 weeks. In some embodiments, liposomes are administered for about or at least about 1, 2, 3, or 4 months.

[0116] In some embodiments, liposomes are administered at the frequency described above for about or at least about two, three, four, five, six, or seven days. In some embodiments, the liposomes are administered at a frequency of about or at least about one, two, three, four, five, or six weeks. In some embodiments, liposomes are administered at the frequency described above for about or at least about one, two, three, or four months. In some embodiments, liposomes are administered at the frequency described above for about one week to about one year (e.g., about one month to about six months, or about six weeks to about four months).

[0117] The liposomes of this application can be administered via any suitable route. In some embodiments, the liposomes are administered parenterally. In some embodiments, the liposomes are administered intravenously, intra-arterially, intraperitoneally, intracapsularly, subcutaneously, intrathecally, intrapulmonaryly, intramuscularly, intratracheally, intraocularly, transdermally, or orally, or by inhalation.

[0118] liver fibrosis

[0119] In some implementations, the methods described herein are suitable for treating, reducing, or reversing liver fibrosis. In the pathogenesis of chronic liver disease, the ongoing liver injury and repair process leads to a significant reduction in available zinc, resulting in a range of functional abnormalities. This promotes collagen synthesis and inhibits its degradation, leading to a net accumulation of collagen fibrils. Insufficient signals triggering cell death or quiescence in myofibroblasts allow for the maintenance of myofibroblasts that produce large amounts of collagen, thus preserving a high level of fibrogenic activity. Hepatocytes will also cease replication, resulting in the replacement of liver tissue with ECM and loss of normal function.

[0120] There are several different assessment methods for staging liver fibrosis. The METAVIR scoring system is one of the most popular. This system assigns a score to "activity" or prediction of fibrosis progression, as well as the level of fibrosis itself. Typically, this score is assigned only after a liver biopsy or tissue sample collection. Liver tissue inflammatory activity is graded from A0 to A3. A0 indicates no activity; A1 indicates mild activity; A2 indicates moderate activity; and A3 indicates severe activity. Fibrosis severity is graded from F0 to F4. F0 indicates no fibrosis. F1 indicates portal fibrosis without septa. F2 indicates portal venous fibrosis with few portal septa. F3 indicates numerous septa without cirrhosis. F4 indicates cirrhosis.

[0121] In some embodiments, liver fibrosis has not yet progressed to cirrhosis. In some embodiments, liver fibrosis has progressed to cirrhosis prior to liposome administration. In some embodiments, liver fibrosis is classified as stage F1. In some embodiments, liver fibrosis is classified as stage F2. In some embodiments, liver fibrosis is classified as stage F3. In some embodiments, liver fibrosis is classified as stage F4. In some embodiments, liver fibrosis is classified as A0 under the MetAVIR scoring system. In some embodiments, liver fibrosis is classified as A1 under the MetAVIR scoring system. In some embodiments, liver fibrosis is classified as A2 under the MetAVIR scoring system. In some embodiments, liver fibrosis is classified as A3 under the MetAVIR scoring system.

[0122] In some implementations, liver fibrosis is reversible. In other implementations, liver fibrosis is irreversible.

[0123] In some implementations, liver fibrosis is associated with alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or viral infection.

[0124] In some implementations, liver fibrosis is associated with drug-induced injury (DILI), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), or other liver diseases.

[0125] Metallothioneins (MTs)

[0126] It has been reported that metallothioneins (MTs) in the hepatocytes of some animals can temporarily store zinc and provide zinc as a reservoir when needed. MTs are a group of small proteins rich in cysteine. They can use their cysteine ​​residues to bind to zinc ions to form a reservoir of zinc-MT complexes. When cellular oxidative stress increases, the cysteine ​​residues are oxidized and can then release the bound zinc ions.

[14] Like a “zinc container,” MTs can accept and protect a portion of serum zinc supplied by the blood and provide these “cargo” when there is a high demand for zinc, such as in cases of cell damage or increased oxidative stress. In this sense, MTs maintain a “available” pool of zinc in the cellular environment and are an important factor in maintaining tissue zinc levels.

[0127] However, MT levels depend on physiological state and vary in liver pathogenesis. MTs have been reported to be almost completely absent in the livers of cirrhotic mice

[15] and rats

[16] . Without MTs, trace amounts of “available” zinc may exist, which can be recruited by required proteins, including key enzymes involved in cell survival and regeneration. Furthermore, additional zinc supplied by the circulatory system may not remain in hepatocytes, and any excess zinc can be transported back into the bloodstream. According to this theory, the absence of MTs leads to a lack of cellular capacity to retain “available” zinc. In this case, hepatocytes remain zinc deficient despite zinc replenishment from the blood.

[0128] On the other hand, both MT transcription

[17] and degradation

[18] have been reported to be influenced by zinc. Together they form a self-regulating system and a balance. This system is able to regulate each other’s levels in response to cellular changes, thereby keeping the amount of available zinc constant within a preferred range. The release of zinc from MT leads to an increase in MT transcription, and thus an increase in its amount in the case of acute liver injury, thereby providing greater capacity for zinc uptake, retention and cell regeneration. However, when the injury persists for a period of time, regeneration requires large amounts of zinc, leading to a decrease in MT transcription, an increase in MT degradation and a decrease in MT, as observed in our previous study

[15] . Without MT, the ability to capture and retain cellular zinc will be impaired, and zinc will be more severely depleted.

[0129] Once available zinc and MT become negligible, meaning the balance shifts to the extremities, the system may lose its self-regulating ability. The more severe the existing conditions, the more widespread the balance shift becomes, and the less effective current zinc supplements are at responding to changes in conditions.

[0130] In some embodiments, the individual has abnormal levels of liver metallothionein (MT) prior to administration of the liposomes. In some embodiments, the individual's MT levels are significantly reduced (e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) instead of normal MT levels. In some embodiments, the reference MT level is the MT level of an individual without liver fibrosis or the average MT level of a group of individuals without liver fibrosis. This can be described, for example, according to Jiang Y., et al., Mol Ther, 2004 Dec; 10(6):1130-9.

[0131] In some implementations, the individual has a mutation in the liver metallothionein gene.

[0132] Patient or individual

[0133] In some implementations, the individual is a mammal. In some implementations, the individual is a primate (e.g., a monkey or a human). In some implementations, the individual is a human.

[0134] In some implementations, the individual suffers from liver fibrosis (e.g., any type or kind of liver fibrosis described in the "Liver Fibrosis" section).

[0135] In some implementations, the individual has a liver disease. In some implementations, the liver disease is selected from alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), drug-induced injury (DILI), primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC).

[0136] Safety, effectiveness and quality of life

[0137] In some embodiments, the liver zinc concentration in the individual after administration is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. In some embodiments, the increased liver zinc concentration persists for at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least about 1, 2, 3, 4, 5, 6, or 7 days after a single liposome administration. In some embodiments, compared to the control group, the individual's liver zinc concentration increases by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% after about 1, 2, 3, 4, 5, 6, or 7 days after liposome administration. In some embodiments, the post-administration zinc concentration in an individual is greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% compared to the pre-administration liver zinc concentration. A reference liver zinc concentration is the liver zinc concentration prior to liposome administration, for example, immediately preceding the administration of the liposome. In some embodiments, the reference liver zinc concentration is the liver zinc concentration of another individual without liver fibrosis or of average level. A group of people without liver fibrosis has liver zinc concentrations. In some embodiments, liposome administration is a single dose. In some embodiments, the increased tissue zinc concentration persists for at least about 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours, or at least for about 1, 2, 3, 4, 5, 6, or 7 days after a single liposome administration. In some embodiments, liposome administration includes multiple doses. In some implementations, the administration of liposomes involves repeated administration of liposomes or liposomes thereof at a frequency of at least once a week (e.g., once every 1, 2, 3, 4, 5, 6 or 7 days) for a duration of at least about 1, 2, 3, 4, 5 or 6 weeks, or at least about 1, 2, 3 or 4 months.

[0138] In some embodiments, after liposome administration, the blood zinc concentration (e.g., serum zinc concentration or plasma zinc concentration) in an individual does not exceed approximately 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1.1 times, or 1 times the reference blood zinc concentration. In some embodiments, approximately 1, 2, 4, 8, 12, 18, 24, 36, or 48 hours after liposome administration, the blood zinc concentration (e.g., serum zinc concentration or plasma zinc concentration) in an individual does not exceed approximately 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1.1 times, or 1 times the reference blood zinc concentration. In some embodiments, approximately 1, 2, 3, 4, 5, 6, or 7 days after liposome administration, the blood zinc concentration (e.g., serum zinc concentration or plasma zinc concentration) in an individual does not exceed approximately 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1.1 times, or 1 times the reference blood zinc concentration. In some embodiments, the peak blood zinc concentration (e.g., serum zinc concentration or plasma zinc concentration) in an individual after liposome administration does not exceed approximately 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1.1 times, or 1 times the reference blood zinc concentration. In some embodiments, the reference blood zinc concentration is the individual's baseline blood zinc concentration before liposome administration. In some embodiments, the reference blood zinc concentration is the blood zinc concentration of an individual without liver fibrosis or the average blood zinc concentration of a group of individuals without liver fibrosis. In some embodiments, liposome administration is a single administration. In some embodiments, liposome administration includes multiple administrations. In some embodiments, liposome administration comprises repeated administration of liposomes or components thereof at a frequency of at least once a week (e.g., every 1, 2, 3, 4, 5, 6, or 7 days) for a duration of at least about 1, 2, 3, 4, 5, or 6 weeks or at least about 1, 2, 3, or 4 months.

[0139] In some embodiments, the half-life of zinc after administration of liposomes is at least about 1, 2, 4, 8, 12, 16, 20, or 24 hours. In some embodiments, the half-life of zinc after administration of liposomes is at least about 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, the half-life of zinc after administration of liposomes is at least about 0.5, 1, 2, 3, 4, 5, 7.5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times the half-life of zinc before administration of liposomes.

