Composition and method for treating fibrosis

Abstract

The present invention relates to a composition comprising phosphatidylcholine and a phosphatidylcholine derivative, such as DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) or DPPC, for treating fibrosis, which generally includes liver fibrosis and related conditions such as fatty liver disease, non-alcoholic steatohepatitis (NASH) and cirrhosis, or pulmonary fibrosis and conditions related to pulmonary fibrosis.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 570,847 filed on October 11, 2017, which is incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a composition comprising a phosphatidylcholine derivative, such as DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) or DPPC, for the treatment of fibrosis, including, in general, liver fibrosis and fatty liver disease, non-alcoholic steatohepatitis (NASH) and cirrhosis. Background Technology

[0005] Fatty liver disease (FLD), whether alcoholic (AFLD) or non-alcoholic (NAFLD), results in unregulated lipid metabolism. While there are many pathways that cause FLD, three pathways critical to disease progression are reactive oxygen species (ROS), sterol regulatory factor-binding protein-1 (SREBP-1), and peroxisome proliferator-activated receptor (PPAR). Regulation of each of these pathways has been shown to have some benefit in clearing FLD. Blocking ROS using vitamin E has shown positive effects in animal models, and there is also some positive data from human studies. Several candidate drugs designed to block PPAR gamma are currently under development for the treatment of non-alcoholic steatohepatitis (NASH).

[0006] Agonists for liver receptor homologue 1 (LRH-1) positively modulate SREBP-1 in vivo. These include 12-carbon free fatty acids attached to phosphocholine, designated as 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and / or 16-carbon free fatty acids attached to phosphocholine, designated as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Long-chain phosphatidylcholine, which binds to PPAR alpha and LRH-1 in cells and tissues, such as fibrous tissue, can be replaced by short-chain DLPC and DPPC species. In animal models, it was confirmed that administering DLPC to Chows slows the progression of liver fibrosis (U.S. Patent No. 5,284,835). DPPC can also bind to PPAR alpha, which eventually blocks PPAR gamma and may prevent disease progression.

[0007] Fibrosis is caused by the formation or development of excessive fibrous connective tissue as a restorative or reactive process in organs or tissues, and occurs in various tissues when this process is not regulated by impaired re-epithelialization (Tomasek et al. Nat Rev Mol Cell Biol 3, 349-363 (2002), Thannickal et al Annu Rev Med 55, 395-417 (2004), Hinz et al. Am J Pathol 170, 1807-1816 (2007)). Also refer to U.S. Patent Publication No. 20120141461.

[0008] The present invention provides a method for delivering phosphotidylcholine, such as 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and / or any version of a combination of free fatty acids that activate LRH-1 and PPAR alpha to control fibrosis in the liver and other tissues. As lipid moiety, DLPC and / or DPPC are considered to be soluble in liposomes, micelles, or emulsions to facilitate targeted delivery to tissues.

[0009] In various embodiments, the present invention provides a method for treating fibrosis comprising the step of administering an effective amount of phosphatidylcholine (PC) within a liposome or nanoparticle to a subject in need of it to treat fibrosis.

[0010] The present invention also provides a composition comprising the liposomes or nanoparticles described herein, comprising phosphatidylcholine in an amount effective for treating fibrosis. In various embodiments, the present invention provides a composition comprising the liposomes described herein for use in treating fibrosis.

[0011] In various embodiments, phosphatidylcholine is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or a choline head with a phospholipid tail of 16 carbons. In various embodiments, phosphatidylcholine is 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or a choline head and a phospholipid tail of 12 carbons. In various embodiments, the liposome or nanoparticle comprises phosphatidylcholine, e.g., DPPC and / or DPLC, and / or a mixture of phosphatidylcholine having a choline head with a phospholipid tail of 16 carbons or 12 carbons.

[0012] In various embodiments, the liposome or nanoparticle further comprises an antioxidant. In various embodiments, the antioxidant is a fat-soluble antioxidant. In various embodiments, the antioxidant is vitamin E. In various embodiments, vitamin E is present in the liposome at a concentration of about 1 to 20 mg / ml, about 2 to 10 mg / ml, about 4 to 10 mg / ml, or about 4 to 8 mg / ml.

[0013] Additional antioxidants are described further in the detailed description.

[0014] In various embodiments, the liposome or nanoparticle further comprises a factor capable of stimulating new cell growth. In various embodiments, the liposome or nanoparticle further comprises a protein or peptide capable of stimulating new cell growth. In various embodiments, the peptide is a GHK peptide capable of stimulating new cell growth.

[0015] In various embodiments, the liposome further comprises a factor capable of stimulating new cell growth and an antioxidant. In various embodiments, the factor stimulating new cell growth is a GHK peptide, and the antioxidant is vitamin E. In various embodiments, the liposome or nanoparticle comprises GHK in combination with DLPC and / or DPPC alone, or in combination with vitamin E.

[0016] In various embodiments, the liposome or nanoparticle further comprises polyethylene glycol (PEG). In various embodiments, PEG is present in the liposome at a concentration of about 2 to 20 mg / ml, about 5 to 15 mg / ml, or about 4 to 10 mg / ml.

[0017] Phosphatidylcholine is present in liposomes and is considered to be administered at concentrations of about 2 to 50 mg / kg, 5 to 40 mg / kg, 10 to 30 mg / kg, 15 to 25 mg / kg, or 5 to 20 mg / kg per dose. DLPC, DPPC and / or other phosphatidylcholines with a 16 or 12-carbon carbon tail are considered to be administered at concentrations of about 2 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 75 mg / kg, or 100 mg / kg. In various embodiments, phosphatidylcholine (PC) is present in liposomes or nanoparticles at a total PC concentration of about 0.1 to 100 mg / ml, about 1 to 80 mg / ml, about 5 to 70 mg / ml, about 10 to 60 mg / ml, about 15 to 100 mg / ml, about 15 to 50 mg / ml, about 20 to 80 mg / ml, about 20 to 70 mg / ml, about 30 to 60 mg / ml, or about 40 to 50 mg / ml. In various embodiments, the total PC concentration is about 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, or 100 mg / ml. In various embodiments, the total PC that can be delivered per single dose delivered IV is about 20,000 mg, 15,000 mg, 10,000 mg, 8,000 mg, 6,000 mg, 4,000 mg, 3,000 mg, 2,000 mg, 1,000 mg, 750 mg, 500 mg, 400 mg, 200 mg / ml, or 100 mg.

