EA047345B1 - CAROTENOID COMPOSITION AND METHOD FOR ITS OBTAINING - Google Patents

Description

Cross reference to related applications

This application claims the benefit of U.S. Provisional Applications No. 62/666699, filed May 3, 2018, and 62/809123, filed February 22, 2019, each of which is incorporated herein in its entirety.

State of the art

Under normal conditions, bacteria and associated toxins (endotoxicosis) should not normally be present in the blood of healthy people. However, there is increasing recognition that bacteria do enter the bloodstream under certain conditions from sources such as the gut (leaky gut syndrome) or the tooth-gum interface (mainly in patients with poor gum health). The consequences of this process, known as bacterial translocation, are a state of chronic bacteremia (bacteria) in the blood stream. Bacterial translocation leads to chronic bacteremia, which generates endotoxins such as bacterial lipopolysaccharides, LPS, which leads to chronic low-grade endotoxemia and the resulting chronic inflammatory state. This mild chronic endotoxemia associated with bacterial translocation has also been associated with the pathogenesis of many diseases.

The more severe form of endotoxemia is associated with sepsis, a life-threatening medical condition caused by a deregulated host inflammatory response to infection. Sepsis is a global health problem that affects approximately 30 million people worldwide each year. The mortality rate from sepsis in adults is approximately 40%. Sepsis occurs when the body's attempt to fight an infection causes the immune system to damage tissues and organs. This uncontrolled response, usually intended to protect the body, causes widespread inflammation, leaky blood vessels and impaired blood clotting, leading to organ damage. In severe cases, blood pressure drops, followed by multiple organ failures, and the patient may quickly die from septic shock.

Management of sepsis is a complex and unmet clinical challenge, requiring early detection and management of infection, hemodynamic problems, and other organ dysfunctions. Infections that cause sepsis are treated with antimicrobial agents, most often a broad spectrum of antibacterial, antiviral and antifungal agents. Current treatment guidelines for the management of hemodynamic problems associated with sepsis and septic shock recommend the use of vasopressors with norepinephrine as first-line treatment.

Despite these measures discussed above, sepsis remains a leading cause of death, and there remains a great need for new treatments for sepsis and related diseases. One observation is that most sepsis-targeted treatments tend to target the infection and individual organ system failure rather than the key underlying pathophysiologic drivers of sepsis. An alternative approach may target the mechanisms underlying sepsis in addition to treating co-infection.

Sepsis, along with many other diseases, is associated with oxygen starvation (hypoxia). The leading causes of death worldwide are related to some extent to hypoxia. Examples include, but are not limited to, coronary artery disease, stroke, chronic and acute respiratory disease. Additionally, hypoxia is a common feature of many cancers and leads to resistance to radiation therapy, chemotherapy, and potentially immunotherapy. Preclinically, relief of hypoxia in cancer has been associated with improved response to treatment. This suggests that clinical strategies to manage hypoxia may lead to improved outcomes in cancer.

Carotenoids are a class of natural fat-soluble pigments found primarily in plants, where they function as accessory pigments and impart tissue protection through their ability to quench singlet oxygen and free radicals. Carotenoids are known to have antioxidant properties and therefore provide numerous beneficial health effects, including reducing the potential risk of cardiovascular disease, cancer, and slowing and/or reversing the degenerative effects of aging in various human physiological processes. However, carotenoids tend to be highly lipophilic compounds, and the clinical use of many carotenoids is limited by their instability and low bioavailability.

Crocetin is a carotenoid with antioxidant properties that is poorly soluble in water. Chemically, crocetin is a 20-carbon apocarotenoid molecule containing seven double bonds and a carboxylic acid group at each end. Administration of pharmaceutical formulations of trans-crocetin (free acid), and its salt, trans-crocetinate sodium, in free form (eg, unencapsulated) has been reported to be promising in the treatment of diseases caused by hypoxia, ischemia, and other diseases. However, none have demonstrated clinical therapeutic efficacy. This is partly due to the fact that

- 1 047345 positions of trans-crocetin and its sodium salt, sodium trans-crocetinate, have so far been limited by instability, low bioavailability and short half-life.

In view of the health benefits provided by carotenoids and the low bioavailability and instability described above, there is a need to provide pharmaceutical compositions containing carotenoids with improved bioavailability and stability. Compositions and methods for eliminating the disadvantages of carotenoids described above are proposed. These compositions and methods will further help overcome the limitations of current therapeutic approaches for disease states associated with endotoxemia and hypoxia, as well as other unmet medical needs. These compositions have use both as single agents and in combination with other treatments.