[0140] In some embodiments, individuals have reduced collagen content after liposome administration compared to a reference collagen content. In some embodiments, after liposome administration, the collagen content in an individual is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, or 35% compared to a reference collagen content. In some embodiments, the collagen content in an individual is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, or 35% over about 6, 5, or 4 months, or over about 10 months. This reduction occurs at weeks 9, 8, 7, or 6 after liposome administration (e.g., after initiation of liposome administration). In some embodiments, the collagen content in an individual is reduced by at least about 50%, 60%, 70%, or 80% after liposome administration (e.g., after initiation of liposome administration) compared to a liposome reference collagen content. In some embodiments, a reduction is observed over about 6, 5, or 4 months, or over about 10, 9, 8, 7, or 6 weeks (e.g., after initiation of liposome administration). In some embodiments, administration of liposomes includes multiple administrations. In some embodiments, administration of liposomes includes repeated administration of liposomes or components thereof at a frequency of at least once a week (e.g., every 1, 2, 3, 4, 5, 6, or 7 days) for a duration of at least about 1, 2, 3, 4, 5, or 6 weeks, or at least about 1, 2, 3, or 4 months. In some embodiments, the reference collagen content is the baseline collagen content of an individual prior to liposome administration, such as immediately preceding the administration of liposomes. In some embodiments, the reference collagen content is the collagen content in another individual with a similar degree of liver fibrosis but who was not given liposomes. Collagen content can be measured using a variety of methods known to those skilled in the art, such as Sirius red staining or hydroxyproline detection.

[0141] In some implementations, no serious adverse events occurred in the individual following liposome administration. In some implementations, no serious adverse events occurred in the individual within 1, 2, 3, 4, 5, or 6 weeks after the initiation of liposome administration. In some implementations, no serious adverse events occurred in the individual within 1, 2, 3, or 4 months after the initiation of liposome administration. In some implementations, a serious adverse event is a life-threatening event as defined by an agency (e.g., the FDA). In some implementations, a serious adverse event is selected from death, life-threatening events, and events that: a) require hospitalization; b) result in prolonged existing hospitalization; c) result in persistent or severe disability / incapacity; d) may cause congenital abnormalities or birth defects; or e) require intervention to prevent permanent damage or harm. In some implementations, serious adverse events are selected from Grade 3, 4, and 5 adverse events as defined in the Common Terminology Standard for Adverse Events v.3.0. In some implementations, liposomes are administered at a frequency of at least once a week (e.g., every 1, 2, 3, 4, 5, 6, or 7 days) for at least about 1, 2, 3, 4, 5, or 6 weeks, or at least about 1, 2, 3, or 4 months.

[0142] In some embodiments, the individual did not experience more than 5, 4, 3, 2, or 1 non-serious adverse events after liposome administration. In some embodiments, the individual did not experience more than 5, 4, 3, 2, or 1 non-serious adverse events within 1, 2, 3, 4, 5, or 6 weeks after the start of liposome administration. In some embodiments, the individual did not experience more than 5, 4, 3, 2, or 1 non-serious adverse events within 1, 2, 3, or 4 months after the start of liposome administration. In some embodiments, non-serious adverse events are selected from Grade 1 and Grade 2 adverse events as defined in the Common Terminology Standard for Adverse Events, version 3.0. In some embodiments, liposome administration is repeated at least weekly (e.g., every 1, 2, 3, 4, 5, 6, or 7 days) for at least about 1, 2, 3, 4, 5, or 6 weeks, or at least about 1, 2, 3, or 4 months.

[0143] In some embodiments, an individual gains weight after liposome administration. In some embodiments, an individual gains at least about 1%, 2%, 3%, 4%, or 5% of their body weight within about 1, 2, 3, 4, 5, or 6 weeks after the initiation of liposome administration. In some embodiments, an individual achieves at least about 2%, 5%, 7%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% of their body weight gain within about 1, 2, 3, 5, 6, or 7 months after the initiation of liposome administration. In some embodiments, liposome administration is repeated at least once a week (e.g., every 1, 2, 3, 4, 5, 6, or 7 days) for at least about 1, 2, 3, 4, 5, or 6 weeks, or for at least 1, 2, 3, or 4 months.

[0144] combination therapy

[0145] The liposome composition of this application can be used in combination with a second therapy in any of the methods described herein (e.g., for treating, reversing, or reducing liver fibrosis or promoting liver regeneration). In some embodiments, the second therapy is a standard or commonly used liver fibrosis therapy. In some embodiments, the second therapy includes antiviral therapy. In some embodiments, the second therapy includes anti-inflammatory therapy. In some embodiments, the second therapy includes antifibrotic therapy.

[0146] In some embodiments, the liposome composition is administered concurrently with a second therapy. In some embodiments, the liposome composition is administered concurrently with a second therapy. In some embodiments, the liposome composition is administered sequentially with a second therapy.

[0147] Composition

[0148] This application provides a composition comprising liposomes, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; and b) an effective amount of zinc, wherein the zinc is encapsulated by the lipid bilayer. In some embodiments, this composition is not suitable for oral administration.

[0149] The liposomes described herein comprise: a) a lipid bilayer composed of phospholipids and cholesterol and b) zinc, with a sufficiently effective amount of zinc encapsulated in the lipid bilayer. In some embodiments, the phospholipids comprise electrically neutral phospholipids. In some embodiments, the electrically neutral phospholipids comprise phosphatidylcholine (PC). In some embodiments, the PC is selected from egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC).

[0150] In some embodiments, a composition comprising liposomes is provided, wherein the liposomes comprise: a) a lipid bilayer composed of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated in the lipid bilayer, wherein the liposomes comprise a phase transition temperature of at least 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C). In some embodiments, a composition comprising liposomes is provided, wherein the liposomes comprise: a) a lipid bilayer composed of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated in the lipid bilayer, wherein the liposomes comprise electrically neutral phospholipids with a phase transition temperature of at least 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C). In some embodiments, the electrically neutral phospholipid refers to hydrogenated soybean phospholipids (HSPC). In some embodiments, the phospholipids further comprise negatively charged phospholipids. In some embodiments, the negatively charged phospholipids comprise or are DSPG. In some embodiments, the molar ratio of neutral phospholipids to negatively charged phospholipids is approximately 50:1 to 10:1 (e.g., approximately 50:1 to 20:1, or 20:1 to 10:1). In some embodiments, the molar ratio of neutral phospholipids to negatively charged phospholipids does not exceed 20:1 or 10:1. In some embodiments, the phospholipids do not contain negatively charged phospholipids. In some embodiments, the molar ratio of phospholipids (e.g., neutral phospholipids, such as PC) to cholesterol is not greater than 8:1 (e.g., not greater than 7:1, 6:1, 5:1, or 4:1). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc. In some embodiments, the phospholipids do not contain positively charged phospholipids. In some embodiments, the phospholipids are not polyethylene glycol-derived phospholipids. In some embodiments, the zinc may be composed of an organic zinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc is readily ionized or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the average particle size of the liposomes does not exceed 200 nm (e.g., about 50-200 nm, or about 90-140 nm). In some embodiments, the PDI of the liposomes is not greater than 1 (e.g., not greater than 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05). In some embodiments, the absolute value of the zeta potential of the liposomes is at least or is 5 mV (e.g., at least about 10 mV, 15 mV, 20 mV, 25 mV, or 30 mV). In some embodiments, the 0.5-hour in vitro release rate of the liposomes is not greater than or less than 20% (e.g., not greater than or less than 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%).In some embodiments, the liposomes exhibit an in vitro release rate of no more than or less than 50% over 48 hours (e.g., no more than or less than 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, at least about 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%) of the liposomes have a lipid bilayer.

[0151] In some embodiments, a composition comprising liposomes is provided, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated by the lipid bilayer, wherein the phospholipids comprise electrically neutral phospholipids, including egg yolk lecithin (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, electrically neutral phospholipids refer to EPC. In some embodiments, electrically neutral phospholipids refer to HSPC. In some embodiments, the phospholipids further comprise negatively charged phospholipids. In some embodiments, the negatively charged phospholipids comprise or are DSPG. In some embodiments, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids is about 50:1 to 10:1 (e.g., about 50:1 to 20:1, or 20:1 to 10:1). In some embodiments, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids does not exceed 20:1 or 10:1. In some embodiments, the phospholipids do not comprise negatively charged phospholipids. In some embodiments, the molar ratio of phospholipids (e.g., electrically neutral phospholipids, such as PC) to cholesterol is no greater than 8:1 (e.g., no greater than 7:1, 6:1, 5:1, or 4:1). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agent other than zinc. In some embodiments, the phospholipid does not contain positively charged phospholipids. In some embodiments, the phospholipid is not a polyethylene glycol-derived phospholipid. In some embodiments, the zinc may be composed of an organic zinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc is readily ionized or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the average particle size of the liposomes does not exceed 200 nm (e.g., about 50-200 nm, for example, about 90-140 nm). In some embodiments, the PDI of the liposomes is no greater than 1 (e.g., no greater than 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05). In some embodiments, the absolute value of the zeta potential of the liposomes is at least or 5 mV (e.g., at least about 10 mV, 15 mV, 20 mV, 25 mV, or 30 mV). In some embodiments, the 0.5-hour in vitro release rate of the liposomes is not greater than or less than 20% (e.g., not greater than or less than 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, the 48-hour in vitro release rate of the liposomes is not greater than or less than 50% (e.g., not greater than or less than 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, at least about 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) of the liposomes have a lipid bilayer.