[0018] In various embodiments, the liposome or nanoparticle further comprises cholesterol. The cholesterol may be in the liposome or nanoparticle in the range of about 1 to 20 mg / kg, about 2 to 15 mg / kg, or about 5 to 10 mg / kg. In various embodiments, the cholesterol is present in the liposome or nanoparticle in the range of about 1 to 20 mg / ml, or about 2 to 15 mg / ml, or about 5 to 10 mg / ml, or about 1 to 5 mg / ml, or about 1 to 3 mg / ml, or about 0.5 to 5 mg / ml. In various embodiments, the cholesterol concentration is about 1.0 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 5 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 7.5 mg / ml, 8.0 mg / ml, 8.5 mg / ml, 9.0 mg / ml, 9.5 mg / ml, 10.0 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml.

[0019] In various embodiments, the liposomes may have the following composition based on weight% composition: DLPC 10 to 50%, DPPC 10 to 50%, cholesterol 0 to 5%, vitamin E 5 to 20%, and optionally PEG 0 to 15%. Combinations of components within these ranges are useful for preparing the liposomes of the present invention and are considered to be specifically provided by the present disclosure. Liposome content may be measured by HPLC or other analytical techniques used to determine the chemical content of the composition. In various embodiments, the liposomes or nanoparticles contain total phosphatidylcholine (PC) at a concentration of 20 to 80 weight% of the composition in the liposomes. In various embodiments, the liposomes or nanoparticles contain total phosphatidylcholine (PC) at a concentration of 1 to 100 mg / ml in the liposomes.

[0020] In various embodiments, liposomes are administered by injection (e.g., intravenously, subcutaneously), topically, orally, by depot, or by inhalation. In various embodiments, topical administration is transdermal. In various embodiments, liposomes are administered intravenously.

[0021] In various embodiments, the subject suffers from a disease or condition that is a precursor to or directly leads to fibrosis or related fibrosis. In various embodiments, the disease or condition is any fibrotic disease, including but not limited to liver fibrosis diseases such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis, or pulmonary fibrosis diseases such as chronic obstructive pulmonary disease (COPD) or interstitial pulmonary fibrosis.

[0022] In various embodiments, administration relieves one or more symptoms of fibrosis, including, but not limited to, liver fibrosis, liver fat content, onset or progression of cirrhosis, onset of hepatocellular carcinoma, increased liver aminotransferase levels, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), increased serum ferritin, elevated levels of gamma-glutamyltransferase (gamma-GT), and elevated levels of plasma insulin, cholesterol, and triglycerides.

[0023] In various embodiments, liposomes or nanoparticles are administered weekly, every two weeks, every three weeks, monthly, or every two months. In various embodiments, the therapy is administered for at least one year and may be administered for the lifetime of the subject or patient.

[0024] In various embodiments, the subject is a mammal. In various embodiments, the subject is a human. In various embodiments, the subject is an adult or adolescent.

[0025] In various embodiments, the composition further comprises pharmaceutically acceptable excipients, carriers, or diluents.

[0026] It should be understood that each feature, embodiment, or combination described herein is a non-limiting and illustrative example of any aspect of the invention and therefore implies that it may be combined with any other feature, embodiment, or combination described herein. For example, where a feature is described in language such as “one embodiment,” “some embodiment,” “specific embodiment,” “additional embodiment,” “specific exemplary embodiment,” and / or “another embodiment,” each of these types of embodiments is a non-limiting example of a feature intended to be combined with any other feature or combination of features described herein without the need to enumerate all possible combinations. Such features or combinations of features apply to any aspect of the invention. Where examples of values ​​falling within a range are disclosed, any such example is considered as an endpoint of a possible range, any numerical value between such endpoints is considered, and any combination of upper and lower limits is anticipated. Brief explanation of the drawing

[0027] Figure 1 shows the reduction of fibrotic pathology in mice treated with liposomes containing phosphatidylcholine. Figure 2 shows representative liver histology from the control group and the liposome-treated group. Figure 3 shows that there was no significant change in liver enzyme levels (ALT, AST, and bilirubin) between the control group and the treatment group. Specific details for implementing the invention

[0028] The present invention provides liposomes for the treatment of fibrosis including liver fibrosis and, to a limited extent, liver diseases including cirrhosis, hepatitis, and non-alcoholic fatty liver disease (NASH). The liposomes disclosed herein are absorbed by the liver and reduce fibrosis and inflammation caused by oxidative tumors and the death of hepatocytes.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which this invention pertains. The following references provide to those skilled in the art general definitions of many terms used in this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).

[0030] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it does not conform to the contents of this disclosure.

[0031] As used in the present invention and the appended claims, the singular forms “one (a),” “and,” and “the” include multiple referents unless the context clearly indicates otherwise. Accordingly, for example, a reference to “one derivative” includes multiple such derivatives, and a reference to “one object” includes a reference to one or more objects, etc.

[0032] Where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in some specific examples, they may alternatively be described using the language of "essentially constituting" or "constituting."

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. Although methods and materials similar or equivalent to those described herein may be used to practice the disclosed methods and compositions, exemplary methods, apparatuses, and materials are described herein.

[0034] As used herein, "liposome" refers to a closed structure comprising an outer lipid bilayer or multilayer membrane surrounding an internal aqueous space. Liposomes may be multilayer or single-layer. Liposomes are considered to have a diameter in the range of 5 to 10 μM to nanoparticle size. In certain embodiments, liposome nanoparticles have a diameter of about 50 to 1000 nm, about 100 nm to 600 nm, about 200 to 600 nm, about 200 to 500 nm, or about 100 to 300 nm, or about 100 to 200 nm. In various embodiments, liposomes or nanoparticles have a diameter of about 200 nm.

[0035] As used herein, "micelle" refers to a roughly spherical lipid-based particle in an aqueous solution, wherein the shape forms aggregates with the hydrophilic "head" region of the lipid upon contact with the surrounding solvent, isolating the hydrophobic single-tail region at the center of the micelle. Micelles contain a single lipid layer compared to liposomes, which are lipid bilayers.

[0036] As used herein, the term “emulsion” refers to a mixture of two or more liquids that are generally immiscible or cannot be mixed in their natural state. In certain embodiments, the emulsion is an oil and water emulsion.