Brief description of the invention

Provided herein are pharmaceutical compositions containing carotenoids, including liposomes that encapsulate ionizable salts of carotenoids such as transcrocetin and trans-norbixin. The present compositions have applications in the treatment of diseases, disorders and conditions associated with, but not limited to, infection, inflammation, sepsis, ischemia, hypoxia, shock, stroke, trauma, cardiovascular disease, kidney disease, liver disease, inflammatory disease, metabolic disease, pulmonary disease, neurodegenerative disease, immune system disease and hyperproliferative diseases such as cancer. Methods for the manufacture, delivery and use of pharmaceutical compositions are also provided, as are kits containing these compositions.

The described pharmaceutical compositions are intended for enhanced delivery of carotenoids, including ionizable polyene carotenoids, such as trans-crocetin, with poor pharmacokinetics and biodistribution. Also provided herein are liposome compositions that exhibit high encapsulation efficiency (>98%), a high drug-to-lipid ratio, and/or increased drug retention. The proposed pharmaceutical compositions have applications in the treatment of diseases, disorders and conditions associated with, but not limited to, infection, inflammation, sepsis, ischemia, hypoxia, anemia, wound, trauma, stroke, shock, diabetes, wound healing, injury (eg, reperfusion injury, nerve injury, kidney damage, liver damage and lung injury) and hyperproliferative diseases such as cancer, as well as conditions associated with the treatment of these diseases and disorders (eg, anemia, neutropenia and immunosuppression). Methods for the manufacture, delivery and use of these compositions are also proposed.

In some embodiments of the invention, this specification provides:

[1] a pharmaceutical composition containing a liposome encapsulating an ionizable carotenoid salt having the formula Q-trans-crocetin-Q or Q-trans-norbixin-Q, where Q is a polyvalent cationic counterion;

[2] the pharmaceutical composition according to claim [1], wherein Q is a divalent metal cation, a divalent organic cation or a trivalent cation such as Fe ³⁺ ;

[3] the pharmaceutical composition according to claim [2], wherein Q is a divalent metal cation selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ ;

[4] a pharmaceutical composition according to any one of claims [1] to [3], wherein the liposome encapsulates an ionizable carotenoid salt having the formula Q-Ίrans-norbixin-Q;

[5] the pharmaceutical composition according to any one of claims [1] to [3], wherein the liposome encapsulates an ionizable carotenoid salt having the formula Q-Ίrans-crocetin-Q;

[6] a pharmaceutical composition according to any one of claims [1] to [3] and [5], wherein the liposome encapsulates magnesium trans-crocetinate (MTS);

[7] a pharmaceutical composition according to any one of claims [1] to [3] and [5], wherein the liposome encapsulates calcium trans-crocetinate (CTC);

[8] the pharmaceutical composition according to any one of claims [1] to [4], wherein the liposome encapsulates magnesium trans-norbixinate (MTN) or calcium trans-norbixinate (CTN);

[9] a pharmaceutical composition according to any one of claims [1] to [8], wherein the ionizable carotenoid/lipid ratio is from about 1 to 1000 g/mol lipid, from about 10 to about 150 g/mol lipid, or from about 20 up to about 100 g/mol lipid or wherein said liposomes contain at least 0.1 to 97% by weight (w/w) of an ionizable carotenoid;

[10] a pharmaceutical composition according to any one of claims [1] to [9], wherein said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, or 80 to 120 nm;

[11] a pharmaceutical composition according to any one of claims [1] to [10], wherein said liposome is formed from liposome components, wherein the liposome components include:

at least one of a cationic lipid, an anionic lipid and a neutral lipid, or at least one selected from the following: distearoylphosphatidyl-1-ethanolamine (DSPE); DSPE-polyethylene glycol (PEG); DSPE-PEG-maleimide; hydrogenated soy phosphatidylcholine (HSPC); HSPC-PEG; cholesterol; cholesterol-PEG and cholesterol-maleimide;

- 2 047345

[12] a pharmaceutical composition according to any one of claims [1] to [11], wherein said liposome contains an oxidized phospholipid such as oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3phosphorylcholine (OxPAPC);

[13] the pharmaceutical composition according to claim [11], wherein the one or more liposome components further includes at least one steric stabilizer selected from:

poly-L-lysine (PLL);

monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP);

poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidones; L-amino acid based polymer; oligoglycerin, a copolymer containing polyethylene glycol and polypropylene oxide, Poloxamer 188 and polyvinyl alcohol;