[0152] In some embodiments, a composition comprising liposomes is provided, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated by the lipid bilayer, wherein the phospholipids comprise i) electrically neutral phospholipids and ii) negatively charged phospholipids. In some embodiments, the electrically neutral phospholipid refers to EPC. In some embodiments, the electrically neutral phospholipid refers to HSPC. In some embodiments, the negatively charged phospholipid comprises or is DSPG. In some embodiments, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids is about 50:1 to 10:1 (e.g., about 50:1 to 20:1, or 20:1 to 10:1). In some embodiments, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids does not exceed 20:1 or 10:1. In some embodiments, the phospholipids do not comprise negatively charged phospholipids. In some embodiments, the molar ratio of phospholipids (e.g., electrically neutral phospholipids, such as PC) to cholesterol is not greater than 8:1 (e.g., not greater than 7:1, 6:1, 5:1, or 4:1). In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agent other than zinc. In some embodiments, the phospholipid does not contain positively charged phospholipids. In some embodiments, the phospholipid is not a polyethylene glycol-derived phospholipid. In some embodiments, the zinc may be composed of an organic zinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc is readily ionizable or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the average particle size of the liposomes does not exceed 200 nm (e.g., about 50-200 nm, for example, about 90-140 nm). In some embodiments, the PDI of the liposomes is not greater than 1 (e.g., not greater than 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05). In some embodiments, the absolute value of the zeta potential of the liposomes is at least 5 mV (e.g., at least about 10 mV, 15 mV, 20 mV, 25 mV, or 30 mV). In some embodiments, the 0.5-hour in vitro release rate of liposomes is no greater than or less than 20% (e.g., no greater than or less than 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, the 48-hour in vitro release rate of liposomes is no greater than or less than 50% (e.g., no greater than or less than 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, at least about 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%) of the liposomes have a lipid bilayer.

[0153] In some embodiments, a composition comprising liposomes is provided, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated in the lipid bilayer, wherein the phospholipids comprise electrically neutral phospholipids, and wherein the molar ratio of the phospholipids (e.g., electrically neutral phospholipids, such as PC) to cholesterol is not greater than 4:1. In some embodiments, the electrically neutral phospholipid refers to EPC. In some embodiments, the electrically neutral phospholipid refers to HSPC. In some embodiments, the phospholipids further comprise negatively charged phospholipids. In some embodiments, the negatively charged phospholipids comprise or are DSPG. In some embodiments, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids is about 50:1 to 10:1 (e.g., about 50:1 to 20:1 or 20:1 to 10:1). In some embodiments, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids does not exceed 20:1 or 10:1. In some embodiments, the phospholipids do not include negatively charged phospholipids. In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc. In some embodiments, the phospholipid does not contain positively charged phospholipids. In some embodiments, the phospholipid is not a polyethylene glycol-derived phospholipid. In some embodiments, the zinc may be composed of an organic zinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc is readily ionized or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the average particle size of the liposomes does not exceed 200 nm (e.g., about 50-200 nm, for example, about 90-140 nm). In some embodiments, the PDI of the liposomes is not greater than 1 (e.g., not greater than 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05). In some embodiments, the absolute value of the zeta potential of the liposomes is at least or is 5 mV (e.g., at least about 10 mV, 15 mV, 20 mV, 25 mV, or 30 mV). In some embodiments, the 0.5-hour in vitro release rate of liposomes is no greater than or less than 20% (e.g., no greater than or less than 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, the 48-hour in vitro release rate of liposomes is no greater than or less than 50% (e.g., no greater than or less than 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, at least about 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%) of the liposomes have a lipid bilayer.

[0154] In some embodiments, a composition comprising liposomes is provided, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated in the lipid bilayer, wherein the phospholipids comprise electrically neutral phospholipids, and wherein the lipid bilayer encapsulation does not contain any therapeutic agent other than zinc. In some embodiments, the phase transition temperature of the electrically neutral phospholipids is at least 10°C (e.g., at least about 20°C, 30°C, 40°C, or 50°C). In some embodiments, the electrically neutral phospholipid refers to EPC. In some embodiments, the electrically neutral phospholipid refers to HSPC. In some embodiments, the phospholipids further comprise negatively charged phospholipids. In some embodiments, the negatively charged phospholipids comprise or are DSPG. In some embodiments, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids is about 50:1 to 10:1 (e.g., about 50:1 to 20:1, or 20:1 to 10:1). In some embodiments, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids does not exceed 20:1 or 10:1. In some embodiments, the phospholipids do not include negatively charged phospholipids. In some embodiments, the molar ratio of the phospholipid (e.g., an electrically neutral phospholipid, such as PC) to cholesterol is not greater than 8:1 (e.g., not greater than 7:1, 6:1, 5:1, or 4:1). In some embodiments, the phospholipids do not include positively charged phospholipids. In some embodiments, the phospholipids are not polyethylene glycol-derived phospholipids. In some embodiments, the zinc may be composed of an organic zinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc is readily ionized or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the average particle size of the liposomes does not exceed 200 nm (e.g., about 50-200 nm, for example, about 90-140 nm). In some embodiments, the PDI of the liposomes is not greater than 1 (e.g., not greater than 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05). In some embodiments, the absolute value of the zeta potential of the liposomes is at least or 5 mV (e.g., at least about 10 mV, 15 mV, 20 mV, 25 mV, or 30 mV). In some embodiments, the 0.5-hour in vitro release rate of the liposomes is not greater than or less than 20% (e.g., not greater than or less than 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%). In some embodiments, the 48-hour in vitro release rate of the liposomes is not greater than or less than 50% (e.g., not greater than or less than 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%).In some embodiments, at least about 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%) of the liposomes have a lipid bilayer.

[0155] In some embodiments, a composition comprising liposomes is provided, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated in the lipid bilayer, wherein the phospholipid is hydrogenated soybean phospholipid (HSPC), wherein the molar ratio of HSPC to cholesterol is approximately 4:1, and the average particle size of the liposomes is approximately 50-200 nm (e.g., approximately 90-140 nm). In some embodiments, the liposomes do not contain negatively charged or positively charged phospholipids. In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agent other than zinc. In some embodiments, the zinc may be composed of an organic zinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc is readily ionizable or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the 0.5-hour in vitro release rate of the liposomes is not greater than or less than 10% (e.g., not greater than or less than 9%, 8%, or 7%). In some embodiments, the 48-hour in vitro release rate of the liposomes is not greater than or less than 12%. In some embodiments, the PDI of the liposomes is not greater than 0.1 (e.g., not greater than 0.09, 0.08, or 0.07).

[0156] In some embodiments, a composition comprising liposomes is provided, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated in the lipid bilayer, wherein the phospholipids comprise hydrogenated soybean phospholipids (HSPC) and negatively charged phospholipids, wherein the molar ratio of HSPC to cholesterol is approximately 4:1, and wherein the average particle size of the liposomes is approximately 50-200 nm (e.g., approximately 90-140 nm). In some embodiments, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids does not exceed 20:1 or 10:1. In some embodiments, the negatively charged phospholipids comprise or are DSPG. In some embodiments, a composition comprising liposomes is provided, wherein the liposomes comprise: a) a lipid bilayer consisting of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated by the lipid bilayer, wherein the phospholipids comprise HSPC and DSPG, wherein the molar ratio of HSPC to cholesterol is approximately 4:1, wherein the molar ratio of HSPC to DSPG is not more than 20:1 (e.g., approximately 10:1), and wherein the average particle size of the liposomes is approximately 50-200 nm (e.g., approximately 90-140 nm) (e.g., at least approximately 85%, 90%, or 95%). In some embodiments, the PDI of the liposomes is not greater than 0.1 (e.g., not greater than 0.09, 0.08, 0.07, 0.06, or 0.05). In some embodiments, the 0.5-hour in vitro release rate of the liposomes is not greater than or less than 5% (e.g., not greater than or less than 4%, 3%, or 2%). In some embodiments, the 48-hour in vitro release rate of the liposomes is not greater than or less than 10% (e.g., not greater than or less than 9%, 8%, 7%, or 6%). In some embodiments, the absolute value of the zeta potential of the liposomes is at least about 20 mV (e.g., at least about 25 mV or 30 mV).

[0157] A) Composition of liposomes

[0158] Phospholipids

[0159] To the judgment of those skilled in the art, any suitable phospholipid can be used in the embodiments of this application. The phospholipids include phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylserine (PS); glycolipids; and sphingomyelins such as sphingosine, ceramides, sphingomyelin, and glycosphingospholipids (e.g., cerebrosides and gangliosides). In some embodiments, the phospholipids comprise natural phospholipids. In some embodiments, the phospholipids comprise synthetic phospholipids.

[0160] Exemplary phospholipids include soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), lecithin (ESM), egg yolk lecithin (EPC), myristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPC), distearyl phosphatidylcholine (DSPC), myristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylglycerol (DOPG), distearyl phosphatidylglycerol (DSPG), distearyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dioleoyl phosphatidylethanolamine (DOPE), myristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), dioleoyl phosphatidylserine (DOPS), and dilauryl phosphatidyl phosphatidyl acid (DLPA).

[0161] In some embodiments, the phospholipid comprises an electrically neutral phospholipid. In some embodiments, the electrically neutral phospholipid comprises or is phosphatidylcholine (PC). In some embodiments, the PC is selected from lecithin choline (EPC) and hydrogenated soybean lecithin (HSPC). In some embodiments, the PC is HSPC.

[0162] In some embodiments, the phospholipid (e.g., an electrically neutral phospholipid, such as PC) has a phase transition temperature (Tc) of at least about 10°C, 20°C, 30°C, 40°C, or 50°C. In some embodiments, the phospholipid (e.g., an electrically neutral phospholipid, such as PC) has a phase transition temperature of about 20°C to 60°C, about 30°C to 60°C, about 40°C to 60°C, about 40°C to 60°C, about 45°C to 58°C, or about 50°C to 54°C.

[0163] As is well known, the temperature range (Tc) of SPC is approximately -30°C to -20°C. The Tc of HSPC is approximately 52°C. The Tc of EPC is approximately -15°C to -5°C. The Tc of DMPC is approximately 23°C. The Tc of DPPC is approximately 41°C. The Tc of DOPC is approximately -22°C. The Tc of DSPC is approximately 55°C. The Tc of DMPC is approximately 23°C. The Tc of DPPG is approximately 41°C. The Tc of DOPG is approximately -18°C. The Tc of DSPG is approximately 55°C. The Tc of DMPE is approximately 50°C. The Tc of DPPE is approximately 60°C. The Tc of DOPE is approximately -16°C. The Tc of DMPS is approximately 38°C. The Tc of DPPS is approximately 51°C. The Tc of DOPS is approximately -10°C. Referring to Li et al., Asian Journal of Pharmaceutical Sciences, Volume 10, Issue 2, April 2015, pp. 81-98. In some embodiments, the phospholipid is selected from one or more of HSPC, ESM, DPPC, DSPC, DPPG, DSPG, DMPE, DPPE, and DPPS. In some embodiments, the phospholipid is selected from one or more of HSPC, DSPC, DSPG, DMPE, and DPPS. In some embodiments, the phospholipid is selected from one or both of HSPC and DSPC.