[0037] As used herein, “therapeutic effective dose” or “effective dose” refers to an amount of compound sufficient to result in an improvement of symptoms, such as the treatment, cure, prevention, or improvement of a related medical condition, or an increase in the rate of treatment, cure, prevention, or improvement of such condition, typically providing statistically significant improvement in a treated patient population. When referring to an individual active ingredient administered alone, the therapeutic effective dose refers to that ingredient alone. When referring to combination therapy, the therapeutic effective dose refers to the combined amount of the active ingredient that produces a therapeutic effect, regardless of whether it is administered sequentially or simultaneously. In various embodiments, a therapeutically effective amount of DLPC or DPPC liposomes or particles improves one or more fibrotic symptoms, including, but not limited to, liver fibrosis, liver fat content, onset or progression of cirrhosis, onset of hepatocellular carcinoma, increased liver aminotransferase levels, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), increased serum ferritin, elevated levels of gamma-glutamyltransferase (gamma-GT), and elevated levels of plasma insulin, cholesterol, and triglycerides.

[0038] As used herein, “one or more symptoms” refer to general signs or indications that the patient is suffering from a specific pathological condition or disease. For example, NAFLD-related symptoms considered herein include, but are not limited to, hepatic fibrosis, hepatic fat content, increased hepatic aminotransferase levels, such as ALT and AST, increased serum ferritin, elevated levels of gamma-glutamyltransferase (gamma-GT), and elevated levels of plasma insulin, cholesterol, and triglycerides. Pulmonary fibrosis-related symptoms considered herein include, but are not limited to, weakness and lethargy, dry cough of unknown etiology, shortness of breath, and reduced exercise capacity. Hepatic fibrosis-related symptoms considered herein include, but are not limited to, yellowing of the skin (jaundice), fatigue, weakness, loss of appetite, itching, and bruising.

[0039] As used herein, “subject” refers to a human or non-human animal, including mammals or primates, to which the liposomes or nanoparticles described herein are administered. The subject may include animals such as dogs, cats, rats, mice, rabbits, horses, pigs, sheep, cattle, and humans and other primates.

[0040] As used herein, the term “pharmaceuticalally acceptable” refers to a compound, substance, composition, and / or form of administration suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications consistent with a reasonable benefit / risk ratio within the scope of sound medical judgment. The term “pharmaceuticalally acceptable component” refers to a vehicle in which a pharmaceutically acceptable diluent, adjuvant, excipient, or antibody-drug conjugate is administered.

[0041] Liposomes

[0042] Liposomes have been used in pharmaceutical development for over 10 years. Liposomal formulations are described in U.S. Patent No. 6,217,899; U.S. Patent Publication No. 20100021531, Lichtenberg et al., Methods Biochem Anal. 33:337-462, 1988; and G. Gregoriadis: "Liposome Technology Liposome Preparation and Related Techniques," 2nd edition, Vol. I-III, CRC Press. Liposomes for pharmaceutical use are described in Mozafari, M., Liposomes, Methods and Protocols Vol. 1, Chapter 2, V. Wessing Ed. 2010, Humana Press.

[0043] Liposomes may be multilayered or single-layered. Liposomes are considered to have a diameter in the range of 5 to 10 μM to nanoparticle size. In certain embodiments, liposome nanoparticles have a diameter of about 50 to 1000 nm, about 100 nm to 600 nm, about 200 to 600 nm, about 200 to 500 nm, or about 100 to 200 nm. Liposome size may be measured using methods known in the art, including the use of a Zetasizer (Malvern Instruments, United Kingdom) which measures particle size as the average diameter value of all particles by a dynamic light scattering method.

[0044] In various embodiments, the liposome comprises lipids, and optionally sterols. In various embodiments, the liposome comprises phosphatidylcholine derivatives, such as DLPC and DPPC, and optionally, cholesterol.

[0045] Phosphatidylcholine is present in liposomes and is considered to be administered at concentrations of about 2 to 50 mg / kg, 5 to 40 mg / kg, 10 to 30 mg / kg, 15 to 25 mg / kg, or 5 to 20 mg / kg per dose. DLPC, DPPC, and / or other phosphatidylcholines with 16 or 12 carbon tails are considered to be administered at concentrations of about 2 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 75 mg / kg, or 100 mg / kg. In various embodiments, phosphatidylcholine (PC) is present in liposomes or nanoparticles at a total PC concentration of about 0.1 to 100 mg / ml, about 1 to 80 mg / ml, about 5 to 70 mg / ml, about 10 to 60 mg / ml, about 15 to 100 mg / ml, about 15 to 50 mg / ml, about 20 to 80 mg / ml, about 20 to 70 mg / ml, about 30 to 60 mg / ml, or about 40 to 50 mg / ml. In various embodiments, the total PC concentration is about 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, or 100 mg / ml. In various embodiments, the total PC that can be delivered per single dose delivered IV is about 20,000 mg, 15,000 mg, 10,000 mg, 8,000 mg, 6,000 mg, 4,000 mg, 3,000 mg, 2,000 mg, 1,000 mg, 750 mg, 500 mg, 400 mg, 200 mg / ml, or 100 mg.

[0046] In various embodiments, the liposome or nanoparticle further comprises cholesterol. The cholesterol may be in the liposome or nanoparticle in the range of about 1 to 20 mg / kg, about 2 to 15 mg / kg, or about 5 to 10 mg / kg. In various embodiments, the cholesterol is present in the liposome or nanoparticle in the range of about 1 to 20 mg / ml, or about 2 to 15 mg / ml, or about 5 to 10 mg / ml, or about 1 to 5 mg / ml, or about 1 to 3 mg / ml, or 0.5 to 5 mg / ml. In various embodiments, the cholesterol concentration is about 1.0 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 5 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 7.5 mg / ml, 8.0 mg / ml, 8.5 mg / ml, 9.0 mg / ml, 9.5 mg / ml, 10.0 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml.

[0047] Vitamin E is a fat-soluble moiety and has shown some efficacy as an antioxidant, but the theory in the literature hypothesizes that vitamin E works better when used in combination with other approaches. In various embodiments, liposomes comprise DLPC and / or DPPC as a lipid-solubilized mixture with vitamin E or any other antioxidant.

[0048] Exemplary antioxidants are, but not limited to, tocopherols, tocotrienols, alpha-tocopherols, beta-tocopherols, gamma-tocopherols, delta-tocopherols, alpha-tocotrienols, beta-tocotrienols, alpha-tocopherolquinones, trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, isoflavones, lycopene, beta-carotene, selenium, ubiquinones, ruetin, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine, citric acid, L-carnitine, BHT, monothioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione, cystamine, and It includes cystathionine and glycine-glycine-histidine (tripeptide). In various embodiments, the antioxidant is a fat-soluble antioxidant.

[0049] In various embodiments, the concentration of vitamin E in the liposomes is about 1 to 20 mg / ml, about 5 to 25 mg / ml, about 10 to 15 mg / ml, or about 2 to 10 mg / ml, about 4 to 10 mg / ml, or about 4 to 8 mg / ml.