[0164] In some embodiments, the phospholipid further comprises a negatively charged phospholipid. In some embodiments, the negatively charged phospholipid comprises or is DSPG. In some embodiments, the molar ratio of the neutral phospholipid to the negatively charged phospholipid is from about 100:1 to about 1:1, for example, about 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1. In some embodiments, the molar ratio of the neutral phospholipid to the negatively charged phospholipid is from about 50:1 to about 10:1, for example, from about 50:1 to 20:1, or from 20:1 to 10:1. In some embodiments, the molar ratio of the neutral phospholipid to the negatively charged phospholipid is at least about 20:1 or 10:1. In some implementations, the molar ratio of electrically neutral phospholipids to negatively charged phospholipids is no greater than about 20:1 or 10:1.

[0165] In some embodiments, the phospholipids do not contain negatively charged phospholipids.

[0166] In some embodiments, the phospholipids do not contain positively charged phospholipids.

[0167] In some embodiments, the molar ratio of the phospholipid (e.g., a neutral phospholipid, such as PC) to cholesterol is about 10:1 to about 2:1 (e.g., about 8:1 to about 2:1, about 6:1 to about 3:1, or about 5:1 to about 4:1). In some embodiments, the molar ratio of the phospholipid (e.g., a neutral phospholipid, such as PC) to cholesterol is not greater than 8:1 (e.g., not greater than 7:1, 6:1, 5:1, or 4:1). In some embodiments, the molar ratio of the phospholipid (e.g., a neutral phospholipid, such as PC) to cholesterol is at least about 1:1, 2:1, or 4:1. In some embodiments, the molar ratio of the phospholipid (e.g., a neutral phospholipid, such as PC) to cholesterol is about 4:1.

[0168] In some embodiments, the phospholipid is not a polyethylene glycol-derived phospholipid.

[0169] Zinc compounds

[0170] In some embodiments, the zinc compound may be an organozinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc compound is readily ionized or ionized in aqueous solution. In some embodiments, the zinc compound is in the form of a salt. In some embodiments, the organozinc salt includes zinc lactate, zinc gluconate, zinc acetate, zinc hexanoate, zinc glycyrrhizinate, zinc citrate, zinc amino acids, etc. In some embodiments, the inorganic zinc salt includes zinc sulfate, zinc chloride, zinc oxide, etc. In some embodiments, the zinc chelate of conjugated zinc includes yeast zinc, protein zinc, etc. In some embodiments, the zinc compound comprises or is zinc sulfate.

[0171] Other components in liposomes

[0172] In some embodiments, the lipid bilayer encapsulation does not contain any therapeutic agents other than zinc.

[0173] B) Liposome characterization

[0174] The liposomes described in this application can be characterized in various ways, including the following.

[0175] In some embodiments, the average particle size of the liposomes described in this application is no more than about 200 nm (e.g., no more than about 190 nm, 180 nm, 170 nm, 160 nm, 150 nm, or 140 nm). In some embodiments, the average particle size of the liposomes described in this application is at least about 50 nm (e.g., at least 60 nm, 70 nm, 80 nm, or 90 nm). In some embodiments, the average particle size of the liposomes is about 50-200 nm (e.g., about 60-180 nm, 70-160 nm, 80-150 nm, or 90-140 nm). In some embodiments, the average particle size of the liposomes described in this application is about 90-100 nm, 100-110 nm, 110-120 nm, 120-130 nm, or 130-140 nm. In some embodiments, the average particle size of the liposomes is about 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, or 140 nm.

[0176] Particle size distribution can be assessed using the polydispersity index (PDI). In some embodiments, the PDI of the liposomes is not greater than about 1, 0.5, 0.2, or 0.1. In some embodiments, the PDI of the liposomes is not greater than about 0.09, 0.08, 0.07, 0.06, or 0.05.

[0177] Many techniques can be used to determine the particle size of liposomes, including microscopy (optical microscopy, negative staining transmission electron microscopy, cryo-transmission electron microscopy, cryofragmentation electron microscopy, and scanning electron microscopy), diffraction and scattering techniques (laser scattering and photon correlation spectroscopy), and hydrodynamic techniques (gel permeation and ultracentrifugation). In some embodiments, the particle size and PDI of the liposomes are determined by dynamic light scattering (e.g., by Zetasizer Nano ZS, Malvern Panalytical Ltd, UK).

[0178] In some embodiments, the absolute value of the zeta potential of the liposome is at least or about 5 mV, 10 mV, 15 mV, 20 mV, 25 mV, or 30 mV. In some embodiments, the absolute value of the zeta potential of the liposome is no more than 30 mV, 25 mV, 20 mV, 15 mV, 10 mV, 5 mV, or 2 mV. In some embodiments, the zeta potential of the liposome is determined by dynamic light scattering (e.g., by Zetasizer Nano ZS, Malvern Panalytical Ltd, UK).

[0179] In some embodiments, the liposomes release rate at 0.5 hours is no more than or not less than 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%.

[0180] In some embodiments, the liposomes release rate over 48 hours is no more than or not less than 60%, 55%, 50%, 40%, 35%, 30%, 25%, 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%.

[0181] According to previous research (Zhang, S., et al. AAPS PharmSciTech, 2014, 15(3): p. 620-9.), the release of drugs from the liposomes can be determined using the dialysis bag method. Zinc liposomes were placed in a dialysis bag and immersed in physiological saline, and cultured in a shaking incubator (37°C, 100 rpm). The zinc concentration in the extradialysis bag fluid was measured at preset time intervals.

[0182] The number of bilayers present in liposomes, i.e., multilayering, can be determined using cryo-fragmentation electron microscopy and... 31 P-NMR analysis is used to determine this. In some embodiments, at least about 50%, 60%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the liposomes have a lipid bilayer.

[0183] In some embodiments, the phase transition temperature of the liposomes is at least about 10°C, 20°C, 30°C, 40°C, or 50°C. In some embodiments, the phase transition temperature of the liposomes is about 10°C to 60°C, for example, about 10°C to about 20°C, about 20°C to about 30°C, about 30°C to about 40°C, about 40°C to about 50°C, or about 50°C to about 60°C.

[0184] C) Other components in the liposome composition

[0185] The liposome compositions described herein can be present in compositions including other reagents, excipients, or stabilizers. For example, stability can be increased by adding certain negatively charged components, such as bile salts of bile acids, including glycocholic acid, cholic acid, chenodeoxycholic acid, taurocholic acid, glucodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, dehydrocholic acid, etc. Negatively charged surfactants or emulsifiers are also suitable as additives, such as sodium cholesterol sulfate.

[0186] In some embodiments, the composition is suitable for human administration. In some embodiments, the composition is suitable for veterinary administration, such as to domestic pets or agricultural animals. A variety of suitable formulations are available for the liposome composition. The following formulations and methods are exemplary only and not limiting. Formulations suitable for oral administration may include (a) liquid solutions, such as dissolving an effective amount of the compound in a diluent, such as water, saline, or orange juice; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient in solid or granular form; (c) suspensions in a suitable liquid; and (d) suitable emulsions. Tablet forms may include lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, gum arabic, gelatin, colloidal silica, croscarmellose sodium, talc, magnesium stearate, stearic acid, and one or more of the following excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. The tablet form may contain the active ingredient in a flavoring agent, typically sucrose and gum arabic or astragalus gum, as well as tablets containing the active ingredient in an inert matrix, such as gelatin and glycerin or sucrose and gum arabic, emulsions, gels, etc., and may contain other excipients known in the art in addition to the active ingredient.

[0187] In some embodiments, a composition comprising liposomes of a drug is provided, wherein the liposomes comprise: a) a lipid bilayer composed of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated in the lipid bilayer, wherein the pharmaceutical composition is administered parenterally or by injection. In some embodiments, a composition comprising liposomes of a drug is provided, wherein the liposomes comprise: a) a lipid bilayer composed of phospholipids and cholesterol; b) an effective amount of zinc, wherein the zinc is encapsulated in the lipid bilayer; c) an excipient for injection.

[0188] Formulations suitable for parenteral or injectable administration include aqueous and non-aqueous isotonic sterile injectable solutions, wherein the solutions may contain blood-compatible antioxidants, buffers, antibacterial agents, etc., and excipients compatible with aqueous and non-aqueous sterile suspensions, including suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Formulations may be presented in single-dose or multi-dose forms in sealed containers, such as ampoules and vials, and may be stored under lyophilized (freeze-dried) conditions, requiring only the addition of sterile liquid excipients, such as water for injection, immediately before use. Temporary injectable solutions and suspensions may be prepared from the aforementioned types of sterile powders, granules, and tablets. Injectable formulations are preferred.

[0189] In some embodiments, the composition is formulated to have a pH range of approximately 4.5 to 9.0, including any pH range of 5.0 to 8.0, 6.5 to 7.5, and 6.5 to 7.0. In some embodiments, the pH of the composition is formulated to be not less than 6, including, for example, any one of not less than 6.5, 7, or 8 (e.g., about 8). The composition can also be made isotonic with blood by adding a suitable tonic modulator such as glycerol.

[0190] Preparation method of zinc liposome composition

[0191] This application also provides a method for preparing liposome compositions, which is described below. The exemplary method is described in embodiments 1 and 3 of this application. Other methods can also be used to prepare liposomes, such as French compression vesicle preparation, solvent dispersion, and reverse-phase evaporation. See Sharma at el, An updated Review on: Liposomes as drug delivery system; Pharmatutor; 2018; 6(2):50-62.