[0050] Another approach to restoring liver function may be to stimulate new cell growth. Lipid particles or liposomes may serve as carriers for aqueous solutions. One growth hormone, referred to as GHK, a liver growth peptide, can stimulate liver stem cell growth and is also a potent antioxidant. In various embodiments, liposomes are useful for delivering stem cell growth factors to tissues, such as the liver. In various embodiments, the growth factor is a liver growth factor. In various embodiments, the stem cell growth factor is a protein or a peptide. In various embodiments, the peptide is a GHK peptide. In various embodiments, the stem cell growth factor is contained within a liposome, micelle, or emulsion.

[0051] In various embodiments, the liposome is considered to be an antifibrotic agent and / or contains an antifibrotic agent. In various embodiments, the antifibrotic agent is lipid-soluble. Antifibrotic agents considered for use herein are described in more detail below.

[0052] Additionally, the liposomes may contain stabilizers or other inert substances, including, but not limited to, polyethylene glycol, PLGA, detergents, and / or surfactants. In various embodiments, PEG in the liposomes is present at a concentration of about 2 to 20 mg / ml, about 5 to 15 mg / ml, or about 4 to 10 mg / ml.

[0053] In various embodiments, the liposomes may have the following composition based on weight % composition: DLPC 10 to 50%, DPPC 10 to 50%, cholesterol 0 to 5%, vitamin E or other antioxidant 5 to 20%, and optionally PEG 0 to 15%. Combinations of components within these ranges are useful for manufacturing the liposomes of the present invention and are considered to be specifically provided by the present disclosure. Liposome content may be measured by HPLC or other analytical techniques used to determine the chemical content of the composition.

[0054] How to use

[0055] The liposomes disclosed herein are considered useful for treating fibrosis and fibrosis-related conditions or pre-fibrotic conditions, such as fatty liver disease, NASH, or other related diseases. Previous studies have described that soybean lecithin extracts containing primarily polyenylphosphatidylcholine (PPC) species prevent alcohol-induced liver cirrhosis in baboons (Leiber et al., Gastroenterology 106:152-159, 1994). Lecithin extracts were administered directly to baboons along with their diet. It is hypothesized that the DLPC liposomes of the present invention target the liver or other tissues due to DLPC or DPPC binding to LRH-1 and / or PPAR alpha.

[0056] DLPC, DPPC, and vitamin E have previously been identified as therapeutic agents for NASH. Clinical trials evaluating oral vitamin E supplementation in NASH have been completed (Galli et al., Free Radical Biology and Medicine 2017; 102 : 16 - 36). Both DLPC and DPPC were evaluated alone and in combination with other phospholipids in preclinical animal models of liver fibrosis (primates, rodents, and baboons). For example, Brady et al., Biochem Biophys Res Com 1998; 248 : 174 - 179; Navder et al., Biochem Biophys Res Com 2002; 291 : 1109 - 1112; Lieber et al ., Hepatology 1990; 12 : 1390 - 98; Lieber et al., Alcohol Clin Exp Res 1997: 21 : 375 - 379; Musille et al. Nat Struct Mol Biol ; 19 :532-S2, and Chakravarthy et al., Cell 2009; 138 Refer to : 476 - 488.

[0057] Animal studies of DLPC have demonstrated that DLPC can alleviate the inflammatory processes that cause liver fibrosis by binding to and modulating the action of an intracellular receptor known to play a pivotal role in fibrosis: liver receptor homologue-1 (LRH-1) (Musille et al. above). In these preclinical studies, DLPC was administered orally with food. DLPC has also been previously studied in human clinical trials using an oral administration route. In these studies, DLPC was found to have no clinically significant effects due to its low oral bioavailability.

[0058] Alpha-tocopherol (Vitamin E) is a fat-soluble vitamin that acts as a chain-breaking antioxidant to terminate free radical-dependent peroxidation reactions. Specifically, Vitamin E has been shown to prevent the peroxidation of polyunsaturated lipids in hepatocyte membranes. Vitamin E has been shown to have cytoprotective effects in models of liver fibrosis and, more recently, has been studied in several randomized trials of patients with non-alcoholic fatty liver disease (NAFLD) and NASH. These trials suggest that biopsy-proven NASH patients may benefit from oral Vitamin E supplementation; however, these patients demonstrated improvements in hepatic steatosis and lobular inflammation in Vitamin E trials, but no improvement in portal vein inflammation or fibrosis. Current guidelines from the American Association for Liver Research recommend Vitamin E supplementation as a first-line therapy for the treatment of biopsy-proven NASH patients (Galli et al. cited above).

[0059] In various embodiments, the subject suffers from fibrosis or a disease or condition that causes or is associated with fibrosis. In various embodiments, the disease or condition is non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis, hepatitis, cirrhosis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), or interstitial pulmonary fibrosis.

[0060] Non-alcoholic fatty liver disease (NAFLD) refers to various conditions that occur in the absence of alcohol abuse. It is characterized by the presence of steatosis (fat in the liver) and can manifest hepatic symptoms of metabolic syndrome (including obesity, diabetes, and hypertriglyceridemia). NAFLD is associated with insulin resistance, which causes liver disease in adults and children and can ultimately lead to cirrhosis (Skelly et al., J Hepatol 2001; 35: 195-9; Chitturi et al., Hepatology 2002; 35(2):373-9). The severity of NAFLD varies from relatively benign isolated, primarily macrovesicular steatosis (i.e., non-alcoholic fatty liver or NAFLD) to non-alcoholic steatohepatitis (NASH) (Angulo et al., J Gastroenterol Hepatol 2002; 17 Suppl:S186-90). NASH is characterized by the histological presence of steatosis, cellular ballooning, scattered inflammation, and pericellular fibrosis (Contos et al., Adv Anat Pathol 2002; 9:37-51). Hepatic fibrosis caused by NASH can progress to cirrhosis of the liver or liver failure, and occasionally lead to hepatocellular carcinoma.

[0061] The mechanism by which NAFLD develops into NASH is unclear. Since insulin resistance is associated with both NAFLD and NASH, it is hypothesized that additional factors are required for NASH to develop. This is referred to as the "2-hit" hypothesis (Day C P., Best Pract Res Clin Gastroenterol 2002; 16(5):663-78) and is associated with the accumulation of hepatic fat and the presence of large amounts of free radicals accompanied by increased oxidative stress. Macrovesicular steatosis indicates the hepatic accumulation of triglycerides, which is ultimately due to an imbalance between the delivery of free fatty acids to the liver and their utilization. During periods of increased caloric intake, triglycerides accumulate and act as a reserve energy source. When dietary calories are insufficient, stored triglycerides (in the form of fat) undergo lipolysis, and fatty acids are released into circulation and absorbed by the liver. The oxidation of fatty acids will generate energy for utilization.