[0192] In some embodiments, a method for producing a liposome composition is provided, the method comprising mixing a formulation containing phospholipids and cholesterol with zinc. In some embodiments, the molar ratio of phospholipids to cholesterol is about 10:1 to about 2:1 (e.g., about 8:1 to about 2:1, about 6:1 to about 3:1, or about 5:1 to about 4:1). In some embodiments, the phospholipids comprise HSPC and / or EPC or consist of HSPC and EPC. In some embodiments, the concentration of phospholipids is about 0.001-5 mol / L (e.g., about 0.01-0.5 mol / L, e.g., about 0.05-0.1 mol / L). In some embodiments, the concentration of cholesterol is about 0.0001-1 mol / L, e.g., about 0.001-0.1 mol / L, e.g., about 0.01-0.05 mol / L. In some embodiments, the zinc may be composed of an organic zinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc is readily ionized or ionized in aqueous solution. In some embodiments, zinc is in the form of a salt. In some embodiments, zinc comprises or is zinc sulfate. In some embodiments, zinc is zinc sulfate heptahydrate. In some embodiments, the concentration of zinc (e.g., zinc sulfate) is from about 0.1 to about 20 mol / L (e.g., from about 0.1 to 10 mol / L, from about 0.5 to about 5 mol / L, from about 0.8 to about 3 mol / L, from about 1 to about 2 mol / L, or from about 1.5 mol / L).

[0193] In some embodiments, a method for producing liposomes is provided, the method comprising: a) a lipid bilayer composed of phospholipids and cholesterol; b) an effective amount of zinc encapsulated in the lipid bilayer, the method comprising: 1) dissolving the phospholipids and cholesterol in ethanol to obtain an organic phase; 2) injecting a zinc solution into the organic phase to obtain a liposome suspension. In some embodiments, step 2) is performed under stirring (e.g., magnetic stirring). In some embodiments, stirring is performed at a temperature at least about 5-10°C higher than the Tc of the liposome composition or the phospholipids (e.g., negatively charged phospholipids). In some embodiments, stirring (e.g., magnetic stirring) is performed at a temperature of about 40-80°C (e.g., about 50-70°C, about 60-70°C, or about 65°C). In some embodiments, the injection as described in step 2) is performed by an infusion pump. In some embodiments, the method further comprises maintaining the liposome suspension under stirring for at least about 5 minutes (e.g., at least about 8, 10, 12, or 15 minutes). In some embodiments, the method further includes evaporation (e.g., rotary evaporation) to remove ethanol. In some embodiments, the method further includes vacuuming or stirring under negative pressure to remove ethanol. In some embodiments, evaporation is performed under reduced pressure. In some embodiments, the method further includes passing the liposome suspension through a membrane with a defined pore size. In some embodiments, the membrane pore size is about 0.1 μm to about 1.0 μm (e.g., about 0.1 μm, 0.2 μm, 0.5 μm, or 1 μm). In some embodiments, the membrane pore size is not greater than about 0.25, 0.22, 0.2, 0.18, or 0.15 μm. In some embodiments, the molar ratio of phospholipid to cholesterol is about 10:1 to about 2:1 (e.g., about 8:1 to about 2:1, about 6:1 to about 3:1, or about 5:1 to about 4:1). In some embodiments, the phospholipid comprises HSPC and / or EPC or is composed of HSPC and EPC. In some embodiments, the concentration of phospholipids is about 0.001-5 mol / L (e.g., about 0.01-0.5 mol / L, or about 0.05-0.1 mol / L). In some embodiments, the concentration of cholesterol is about 0.0001-1 mol / L, or about 0.001-0.1 mol / L, or about 0.01-0.05 mol / L.

[0194] In some embodiments, a method for producing liposomes is provided, the method comprising: a) a lipid bilayer composed of phospholipids and cholesterol; b) an effective amount of zinc encapsulated in the lipid bilayer, the method comprising: 1) dissolving phospholipids and cholesterol in chloroform to obtain an organic phase; 2) removing the organic solvent under reduced pressure to obtain a lipid film; 3) hydrating the lipid film with a zinc solution to obtain liposomes. In some embodiments, the zinc may be composed of an organic zinc salt, an inorganic zinc salt, or a zinc chelate of conjugated zinc. In some embodiments, the zinc is readily ionized or ionized in aqueous solution. In some embodiments, the zinc is in the form of a salt. In some embodiments, the zinc comprises or is zinc sulfate. In some embodiments, the zinc is zinc sulfate heptahydrate. In some embodiments, step 2 is carried out at a temperature of about 40-80°C (e.g., about 50-70°C, about 60-70°C, or about 65°C). In some embodiments, the zinc solution is a NaCl salt solution (e.g., a 0.9% NaCl salt solution) containing zinc (e.g., zinc sulfate or zinc sulfate heptahydrate). In some embodiments, the concentration of zinc (e.g., zinc sulfate) is from about 0.1 to about 20 mol / L (e.g., about 0.1 to 10 mol / L, about 0.5 to about 5 mol / L, about 0.8 to about 3 mol / L, about 1 to about 2 mol / L, or about 1.5 mol / L). In some embodiments, the method further includes thoroughly stirring the lipid film at a temperature of about 40-80°C (e.g., about 50-70°C, about 60-70°C, or about 65°C) after or during step 3. In some embodiments, the method further includes treating the lipid vesicles under high-pressure homogenization to obtain monolayer liposomes. In some embodiments, the homogenization pressure is about 400-1200 bar (e.g., about 500-1100 bar, about 600-1000 bar, about 700 to 900 bar, about 750 to 850 bar). In some embodiments, the homogenization is performed at least twice (e.g., at least 3, 4, 5, 6, 7, 8, or 9 times). In some embodiments, the homogenization temperature is approximately 0-20°C (e.g., approximately 2-15°C, approximately 4-10°C, or approximately 6-8°C). In some embodiments, the method further includes purifying the monolayer liposomes using filtration. In some embodiments, filtration is performed by size exclusion chromatography. In some embodiments, the method further includes passing the liposome suspension through a membrane with a defined pore size. In some embodiments, the membrane pore size is approximately 0.1 μm to approximately 1.0 μm (e.g., approximately 0.1 μm, 0.2 μm, 0.5 μm, or 1 μm). In some embodiments, the membrane pore size is not greater than approximately 0.25, 0.22, 0.2, 0.18, or 0.15 μm. In some embodiments, the molar ratio of phospholipids to cholesterol is approximately 10:1 to approximately 2:1 (e.g., approximately 8:1 to approximately 2:1, approximately 6:1 to approximately 3:1, or approximately 5:1 to approximately 4:1).In some embodiments, the phospholipid comprises HSPC and / or EPC or is composed of HSPC and EPC. In some embodiments, the concentration of the phospholipid is about 0.001-5 mol / L (e.g., about 0.01-0.5 mol / L, e.g., about 0.05-0.1 mol / L). In some embodiments, the concentration of cholesterol is about 0.0001-1 mol / L, e.g., about 0.001-0.1 mol / L, e.g., about 0.01-0.05 mol / L.

[0195] In some embodiments, the encapsulation efficiency (i.e., the encapsulation percentage) of the liposomes produced according to the above method is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the encapsulation efficiency (or encapsulation percentage) can be calculated using the following equation: Encapsulation efficiency = Zinc content in liposomes / (Zinc content in liposomes + Zinc content in free zinc) × 100%. Zinc liposomes and free zinc can be separated by size exclusion chromatography. (For example, in...) (This is done on the Desalting Column, for example, GE Healthcare, USA).

[0196] reagent kits and manufactured products

[0197] This application also provides kits, pharmaceuticals, compositions, and unit dosage forms required for any of the methods described herein.

[0198] The kits provided herein include one or more containers containing any of the liposome compositions (including pharmaceutical compositions) and / or other reagents described herein, and in some embodiments, include instructions for use according to the methods described herein. The kits also include descriptions of personalized treatment. The instructions provided herein are typically written instructions on a label or packaging insert (e.g., paper included in the product), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0199] For example, in some embodiments, the kit includes: a) a composition comprising liposomes, said liposomes comprising a lipid bilayer and zinc, wherein the zinc is encapsulated in the lipid bilayer or other pharmaceutically acceptable salts and carriers; alternatively b) instructions for administering the liposome composition to treat a disease or condition associated with liver fibrosis.

[0200] The kit described in this invention is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth flasks, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally provide other components, such as buffer solutions and explanatory information. Therefore, this application also provides an article of manufacture including vials (e.g., sealed vials), bottles, wide-mouth flasks, flexible packaging, etc.

[0201] In some embodiments, the kit contains one or more components that facilitate the delivery of the liposome composition and / or other therapeutic agents to an individual. In some embodiments, the kit includes, for example, syringes and needles suitable for delivering cells to an individual. In these embodiments, the liposome composition may be contained in a bag within the kit, or in one or more vials. In some embodiments, the kit contains components that facilitate the intravenous or intra-arterial delivery of the liposome composition to an individual. In some embodiments, the liposome composition may be contained, for example, in a bottle or bag (e.g., a blood bag or similar bag capable of holding up to about 1.5 L of a solution containing cells), and the kit also includes tubes and needles suitable for delivering the liposome composition to an individual.

[0202] Instructions for use related to the composition typically include information about the dosage, dosing regimen, and route of administration for the intended treatment. Containers may be unit doses, bulk (e.g., multi-dose packs), or subunit doses. For example, a kit may be provided containing a sufficient dose of zinc, as disclosed herein, to provide effective treatment for an individual for a duration such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer. Kits may also include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies such as hospital pharmacies and mixed pharmacies.

[0203] This document also provides the pharmaceuticals, compositions, and unit dosage forms required for the described method.

[0204] In one embodiment, this application relates to a liposomal composition for treating, reversing, or reducing individual liver fibrosis, the liposomal composition comprising liposomes containing a) a lipid bilayer and b) a zinc compound encapsulated in the lipid bilayer, wherein the lipid bilayer comprises phospholipids and cholesterol, the molar ratio of the phospholipids to cholesterol being 1:1 to 4:1, for example greater than or equal to 1:1, greater than or equal to 1.5:1, or greater than or equal to 2:1, or less than or equal to 4:1, or less than or equal to 3.5:1, or less than or equal to 3:1.

[0205] In one embodiment, this application relates to a method of composing a liposome composition, comprising:

[0206] 1) Dissolve phospholipids and cholesterol in ethanol to obtain an organic phase;

[0207] 2) A solution containing a zinc compound is injected into the organic phase to obtain a liposome composition.

[0208] In one embodiment, this application also relates to the use of the liposome composition of this application in the preparation of a medicament for treating, reversing or reducing individual liver fibrosis.

[0209] The following non-limiting examples further illustrate the compositions and methods of the present invention. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the invention. The invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the invention, but, of course, should not be construed as limiting the scope of the invention in any way.

[0210] Example

[0211] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the invention. The invention will now be described in more detail with reference to the following non-limiting embodiments. The following embodiments further illustrate the invention, but, of course, should not be construed as limiting the scope of the invention in any way.

[0212] Example 1: Preparation of zinc liposomes

[0213] Preparation of zinc liposomes by thin film method

[0214] According to Table 1, a certain amount of phospholipids and cholesterol were dissolved in a certain amount of chloroform. The solution was then evaporated under vacuum at 65°C to form a dry thin film for later use. A zinc solution was prepared by dissolving a certain amount of zinc sulfate heptahydrate in 0.9% physiological saline according to Table 1 for later use. The zinc solution was poured into the lipid film, and the film was hydrated by slow stirring at 65°C to obtain a suspension. The resulting suspension was homogenized at 750-850 bar (6°C, 9 times) to obtain liposomes with the desired particle size. Excess free zinc ions were removed by size exclusion chromatography using a Sephadex-G25 column. Finally, the obtained lipid vesicles were sterilized by 0.22 μm filtration and stored at 4°C.

[0215] Liposome characterization

[0216] exist Liposomes and free zinc were separated by size exclusion chromatography on a desalting column (GE Healthcare, USA). The zinc content in zinc liposomes and free zinc was determined.

[0217] Based on previous research (Shi, F., et al. Exp Biol Med (Maywood), 2015, 240(9): p. 1197-204.), zinc concentration was determined by atomic absorption spectrometry (Thermo Scientific iCE3500) after nitration with nitric acid. Encapsulation efficiency (EE) was calculated according to the following equation: EE = Zinc content in liposomes / (Zinc content in liposomes + Unencapsulated zinc content) × 100%; liposome particle size, PDI, and zeta potential were determined by dynamic light scattering (Zetasizer Nano ZS, Malvern Panalytical Ltd, UK). The results are shown in Table 2.

[0218] Based on previous research (Zhang, S., et al. AAPS PharmSciTech, 2014, 15(3): p. 620-9.), drug release from liposomes was determined using the dialysis bag method. Liposomes were placed in dialysis bags and immersed in physiological saline, then cultured in a shaking incubator (37°C, 100 rpm). Zinc concentration in the extradialysis bag fluid was measured at preset time intervals.

[0219] Table 1 Formulations F1 and F2

[0220] Formulation number F1 F2 Types of phospholipids (PC) EPC HSPC PC (mol / L) 0.05 0.05 Cholesterol (mol / L) 0.0125 0.0125 Zinc sulfate (mol / L) 1.5 1.5

[0221] As shown in Table 2, both EPC and HSPC can produce liposomes that meet the particle size requirements. Surprisingly, the encapsulation efficiency of liposomes produced using HSPC is 60% higher than that produced using EPC.

[0222] Table 2 Characterization of formulations F1 and F2

[0223] Formulation number F1 F2 Types of phospholipids (PC) EPC HSPC Particle size (nm) 90 132.2 Encapsulation efficiency (%) 51 85.7

[0224] Example 2: Preparation of zinc liposomes

[0225] Zinc liposomes were prepared using the formulations listed in Table 3, following the method described in Example 1. Different molar ratios of cholesterol and HSPC were screened, and the liposomes were characterized according to the method described in Example 1.

[0226] Table 3 Formulations F2-F6

[0227] Formulation number F3 F2 F4 F5 F6 HSPC (mol / L) 0.05 0.05 0.05 0.05 0.05 Cholesterol (CHO) (mol / L) 0 0.0125 0.025 0.05 0.1 CHO:HSPC 0 1:4 1:2 1:1 2:1 Zinc sulfate (mol / L) 1.5 1.5 1.5 1.5 1.5

[0228] Table 4 shows that formulation F3 failed to form liposomes, formulation F6 produced a large amount of precipitation, and the liposomes produced by all other formulations had a particle size range of 50-200 nm, an encapsulation efficiency of over 80%, and an in vitro release rate of less than 40% after 0.5 h. This indicates that the molar ratio of cholesterol to phospholipid is important for the formation of liposomes in this application and needs to be between 1:1 and 1:4.

[0229] Table 4 Characterization of formulations F2-F6

[0230]

[0231]

[0232] Example 3: Preparation of zinc liposomes

[0233] Preparation of zinc liposomes by ethanol injection

[0234] A certain amount (Table 1, Formulation F2) of phospholipids and cholesterol was dissolved in a certain amount of ethanol to obtain an organic phase, which was then set aside. Under magnetic stirring at 65°C, the obtained organic phase was injected into a certain volume of zinc solution dissolved in distilled water or physiological saline using a peristaltic pump. Liposomes spontaneously form upon contact between the organic phase (composed of ethanol solution) and the aqueous phase. The resulting liposome suspension was then stirred for 15 minutes at room temperature. Finally, ethanol and some water were removed by vacuum distillation. The resulting liposome suspension was extruded through membranes of different pore sizes for particle size control and then purified.

[0235] The same formulation (F2) was prepared using either the thin-film method described in Example 1 or the ethanol injection method described in Example 3, and the zinc liposomes were characterized according to the method described in Example 1.

[0236] Table 5 Characterization of Formulation F2 prepared using different methods

[0237] Formulation number F2 F2 Preparation method Thin film method Ethanol injection method Particle size (nm) 132.2 138.2 Encapsulation efficiency (%) 85.7 96 In vitro release rate (%) after 0.5 hours 6.69 1.52 In vitro release rate (%) after 48 hours 10.86 3.54

[0238] Example 4: Preparation of zinc liposomes

[0239] Some literature (Manaia, EB, et al., Int J Nanomedicine, 2017.12: pp.4991-5011) suggests adjusting the zeta potential of liposomes to increase their stability, with a suggested range of >= 30 mV or <= -30 mV. DSPG was used to adjust the zeta potential of the liposomes. Zinc liposomes were prepared using the formulations listed in Table 6, as described in Example 3, and characterized according to the method described in Example 1.

[0240] Table 6 Formulations F2, F7 and F8

[0241]

[0242]

[0243] Table 7 Characterization of formulations F2, F7 and F8

[0244] Formulation number F2 F7 F8 DSPG:HSPC 0 1:20 1:10 Zeta potential (mV) 1.12 -18.7 -33.67 Particle size (nm) 132.2 142.4 143.9 Encapsulation efficiency (%) 85.7 97 98 In vitro release rate (%) after 0.5 hours 6.69 4.81 3.92 In vitro release rate (%) after 48 hours 10.86 16.2 8.81 PDI 0.072 0.058 0.036

[0245] The zeta potential of liposomes prepared without the addition of DSPG (F2) was outside the recommended range (1.12 mV for F2). As the DSPG:HSPC ratio increased from 1:20 to 1:10 (F7 and F8), the zeta potential decreased from -18.7 mV to -33.67 mV, which is within the optimal range.

[0246] Example 5: Preparation of zinc liposomes

[0247] Zinc liposomes were prepared using the formulations listed in Table 8-1 as described in Example 3, and the zinc liposomes were characterized according to the method described in Example 1 to explore the feasibility of different types of phospholipids.

[0248] Table 8-1 Formulations F9, F10 and F11

[0249]

[0250]

[0251] DLPA stands for dilauroyl phosphatidyl acid.

[0252] The liposomes of all the formulations prepared in Table 8-1 were characterized, and the results are shown in Table 8-11:

[0253] Table 8-11 Characterization of Formulations F9, F10 and F11

[0254] Formulation number F9 F10 F11 Zeta potential (mV) -11.2 0.56 -22.6 Particle size (nm) 205.3 189.6 178.5 Encapsulation efficiency (%) 60 45.6 70.5 In vitro release rate (%) after 0.5 hours 5.56 9.87 6.61 In vitro release rate (%) after 48 hours 12.32 19.93 16.47 PDI 0.046 0.079 0.053

[0255] According to the liposomes of all the preparations obtained from Table 8-1, zinc liposomes can be prepared from different types of phospholipids.

[0256] Similarly, zinc liposomes were prepared using the formulations listed in Table 8-2 according to the method described in Example 1, and different molar ratios of cholesterol with different phospholipids and zinc salts were screened. The liposomes were then characterized according to the method described in Example 1.

[0257] Table 8-2 Formulations F21, F22, F23, F24, F25

[0258]

[0259]

[0260] *DF1 is a contrast agent; the molar ratio of phospholipids to cholesterol is not in the range of 1:1 to 4:1.

[0261] The liposomes of all the formulations prepared in Table 8-2 were characterized, and the results are shown in Table 8-21.

[0262] Table 8-21 Characterization of formulations F21, F22, F23, F24, F25 and DF1

[0263] Formulation number F21 F22 F23 F24 F25 DF1* Zeta potential (mV) -28.6 -18.6 -3.8 -19.2 -29.5 -6.9 Particle size (nm) 128.6 203.9 251.5 236.7 305.9 286.4 Encapsulation efficiency (%) 45.9 59.5 38.4 23.5 5.6 3.9 In vitro release rate (%) after 0.5 hours 9.6 8.5 13.9 15.7 18.9 20.6 In vitro release rate (%) after 48 hours 19.8 15.3 23.3 39.6 42.3 49.5 PDI 0.054 0.034 0.064 0.012 0.026 0.073

[0264] According to the liposomes of all the preparations obtained from Table 8-2, zinc liposomes can be prepared using different types of phospholipids with a molar ratio of phospholipid to cholesterol ranging from 1:1 to 4:1; however, when the molar ratio of phospholipid to cholesterol is greater than 4:1, the stability of the lipid molecule arrangement within the liposome decreases.

[0265] Example 6: Study on the safe dosage range of zinc liposomes

[0266] To determine the safe dosage range for the composition, zinc liposomes (LZ) prepared in the selected formulation were evaluated in mice.

[0267] method

[0268] Test animals. Male Kunming mice were given free access to standard AIN-76 rodent feed and mineral-free double-distilled water. On the day of administration, the mice were randomly divided into four groups: (1) 2.5 mg / kg LZ, (2) 5 mg / kg LZ, (3) 10 mg / kg LZ, and (4) 20 mg / kg LZ.