[0062] Treatment of NASH currently focuses on reducing two major pathogenic factors: fat accumulation in the liver and the excessive accumulation of free radicals that cause oxidative stress. Reduction of oxidative stress through antioxidant treatment has been shown to be effective in some studies. For example, obese children with steatosis were treated with vitamin E (400–1000 IU / day) for 4 to 10 months (Lavine J Pediatr 2000; 136(6):734-8). Despite any significant change in BMI, mean ALT levels decreased from 175 ± 0.106 IU / L to 40 ± 0.26 IU / L (P<0.01), and mean AST levels decreased from 104 ± 0.61 IU / L to 33 ± 0.11 IU / L (P<0.002). In patients who chose to discontinue vitamin E therapy, liver transaminases increased. A study of adults using vitamin E for one year showed a similar reduction in liver transaminases as well as the fibrosis marker TGFβ levels (Hasegawa et al., Aliment Pharmacol Ther 2001; 15(10):1667-72). However, not all treatments with ROS scavengers were effective.

[0063] Steatosis can also develop into steatohepatitis through oxidative stress caused by reactive oxygen species (ROS) and reduced antioxidant defenses (Sanyal et al., Gastroenterology 2001; 120(5):1183-92). ROS can be generated in the liver through various pathways involving mitochondrial oxidation, peroxisomes, cytochrome P450, NADPH oxidase, and lipooxygenase (Sanyal et al., Nat Clin Pract Gastroenterol Hepatol 2005; 2(1):46-53). Insulin resistance and hyperinsulinism have been shown to increase hepatic oxidative stress and lipid peroxidation through increased hepatic CYP2EI activity (Robertson et al., Am J Physiol Gastrointest Liver Physiol 2001; 281(5):G1135-9; Leclercq et al., J Clin Invest 2000; 105(8):1067-75).

[0064] NAFLD can be distinguished from NASH by the NAFLD activity score (NAS), which is the sum of the histopathological scores of a liver biopsy for steatosis (0 to 3), lobular inflammation (0 to 2), and hepatocellular ballooning (0 to 2). A NAS of less than 3 corresponds to NAFLD, 3 to 4 corresponds to borderline NASH, and 5 or more corresponds to NASH. A score for fibrosis (0 to 4) is also assigned to the biopsy.

[0065] Fibrosis can also be measured by non-invasive methods using magnetic resonance elastography (MRE) and FIBROSCAN® ultrasound / transient elastography.

[0066] In various embodiments, administration slows the progression or reduces the occurrence of steatosis, lobular inflammation, portal vein inflammation, and / or hepatic ballooning in subjects with NASH.

[0067] Fibrosis is a pathological process that occurs when the body's natural healing process deteriorates, resulting in the overproduction of extracellular matrix (ECM) and scar formation in response to tissue damage. Fibrosis formation involves interactions between many cell types and cytokines, and fibrosis develops when this balance is disrupted. In some embodiments, fibrosis is fibrosis of the lungs, heart, blood vessels, liver, gallbladder, kidneys, skin, muscles, pancreas, eyes, adrenal glands, thyroid, or other organs of the body. The causes of some fibrotic diseases are currently unknown or not well understood. Fibrosis is typically considered an irreversible process.

[0068] Chronic pulmonary fibrosis results from scarring of the entire lung, which can be triggered by numerous conditions including chronic inflammatory processes (sarcoidosis, Wegener's granulomatosis), infections, environmental agents (exposure to asbestos, silica, and certain gases), exposure to ionizing radiation (e.g., radiation therapy to treat chest tumors), chronic diseases (lupus, rheumatoid arthritis), and even certain medications. In a condition known as hypersensitivity pneumonitis, pulmonary fibrosis can occur after an enhanced immune response to inhaled organic dust or occupational chemicals. These conditions are mostly caused by the inhalation of dust contaminated with bacteria, fungi, or animal products. Currently, treatment options for fibrotic diseases are limited. While prednisone and azathioprine are standard clinical practice, there is no data demonstrating that these drugs provide therapeutic benefits.

[0069] Additional fibrotic disorders include chronic obstructive pulmonary disease, hepatic fibrosis, rheumatoid arthritis, congestive heart failure, chronic renal failure, hypersensitivity pneumonitis, respiratory bronchiolitis / interstitial lung disease, Schistosomiasis mansoni infection, primary pulmonary arterial hypertension (prevention of pyloric lesion formation), herpes virus-associated disease, lung and skin signs; keloid scars, lupus, nephrogenic fibrodermal disease, fibrotic lesions associated with Schistosomiasis japonica infection, autoimmune diseases, pathogenic fibrosis, Lyme disease, stromal modification and stromal fibrosis in pancreatitis, uterine fibrosis, ovarian fibrosis, corneal fibrosis, congestive heart failure and other post-ischemic conditions, abdominal adhesions, wide-angle glaucoma, postoperative scars including trabeculotomy and any combination thereof, or scars caused by drugs or radiation therapy.

[0070] In various embodiments, administration improves one or more symptoms of the fibrotic disease described herein, including, but not limited to, liver fibrosis, liver fat content, increased liver aminotransferase levels, e.g., ALT and AST, increased serum ferritin, elevated levels of gamma-glutamyltransferase (gamma-GT), and elevated levels of plasma insulin, cholesterol and triglycerides, dry cough of unknown etiology, shortness of breath, reduced exercise load, yellowing of the skin (jaundice), fatigue, weakness, loss of appetite, itching, and bruising.

[0071] The lipid particle composition is further considered to be administered together with a second agent useful for treating NASH or other aspects of fibrosis. The second agent may be other therapeutic agents, such as antidiabetic agents, cytokines, growth factors, other anti-inflammatory agents, anticoagulants, agents that lower or reduce blood pressure, agents that reduce cholesterol, triglycerides, LDL, VLDL, or lipoproteins or increase HDL, agents that increase or decrease levels of cholesterol-regulating proteins, or anti-neoplastic drugs or molecules.

[0072] An exemplary second agent comprises a agent used to treat NASH or diabetes mellitus, cyclophosphamide, alone or in combination with mycophenolate mofetil (MMF) or prednisolone, or other corticosteroids, anti-inflammatory agents, azathioprine, or antifibrotic agents, to a non-limiting extent.