[0269] Treatment in mice. Zinc liposomes (prepared using the ethanol injection method, formulation F2) were administered intravenously in a single large dose at their respective concentrations. Morbidity and mortality were observed 14 days post-administration. Body weight was monitored and adverse events were recorded periodically. Mean ± standard deviation is used to represent data from one group.

[0270] result

[0271] No morbidity or mortality was observed in animals receiving 2.5 mg / kg or 5 mg / kg zinc liposomes during the observation period. Furthermore, no significant decrease in body weight or adverse reactions was observed.

[0272] For animals receiving 10 mg / kg liposomal zinc, although no animals died during the observation period, a significant decrease in body weight was observed on day 1.

[0273] Of the animals that received 20 mg / kg liposomal zinc, three died during the observation period. Figure 1 and Figure 2 )

[0274] Example 7: Zinc liver preservation after a single dose of zinc liposomes

[0275] To evaluate the role of zinc liposomes in retaining zinc in liver tissue, liver tissue zinc concentration was monitored after mice were given a single dose of zinc liposomes.

[0276] method

[0277] Test animals. Six-week-old male Kunming mice weighing 18–22 g were allowed free access to standard AIN-76 rodent feed and mineral-free double-distilled water. On the day of administration, the mice were randomly assigned to three groups: (1) zinc sulfate, (2) LZ (F2), and (3) LZ (F6).

[0278] Treatment in mice. Mice were administered zinc liposomes (using the specified formulation F2 or F6) or zinc sulfate via a single intravenous bolus injection of 5 mg / kg. At specified time points (before administration / no treatment, 5 min, 15 min / 0.25 h, 30 min / 0.5 h, 2 h, 4 h, 8 h, 24 h, 48 h, 168 h), three mice from each group were randomly selected and sacrificed, and their livers were collected.

[0279] Determination of zinc concentration. The collected liver was homogenized and digested in nitric acid. The zinc concentration in the digestate was determined using the method described in Example 1.

[0280] result

[0281] like Figure 3 As shown, administration of zinc liposomes significantly and continuously increased the concentration of zinc in liver tissue. Eight hours after injection of F2 or F6, the peak concentration was approximately 160 μg / g, 60% higher than baseline. Forty-eight hours after administration, the liver zinc concentration was approximately 140 μg / g, 40% higher than baseline. In contrast, administration of non-liposomal zinc sulfate at the same concentration only transiently increased liver zinc concentration. Liver zinc concentration returned to baseline within just 15 minutes after administration.

[0282] The results showed that zinc liposomes could efficiently deliver zinc to the liver, and that liver zinc concentrations sustained a significant increase (e.g., from 48 hours post-administration, liver zinc concentrations increased by approximately 40% from baseline). This sustained and significant increase in liver zinc was entirely unexpected. As Chen et al. demonstrated, when liposomes were administered specifically to deliver glycyrrhetinic acid (GA) to the liver, GA concentrations increased rapidly within minutes after administration, but then decreased sharply over several hours, recovering to approximately zero only after 12 hours. See Chen et al. Figure 7Oncotarget, 2017, Vol. 8 (No. 60), pp: 102046-102066. Therefore, the zinc liposome formulation of this application not only facilitates the delivery of zinc to liver tissue but also significantly increases the zinc retention in the liver, which is entirely unexpected.

[0283] Example 8: Treatment of liver fibrosis in Kunming mice with zinc liposomes after bile duct ligation (BDL)

[0284] To demonstrate the therapeutic potential of zinc for liver fibrosis, and more importantly, to demonstrate the potential advantages of zinc liposome (LZ, F1) supplementation over conventional zinc supplementation, intravenous injection of zinc liposomes into a mouse model of irreversible liver fibrosis was compared with intravenous injection of zinc sulfate solution. Animal quality of life was monitored using survival and weight data, serum and liver zinc levels were recorded, and the degree of liver fibrosis was assessed using histological examination as the gold standard.

[0285] Materials and methods

[0286] Animals and the establishment of a liver fibrosis model using bile duct ligation (BDL)

[0287] Eight-week-old male Kunming mice, weighing 30-35g, were given free access to standard AIN-76 rodent food and mineral-free double-distilled water. The BDL / sham procedure was performed as previously described. (Zeybel, M. et al., Nat Med, 2012. 18(9): p. 1369-77.) Briefly, mice were anesthetized by inhalation using 2% to 3% isoflurane mixed with oxygen. The abdomen was opened via midline laparotomy, the bile ducts were separated from the portal vein and hepatic artery, and then ligated with three 5.0 ethereal sutures. Two ligatures were placed upstream of the bile duct and one downstream. The abdomen was then closed with inverted interrupted sutures. In the sham control group, the bile ducts were not ligated or transversely incised, and other procedures were the same as in the BDL group.

[0288] Treatment of fibrotic mice

[0289] Four weeks after BDL surgery, mice were divided into six groups: (1) sham surgery + saline, (2) sham surgery + conventional zinc, (3) sham surgery + zinc liposomes, (4) BDL + saline, (5) BDL + conventional zinc, and (6) BDL + zinc liposomes. These mice were intravenously injected every two days with 0.9% NaCl saline (groups 1 and 4), conventional zinc (i.e., zinc sulfate in 0.9% NaCl saline) (groups 2 and 5), or zinc liposomes (i.e., LZ) (groups 3 and 6). A total of seven injections were administered over two weeks. The dosage used was 1 mg of elemental zinc per kilogram of animal body weight, and the injection dose was the same. One day after the last injection, all mice were sacrificed, and liver and blood samples were collected for analysis. (For study design, please see [link to study design]). Figure 4。 )

[0290] Zinc concentration in blood and liver

[0291] After lyophilization and digestion with nitric acid according to the aforementioned procedure, the concentrations of zinc in serum and liver were determined by atomic absorption spectrometry (Thermo Scientific iCE 3500). (Zuo, X., et al., PLoS One, 2013.8(6):p.e67549.)

[0292] Analysis of liver fibrosis

[0293] Collagen deposition was observed in formalin-fixed, paraffin-embedded liver tissue sections after Sirius red staining (with Fast Green co-staining). Semi-quantitative analysis of collagen deposition was performed by randomly selecting five non-overlapping regions from each slide at x100 magnification under a Nikon Eclipse 80i microscope, and the acquired images were analyzed using Image-pro plus 6.0 software.

[0294] Determination of hepatic hydroxyproline concentration

[0295] The hydroxyproline content in liver tissue was determined according to the manufacturer's instructions using a hydroxyproline assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing). The absorbance of the formed purple complex was measured at 550 nm using a standard spectrophotometer. The content of free hydroxyproline in the sample was calculated using hydroxyproline as a standard. The value is expressed as hydroxyproline (μg) / liver tissue (mg).

[0296] Statistical methods

[0297] Data were analyzed using SPSS / 19.0 software, and one-group comparisons were performed using ANOVA and Dunnett's T3 test. A p-value < 0.05 was considered statistically significant.

[0298] result

[0299] Interestingly, in Figure 5-6 Among the mice, those that did not undergo BDL surgery but received routine zinc treatment had the lowest survival rate, consistent with the greatest weight loss among all groups. This group of mice had the highest serum zinc concentration and decreased liver zinc concentration. Figure 7 The group that underwent BDL surgery followed by the same amount of conventional zinc had relatively higher survival rates, smaller weight loss, relatively higher liver zinc concentrations, and relatively lower serum zinc concentrations. See [link to relevant documentation]. Figure 5-8 The results indicate that high concentrations of zinc in the blood may be harmful to animals.

[0300] Surprisingly, both groups treated with zinc liposomes, regardless of whether they had undergone BDL surgery, achieved 100% survival and gained weight comparable to, or even more than, the control group. Importantly, in the group that received zinc liposomes but did not undergo BDL surgery, serum and liver zinc concentrations were comparable to the control group, indicating that administration of zinc liposomes did not affect zinc concentrations in either the blood or liver (in the absence of zinc deficiency). This contrasts with the effect shown in the group that received conventional zinc treatment but did not undergo BDL surgery, where both blood and liver zinc concentrations were adversely affected. On the other hand, administration of zinc liposomes in the BDL surgery group restored liver concentrations more effectively compared to conventional zinc. As stated above, these results indicate that zinc liposome treatment 1) has a significant protective effect in mice undergoing BDL or sham surgery, and 2) delivers zinc to the liver more effectively in cases of zinc deficiency. These results provide further evidence that zinc is preferentially retained in the liver in the presence of zinc deficiency.

[0301] The effect of zinc liposomes on liver fibrosis

[0302] Figures 9A-9C This study summarizes the combined results of the effects of different forms of zinc on the progression of BDL-induced liver fibrosis. Figure 9A Representative images from histological examinations of each group of animals are shown. Sirius red staining highlights collagen deposition, while Fast Green co-staining provides background staining for liver tissue. Figure 9B This involves a quantitative analysis of all histological findings, using computer software to obtain the percentage area stained with Sirius red and performing statistical analysis. For example... Figure 9C The results show the hydroxyproline content (a key component of deposited fibrin) measured in each test group. Based on these results, BDL induced significant liver fibrosis in the affected animals. On the other hand, the sham-operated group showed no signs of liver fibrosis. Collagen content in the livers of mice treated with zinc liposomes decreased by approximately 20%. Conversely, mice treated with conventional zinc showed no observable effect on liver fibrosis, regardless of increases in liver or serum zinc levels.

[0303] Example 9: Treatment of rhesus monkey liver fibrosis with zinc liposomes after BDL

[0304] As demonstrated in the examples above, zinc liposomes have proven their potential in treating liver fibrosis. Compared to conventional zinc, zinc liposomes exhibit similar capabilities in supplying and restoring serum and hepatic zinc levels. However, they are clearly more effective in inhibiting the progression of liver fibrosis. To further validate this potential in a more human-like environment, non-human primates, rhesus monkeys, underwent the same BDL surgery to induce liver fibrosis and were treated with zinc liposomes (LZ, F2). Liver fibrosis in individual animals was monitored periodically and assessed as a continuous process. Modeling methods were employed using animals that underwent BDL surgery but received no treatment, ensuring that the induced liver fibrosis was irreversible.