[0073] Exemplary antidiabetic agents are, without limitation, sulfonylureas (e.g., glimepiride, glycentide, sulfonylurea, AY31637); biguanides (e.g., metformin); alpha-glucosidase inhibitors (e.g., acarbose, miglitol); Thiazol-idinediones (e.g., troglitazone, pioglitazone, rosiglitazone, glipizide, valaglitazone, riboglitazone, netoglitazone, troglitazone, englitazone, AD 5075, T 174, YM 268, R 102380, NC 2100, NIP 223, NIP 221, MK 0767, siglitazone, adaglitazone, CLX 0921, darglitazone, CP 92768, BM 152054); glucagon-like peptides (GLP) and GLP analogs or agonists of GLP-1 receptors (e.g., exendin) or stabilizers thereof (e.g., DPP4 inhibitors such as sitagliptin); Insulin or its analogs or mimics (e.g., LANTUS), or thyroid hormone receptor beta agonists (including Sobetirome, Eprotirome, MB07811 (2R, 4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl l)phenoxy)methyl]-2-oxydo-[1-3]-dioxaphosphonan, MGL3196 2-[3,5-dichloro-4-(5-isopropyl-6-oxo-1,6-dihydropyridazine-3-yloxy)phenyl]-3,5-dioxo-2,3,4,5-tetrahydro[1,2,4]triazine-6-carbonitrile, e.g. Jakobsson et al., Drugs. 2017; See 77(15): 1613-1621).

[0074] Antifibrotic agents considered for use in the method of the present disclosure may be any agent affecting fibrosis. Agents considered include, but are not limited to, those that reduce the activity of transforming growth factor-beta (TGF-β) (but include, but are not limited to, GC-1008 (Genzyme / MedImmune); ledelimumab (CAT-152; Trabio, Cambridge Antibody); metelimumab (CAT-192, Cambridge Antibody); LY-2157299 (Eli Lilly); ACU-HTR-028 (Opko Health), comprising antibodies targeting one or more TGF-β isoforms, inhibitors of TGF-β receptor kinases TGFBR1 (ALK5) and TGFBR2, and modulators of post-receptor signaling pathways; chemokine receptor signaling; Endothelin receptor anti-agonists, including inhibitors targeting both endothelin receptors A and B and those selectively targeting endothelin receptor A (including, but not limitedly, ambrisentan; avosentan; bosentan; clazosentan; darusentan; BQ-153; FR-139317, L-744453; macitentan; PD-145065; PD-156252; PD163610; PS-433540; S-0139; citaxentan sodium; TBC-3711; and gibbotentan); agents that reduce the activity of connective tissue growth factor (CTGF) (including, but not limitedly, FG-3019 and FibroGen), and also other CTGF-neutralizing antibodies; matrix metalloproteinase (MMP) inhibitors (including, but not limitedly, MMPI-12, PUP-1, and tigapotide trilutetate); Agents that reduce the activity of the epidermal growth factor receptor (EGFR), including, but not limitedly, erlotinib, gefitinib, BMS-690514, cetuximab, EGF receptor-targeting antibodies, EGF receptor kinase inhibitors, and post-receptor signaling pathway modulators;Agents that reduce the activity of platelet-derived growth factor (PDGF) (including, but not limited to, imatinib mesylate (Novartis)) and also include PDGF neutralizing antibodies, PDGF receptor (PDGFR) targeting antibodies, PDGFR kinase activity inhibitors, and post-receptor signaling pathways; agents that reduce the activity of vascular endothelial growth factor (VEGF) (including, but not limited to, axitinib, bevacizumab, BIBF-1120, CDP-791, CT-322, IMC-18F1, PTC-299, and ramucirumab) and also include VEGF-neutralizing antibodies, antibodies targeting VEGF receptor 1 (VEGFR1, Flt-1) and VEGF receptor 2 (VEGFR2, KDR), which is the soluble form of VEGFR1 (sFlt), and derivatives thereof that neutralize VEGF, and VEGF receptor kinase activity inhibitors; Multiple receptor kinase inhibitors, such as BIBF-1120 (including, but not limited to, STX-100 and IMGN-388), which inhibit receptor kinases for vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor, and also including integrin-targeted antibodies; Agents that inhibit the pre-fibrotic activity of IL-4 (including, but not limited to, AER-001, AMG-317, APG-201, and sIL-4Rα) and IL-13 (including, but not limited to, AER-001, AMG-317, anlukinzumab, CAT-354, syntredekin vesodotox, MK-6105, QAX-576, SB-313, SL-102, and TNX-650), and also include antibodies against cytokines, antibodies targeting the IL-4 receptor or the IL-13 receptor which is a soluble form of the IL-4 receptor, or chimeric proteins and toxins containing both IL-4 and IL-13, in whole or in part of IL-13, particularly derivatives thereof reported to bind to and neutralize Pseudomonas endotoxins signaled via the JAK-STAT kinase pathway; Agents that interfere with epithelial-mesenchymal transition, including mTor inhibitors (including AP-23573 to a non-limiting extent);Agents that reduce levels of copper, e.g., tetrathiomolybdate; agents that reduce oxidative stress, including N-acetylcysteine ​​and tetrathiomolybdate; and interferon gamma are also considered. Also considered are phosphodiesterase 4 (PDE4) inhibitors (including, but not limited to, roflumilast); phosphodiesterase 5 (PDE5) inhibitors (including, but not limited to, mirodenafil, PF-4480682, sildenafil citrate, SLx-2101, tadalafil, udenafil, UK-369003, vardenafil, and zaprinas); or agents that are modifiers of the arachidonic acid pathway, including cyclooxygenase and 5-lipoxeganase inhibitors (including, but not limited to, Zileuton). Additional consideration includes propyl hydrolase inhibitors (including, to a non-limiting degree, 1016548, CG-0089, FG-2216, FG-4497, FG-5615, FG-6513, fibrostatin A (Takeda), rupironil, P-1894B, and sapironil) and compounds that reduce tissue modification or fibrosis, including peroxisome proliferator-activated receptor (PPAR)-gamma agonists (including, to a non-limiting degree, pioglitazone and rosiglitazone).;