[0305] LZ was intravenously injected into a monkey model of irreversible liver fibrosis. Body weight data were recorded, and liver fibrosis status was assessed using histological examination as the gold standard. Liver biopsy was performed for sampling, avoiding animal sacrifice.

[0306] Materials and methods

[0307] Animal and BDL liver fibrosis models

[0308] Animals were allowed free access to food, and all animal experimental procedures were approved by the Laboratory Animal Management and Use Committee. BDL procedures were performed as described above. [4] Briefly, animals were anesthetized by inhalation of oxygen mixed with 2–3% isoflurane. The abdomen was opened by midline laparotomy, the bile ducts were separated from the portal vein and hepatic artery, and then ligated with three 5.0 silk (Ethicon) sutures. Two ligatures were placed upstream of the bile duct and one downstream. The abdomen was then closed with inverted interrupted sutures.

[0309] Treatment of fibrotic rhesus monkeys

[0310] Each animal was treated individually without grouping. LZ was administered intravenously at doses of 0 (zinc-free liposome treatment), 0.35 mg / kg (zinc per kilogram of body weight), 0.7 mg / kg, and 1.4 mg / kg, every two days for four months. Figure 10 The weight of individual animals is monitored regularly, and liver biopsies are performed monthly for histological examination.

[0311] Analysis of liver fibrosis

[0312] Collagen deposition was observed in formalin-fixed, paraffin-embedded liver biopsy sections after Sirius red staining (with Fast Green co-staining). Semi-quantitative analysis of collagen deposition was performed by randomly selecting five non-overlapping regions from each slide at x100 magnification using a Nikon Eclipse 80i microscope, and the acquired images were analyzed using Image-pro plus 6.0 software.

[0313] result

[0314] Effects of zinc liposomes on body weight changes in rhesus monkeys

[0315] Figure 11 Weight information from all test animals was summarized. Weight data at the end of four months of treatment were compared with pre-treatment weight data, and the percentage change in weight was calculated as weight change / pre-treatment weight × 100%. The results showed a positive correlation between zinc liposome dosage and weight change. Weight change increased with increasing zinc liposome dosage. Positive weight change does not necessarily equate to better recovery from liver fibrosis, but it does indicate a better overall quality of life for the animals. These results are consistent with findings from histological examination.

[0316] Effects of zinc liposomes on liver fibrosis in rhesus monkeys

[0317] like Figure 12 and 13 As shown, fibrosis continued to progress during the 4-month treatment period, and the fibrotic area doubled in animals that underwent only BDL without treatment. Progression of fibrosis was still observed in the groups treated with 0.35 mg / kg or 0.7 mg / kg zinc liposomes, but to a lesser extent, indicating that zinc liposomes have a protective effect at these doses. Unexpectedly, a significant reduction in the fibrotic area in liver biopsy tissue was observed in rhesus monkeys treated with 1.4 mg / kg zinc liposomes. These results clearly demonstrate that the therapeutic effect of this dose of zinc liposomes not only protects liver tissue from further fibrotic development but also reverses prior liver fibrosis.

[0318] Example 10 : Following bile duct ligation (BDL), F3-F4 fibrosis staging was observed in Kunming mice treated with zinc liposomes. Treatment of liver fibrosis

[0319] As described in Example 7, a fibrosis animal model was established using Kunming mice via BDL surgery. Six weeks after BDL surgery, mice identified as having F3-F4 fibrosis stages were divided into four groups: (1) sham surgery + saline, (2) BDL + saline, (3) BDL + conventional zinc (5 mg / kg), and (6) BDL + zinc liposomes (5 mg / kg). These mice received intravenous injections every two days of 0.9% NaCl saline (Groups 1 and 2), conventional zinc (i.e., zinc sulfate in 0.9% NaCl saline) (Group 3), or zinc liposomes (F2, i.e., LZ) (Group 4). Seven injections were administered over two weeks. The dosage used was 1 mg of elemental zinc per kilogram of animal body weight, and the injection dose was the same. The survival rates of the four groups of mice were shown in... Figure 14 See Table 9. The results indicate that zinc liposomes have a significant protective effect in patients with severe liver fibrosis (F3-F4 stage).

[0320] Table 9

[0321]

[0322] Example 11: Treatment of rhesus monkey liver fibrosis with zinc liposomes after BDL

[0323] As described in Example 9, a liver fibrosis animal model was established in rhesus monkeys using a biochemical lymph node dissection (BDL) procedure. Following the BDL procedure, the animals were divided into three groups and administered medication intravenously. Group 1 received saline, Group 2 received 0.7 mg / kg zinc liposomes, and Group 3 received 1.4 mg / kg zinc liposomes, for a period of 4 months. Collagen deposition was assessed using Sirius red staining, as described in Example 9. Results showed… Figure 15 And see Table 10 below. The results again demonstrate that zinc liposomes effectively reduce and reverse liver fibrosis.

[0324] Table 10

[0325]

[0326] References:

[0327] 1.Bartholomay,AF,et al.,Zinc metabolism in hepaticdysfunction.I.Serum zincconcentrations in Laennec's cirrhosis and their validation by sequential analysis.N Engl J Med,1956.255(9):p.403-8.

[0328] 2.Vallee,BL,et al.,Zinc metabolism in hepaticdysfunction.II.Correlation of metabolic patterns with biochemical findings.NEngl J Med, 1957.257(22):p.1055-65.

[0329] 3.Grungreiff,K.,D.Reinhold,and H.Wedemeyer,The role of zinc in livercirrhosis.Ann Hepatol,2016.15(1):p.7-16.

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[0337] 11.Atsushi Hosui,N.H.,Oral zinc supplementation improves liverfunction and decreases the risk of hepatocellular carcinomadevelopment.Hepatology,2016.64(S1).

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[0339] 13.McClain,C.,V.Vatsalya,and M.Cave,Role of Zinc in the Development / Progression of Alcoholic Liver Disease.Curr Treat Options Gastroenterol,2017.15(2):p.285-295.

[0340] 14.Gavino,F.,et al.,Zinc in gastrointestinal and liverdisease.Coordination Chemistry Reviews,2008.252:p.1257-1269.

[0341] 15.Jiang,Y.and Y.J.Kang,Metallothionein gene therapy for chemical-induced liver fibrosis in mice.Mol Ther,2004.10(6):p.1130-9.

[0342] 16.Sato,S.,et al.,Distribution of zinc-binding metallothionein incirrhotic liver of rats administered zinc.Pharmacol Toxicol,2000.87(6):p.292-6.

[0343] 17.Stuart,G.W.,P.F.Searle,and R.D.Palmiter,Identification of multiplemetal regulatory elements in mouse metallothionein-I promoter by assayingsynthetic sequences.Nature,1985.317(6040):p.828-31.

[0344] 18.Klaassen,C.D.,et al.,In vitro and in vivo studies on thedegradation of metallothionein.Environ Health Perspect,1994.102 Suppl 3:p.141-6.

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[0347] 21.Litzinger,D.C.,et al.,Effect of liposome size on the circulationtime and intraorgan distribution of amphipathic poly(ethylene glycol)-containing liposomes.Biochim Biophys Acta,1994.1190(1):p.99-107.

[0348] 22.Chonn,A.,P.R.Cullis,and D.V.Devine,The role of surface charge inthe activation of the classical and alternative pathways of complement byliposomes.J Immunol,1991.146(12):p.4234-41.

[0349] 23.Friedman,S.L.,Liver fibrosis--from bench to bedside.J Hepatol,2003.38 Suppl 1:p.S38-53.

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Claims

1. Use of the liposome composition in the preparation of a medicament for treating individual liver fibrosis, wherein, The drug is an injectable preparation. The liposome composition comprises liposomes, the liposomes comprising a) a lipid bilayer, b) a zinc compound encapsulated in the lipid bilayer, and c) an excipient for injection, wherein the lipid bilayer comprises phospholipids and cholesterol. The lipid bilayer encapsulation does not contain any therapeutic agents other than zinc compounds. The medication shall be administered at least once a week; Each dose of zinc liposome contains less than 1.6 mg / kg of zinc. The zinc compound includes zinc sulfate. The molar ratio of phospholipids to cholesterol is 1:1 to 4:

1.

2. The use according to claim 1, characterized in that, The phospholipid is selected from one or more of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin.

3. The use according to claim 2, characterized in that, The phospholipids include electrically neutral phospholipids, selected from phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. The phosphatidylcholine is selected from lecithin, hydrogenated soybean lecithin, or a combination thereof.

4. The use according to claim 1, characterized in that, The average particle size of the liposomes is 50 nm to 200 nm.

5. The use according to claim 1, wherein the average particle size of the liposomes is 90 nm to 140 nm.

6. The use according to claim 3, characterized in that, The phospholipids also include negatively charged phospholipids.

7. The use according to claim 6, wherein the negatively charged phospholipid is selected from one or more of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine.

8. The use according to claim 7, characterized in that, The molar ratio of the negatively charged phospholipid to the electrically neutral phospholipid is at least 1:

10.

9. The use according to any one of claims 1 to 8, characterized in that, The absolute value of the zeta potential of the liposomes is ≥25.

10. The use according to claim 1, wherein, The aforementioned liver fibrosis is a condition caused by liver disease.

11. The use according to claim 10, wherein, The liver diseases mentioned are selected from alcoholic liver disease and non-alcoholic fatty liver disease.

12. The use according to claim 11, wherein, The non-alcoholic fatty liver disease mentioned is selected from non-alcoholic steatohepatitis.

13. The use according to any one of claims 10 to 12, characterized in that, Each dose of zinc liposome contains less than 0.8 mg / kg of zinc.

14. The use according to claim 13, characterized in that, Each administration of zinc liposomes contains at least 0.08 mg / kg of zinc.

15. The use according to claim 13, characterized in that, Each administration of zinc liposomes contains at least 0.4 mg / kg of zinc.

16. The use according to claim 1, wherein the liposome composition is prepared by the following method: 1) Phospholipids and cholesterol are dissolved in ethanol to obtain an organic phase; 2) A solution containing a zinc compound is injected into the organic phase to obtain a liposome composition.

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