[0075] Other specific antifibrosis agents under consideration include relaxin, pirfenidone, upironil, surifonil, TGF-β antibodies, CAT-192, CAT-158; ambresentan, thelin; FG-3019, CTGF antibodies; anti-EGFR antibodies; EGFR kinase inhibitors; tarceva; gefitinib; PDGF antibodies, PDGFR kinase inhibitors; Gleevec; BIBF-1120, VEGF, FGF, and PDGF receptor inhibitors; anti-integrin antibodies; IL-4 antibodies; tetrathiomolybdate, copper chelating agents; interferon-gamma; NAC, cysteine ​​prodrugs; hepatocyte growth factor (HGF); KGF; angiotensin receptor blockers, ACE inhibitors, renin inhibitors; COX and LO inhibitors; zileutone; monteleucast; Avastin; statins; PDE5 inhibitors, e.g., sildenafil, udenafil, tadalafil, vardenafil, or zaprinast; rofumilast; etanercept (Enbre); anticoagulants; prostaglandins, e.g., PGE2, PRX-08066, 5HT2B receptor antagonists; syntredegin vesodotox, chimeric human IL13 conjugated to genetically engineered Pseudomonas endotoxin; roflumilast, PDE4 inhibitors; FG-3019, anti-connective tissue growth factor human monoclonal antibody; GC-1008, TGF-β human monoclonal antibody; treprostinil, prostacyclin homologues; interferon-α; QAX-576, IL13 modulators; WEB 2086, PAF receptor antagonists; imatinib mesylate; FG-1019; suramin; Includes bosentan; IFN-1b; anti-IL-4; anti-IL-13; taurine, niacin, NF-κB antisense oligonucleotide; and nitric oxide synthase inhibitor.

[0076] The lipid particles considered herein, such as liposomes, micelles, or emulsions, may be administered by several routes, such as parenteral, topical, intranasal, inhalation, or oral administration. In one embodiment, the composition is suitable for parenteral, such as intravenous administration. In one embodiment, the composition is suitable for topical administration, such as by a transdermal patch. The lipid particles may also be administered via a drug depot.

[0077] manufacturing

[0078] A method for manufacturing liposomes or nanoparticles as described herein is also provided.

[0079] In various embodiments, liposomes are prepared from a natural mixture of phosphatidylcholine or purified synthetic phosphatidylcholine. Liposomes can be manufactured by commercial laboratories. In certain methods, liposomes are prepared by a microfluidic ethanol injection version, which enables optimal control of liposome production. For example, the process may be a single-step sterile process. Liposome size is adjusted by controlling process parameters during vesicle formation. A closed system is used for production. All components can be added via sterile filtration. Subsequent concentration by cross-flow filtration is also possible.

[0080] This method provides homogeneous and uniform parcels with excellent consistency between batches.

[0081] All ingredients used during the manufacturing process are tested to determine purity, and analytical tests such as endotoxin, pH, and visual inspection are performed to verify quality control. These include using HPLC to determine the amount of each phospholipid in the liposome wall.

[0082] Formulation

[0083] Various delivery systems may be used to administer the liposomes or nanoparticles of the present invention. In various embodiments of the present invention, the administration of the liposome composition is by intravenous infusion. In some embodiments, the administration is by intravenous infusion over 30 minutes, 1 hour, or 2 hours.

[0084] The liposomal composition may be administered as a pharmaceutical composition comprising one or more pharmaceutically compatible components. For example, the pharmaceutical composition typically comprises one or more pharmaceutically acceptable carriers, such as aqueous carriers (e.g., sterile liquids).

[0085] If desired, the composition may also contain, for example, saline solution, buffer solution, salt, nonionic detergent, and / or sugar. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0086] The present invention provides a pharmaceutical composition comprising, for example, a therapeutically effective amount of liposomes or nanoparticles, a buffer, optionally a cryopreservative, optionally an extender, optionally a salt, and optionally a surfactant. Additional agents may be added to the composition. A single agent may provide multiple functions. For example, a sugar, such as trehalose, may act as both a cryopreservative and an extender. Any suitable pharmaceutically acceptable buffer, surfactant, cryopreservative, and extender may be used according to the present invention.

[0087] The formulation of the present invention may be used in combination with the method described herein or other methods for treating diseases.

[0088] Kit

[0089] In a further aspect, the present invention comprises a kit comprising one or more liposomes, nanoparticles, or compositions packaged in a manner that facilitates their use in carrying out the method of the present invention. In one embodiment, such a kit comprises the compound or composition described herein (e.g., a composition comprising liposomes or nanoparticles alone or in combination with an antifibrotic agent) packaged in a container such as a sealed bottle or container, which includes a label attached to the container or included in the package describing the use of the compound or composition in carrying out the method. Preferably, the compound or composition is packaged in a unit dose form. The kit may further comprise a device suitable for administering the composition or performing screening analysis according to a specific route of administration. Preferably, the kit includes a label describing the use of the liposomes or nanoparticles.

[0090] Further aspects and details of the serum replacement of the present invention will become apparent from the following examples, which are intended to be exemplary rather than limiting.

[0091] Examples

[0092] Example 1 - Liposomes containing phosphatidylcholine reduced hepatic erythematosus in mice

[0093] While there are several mouse models for liver disease, no single model perfectly mimics human liver disease. Feeding animal models alter the diet of subject animals, for example, by feeding a high-fat diet or alcohol, or by causing cirrhosis over time. Hepatotoxicity animal models, such as carbon tetrachloride (CCl4), induce liver damage and fibrosis due to the toxic effects of the administered agent (Trautwein et al., J Hepatology 62:S15-S24, 2015; Popov and Schuppan, Hepatology 50:1294-1305, 2009). There are also genetic animal models that simulate NASH when combined with dietary therapy. In any mouse model, measurements of fibrosis and / or lipid dysregulation are indicators of the disease. Current treatments are expected to reduce fibrosis and decrease liver enzymes.

[0094] Rat and rabbit models for NASH also exist. Both of these animal models utilize a dietary method to induce liver disease. In the dietary model, animals gain body weight and exhibit fibrosis and elevated liver enzymes due to a high-fat diet. The liposome treatment of the present invention will reduce fibrosis and decrease liver enzymes in these animals.

[0095] Materials and Methods:

[0096] Animal Information: BALB / C male mice were fed a standard rodent diet (Harlan irradiated 2918) consisting of randomly supplied water, 18% protein, 5% fat, and 5% fiber. Microscopic and macroscopic environmental factors, such as ventilation, temperature, and humidity, were recorded daily by various staff members and tracked in documentation. Starting two weeks after CCl4 treatment, animals were administered either a vehicle control or liposomes. The experiment was terminated within 24 hours after 28 days of administration.

[0097] CCl4 Disability: To induce liver fibrosis, a 20% solution of 0.5 μl / g was administered to corn or olive oil-formulated carbon tetrachloride (CCl4) 2 to 3 times a week for 6 weeks.

[0098] Liposome: Liposomes were prepared using the ethanol drop method commonly used in the industry. Briefly, liposome formation was induced by adding a mixture of DPPC and DLPC dropwise with ethanol. The liposomes consisted of phosphatidylcholine and vitamin E. Optionally, the liposomes may contain 5 mg / kg cholesterol.

[0099] Therapeutic administration: To induce fibrosis, 2 weeks after CCl4 administration, mice were divided into n=8 / group: untreated control animals in CCl4+ saline, treatment group I, which received liposome treatment 2x / week via intraperitoneal (ip), and treatment group II, which received liposome treatment 3x / week via ip. Mice were administered a total of 20 mg / kg / week or 30 mg / kg / week at a dose of 10 mg / kg liposomes (containing 5 mg / kg DPPC and 5 mg / kg DLPC).

[0100] Research End: After 4 weeks of administration, mice were sacrificed using the CO2 inhalation method. The liver was isolated, weighed, and cut into four large sections of 200 to 300 mg each and two small sections of 50 to 100 mg each. For analysis, the large lobes were fixed in formalin and the smaller sections were snap-frozen.

[0101] Blood collection: At the start of the study, n=4 / 24 (baseline) blood was collected via intraperitoneal or submandibular venipuncture (estimated serum volume 30 to 50 μl). Terminal blood collected by cardiac perforation.

[0102] Biomarker Analysis and Tissue Scoring:ALT and AST enzyme levels were measured in serum from animals. Liver samples (formalin-fixed tissue blocks) were treated with H&E and Picrosirius Red staining and then scored for fibrotic pathology.

[0103] result

[0104] Analysis of pathology scores for treated and untreated CCl4 animals showed that administration of liposomes containing DLPC (Bastet101) therapeutically reduced the severity of fibrosis in treated animals (Fig. 1). Untreated animals showed an average histological score of approximately 2, while liposome-treated animals had a pathology score of approximately 1.5, showing a statistically significant difference between the treated and untreated groups (p value ≤ 0.0009). Fig. 2 shows representative histological slides of Sirius Red (a stain specific to collagen deposition in fibrosis) in control or treated liver tissues. Treatment with the liposomes of the present invention was shown to prevent CCl4-induced liver fibrosis without causing any hepatotoxicity, as indicated by the levels of ALT, AST, and total bilirubin in control and treated animals (Fig. 3).

[0105] Example 2 - Therapeutic use in human subjects with NALFD or liver fibrosis

[0106] The liposome therapy of the present invention is designed for human use. The therapy is useful, but not limited to, for individuals with liver diseases, including NASH and alcoholic cirrhosis. Disease markers are similar for both increased fibrosis and liver stiffness measured by Fibroscan. Liver biopsy is also used to diagnose liver disease.

[0107] To evaluate the safety and tolerability of liposomes, subjects receiving treatment are 18 years of age or older with biopsy-proven non-alcoholic steatohepatitis (NASH) with a histopathological NASH stage greater than 1 and a fibrosis score greater than 3. Subjects are divided into the following groups: 1) Subjects will receive a placebo infusion of 130 mL of 5% dextrose every 4 weeks; 2) Subjects will receive an IV infusion of 130 mL of liposomes as a 1x dose once every 4 weeks during the study period; 3) Subjects will receive 130 mL of liposomes as a 2x dose once every 4 weeks during the study period. Every 6 weeks, each subject will undergo a medical history, physical examination, and repeated blood counts at baseline. Every 4 months, magnetic resonance elastography (MRE) of the liver and FIBROSCAN® of the liver will be repeated.

[0108] It is hypothesized that targeted therapy reduces fibrosis and associated inflammation in human subjects, which can be directly measured by MRE, FIBROSCAN®, or liver biopsy. For example, treatment with the liposomes of the present invention is expected to slow or reduce the progression of steatosis, lobular inflammation, portal vein inflammation, and / or hepatocellular ballooning in subjects with NASH. The therapy may also reduce liver enzymes (ALT / AST, bilirubin) and potentially increase serum albumin. In some severe cases, the therapy will also reduce associated abdominal ascites.

[0109] Example 3 - Liposome Preparation

[0110] Additional liposomal compositions have been developed for therapeutic administration. The liposomal formulations comprise two phospholipids, cholesterol and alpha-tocopherol (vitamin E, VITE), and optionally polyethylene glycol (PEG). The phospholipids are 1,2-dilauryl-sn-glycerol-3-phosphocholine (DLPC) and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC). In one embodiment, the aqueous interior of the liposome contains 0.9% normal saline. The liposome may contain components in the following proportions:

[0111]

[0112] The content of the liposome of the present invention is compared below with that of other liposomes currently used for drug delivery, indicating that the liposome of the present invention has a composition different from that of previously known liposomes, namely AMBISOME® used for the delivery of amphotericin B and DOXIL® used for the delivery of doxorubicin.

[0113]

[0114] Many modifications and variations of the present invention, as set forth in the exemplary embodiments above, are expected to occur to those skilled in the art. Consequently, only these limitations set forth in the appended claims should apply to the present invention.

Claims

Claim 1 A composition for treating fibrosis comprising phosphatidylcholine (PC) at a concentration of 20 mg / ml to 100 mg / ml in liposomes or nanoparticles, wherein the liposomes or nanoparticles comprise (i) 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or a choline head with a phospholipid tail of 12 carbons in 10 to 50 weight% of the composition; and (ii) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or a choline head with a phospholipid tail of 16 carbons in 10 to 50 weight% of the composition. Claim 2 A composition according to claim 1, wherein i) the phosphatidylcholine is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or ii) the phosphatidylcholine is 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). Claim 3 A composition according to claim 1 or 2, wherein the liposome or nanoparticle further comprises an antioxidant. Claim 4 The composition of claim 1 or 2, wherein the liposome or nanoparticle further comprises: i) vitamin E; ii) GHK peptide; and / or iii) cholesterol. Claim 5 A composition according to claim 1 or 2, wherein phosphatidylcholine binds to LRH-1 and / or PPAR alpha. Claim 6 A composition according to claim 1 or 2, wherein the liposomes or nanoparticles are administered by injection, transdermal, oral, depot, or inhalation. Claim 7 A composition according to claim 1 or 2, wherein the subject has a fibrotic disease, non-alcoholic fatty liver disease (NASH), non-alcoholic fatty liver disease (NAFLD), hepatitis, liver cirrhosis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), or interstitial pulmonary fibrosis. Claim 8 A composition according to claim 1 or 2, wherein the liposomes or nanoparticles are administered weekly, once every two weeks, once every three weeks, monthly, or once every two months. Claim 9 A composition according to claim 1 or 2, wherein the liposome or nanoparticle further comprises a pharmaceutically acceptable excipient, carrier, or diluent. Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete

Images