[14] a pharmaceutical composition according to any one of claims [1] to [13], wherein said liposome is anionic, neutral or cationic;

[15] a pharmaceutical composition according to any one of claims [1] to [14], wherein said liposome has a zeta potential of -150 to 150 mV or -50 to 50 mV;

[16] a pharmaceutical composition according to any one of claims [1] to [14], wherein said liposome has a zeta potential of -150 to 0 mV or -50 to 0 mV;

[17] the pharmaceutical composition according to any one of claims [1] to [16], further comprising a pharmaceutically acceptable carrier which includes an isotonic agent such as dextrose, mannitol, glycerin, potassium chloride or sodium chloride;

[18] a pharmaceutical composition according to any one of claims [1] to [17], wherein said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000 or less than 5,000 molecules or from 10 to 100,000 , from 100 to 10,000 or from 500 to 5,000 molecules of ionizable carotenoid;

[19] a pharmaceutical composition according to any one of claims [1] to [18], wherein said liposome further comprises a targeting moiety, and wherein said targeting moiety has a specific affinity for a surface antigen on a target cell of interest;

[20] the pharmaceutical composition according to claim [19], wherein said targeting fragment is a polypeptide, antibody, humanized antibody, antigen binding fragment of an antibody, single chain antibody, single domain antibody, bispecific antibody, synthetic antibody, pegylated antibody or multimeric antibody;

[21] pharmaceutical composition according to claim [19] or [20], in which the specified liposome contains from 1 to 1000, from 50 to 750, from 100 to 500 or from 30 to 200 targeting fragments;

[22] a pharmaceutical composition according to any one of claims [1] to [21], further comprising one or more of FABP, an immunostimulating agent, an immunosuppressive agent, a detectable marker and a maleimide, wherein said FABP, an immunostimulating agent, an immunosuppressive agent, a detectable marker and the maleimide is attached to said PEG or the outer part of the liposome;

[23] a pharmaceutical composition according to any one of claims [1] to [22], further containing at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol and sucrose;

[24] a pharmaceutical composition according to any one of claims [1] to [23], intended for use in treating a disease or condition in a subject;

[25] the pharmaceutical composition according to claim [24], wherein said disease or condition is a disease or condition associated with endotoxemia; sepsis; infection; bacteremia; liver disease or condition; or a lung disease or condition such as acute respiratory distress syndrome (ARDS), pulmonary fibrosis, pulmonary hemorrhage, lung injury, lung cancer or chronic obstructive pulmonary disease (COPD); kidney disease; autoimmune disorder; sclerosis; inflammation; inflammatory bowel disease; metabolic disease; insulin resistance; diabetes, such as type 2 diabetes, or a related condition; cardiovascular disease; disease or condition characterized by ischemia or hypoxia; heart attack or stroke; shock; or a condition associated with nitric oxide deficiency;

[26] a method for producing a pharmaceutical composition according to any one of claims [1] to [23], including:

(a) preparing a liposome solution containing the liposomes in a solution of a weak acid salt of a polyvalent metal or polyvalent organic cations such as a protonated amine;

(b) adding an ionizable carotenoid to said liposome solution, wherein the ionizable carotenoid is trans-crocetin or trans-norbixin; and (c) maintaining the ionizable carotenoid in the liposome solution for a sufficient time for the carotenoid to penetrate into the liposomes;

[27] the method of claim [26], wherein said weak acid is an organic acid such as an organic acid selected from acetic acid, gluconic acid, tartaric acid, glutamic acid, citric acid, formic acid and glycic acid, wherein uka

- 3 047345 the polyvalent metal in question is a divalent metal such as a divalent metal selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ or a trivalent metal such as Fe ^{3 +} ;

[28] method according to paragraph [26] or [27], in which the added ionizable carotenoid is trans-crocetin. However, other features and advantages of the compositions and methods described herein will become more apparent from the following detailed description when read in conjunction with the accompanying drawings.

Brief description of graphic materials

In fig. Ία-ID illustrates typical ionizable polyene carotenoids of the proposed pharmaceutical compositions.

In fig. 2 illustrates the stability of calcium trans-crocetinate liposome (CTC-LP) at 4°C for 6 months. These CTC-LP test formulations have drug/lipid (D/L) ratios of 80, 60, and 40. Each CTC-LP test formulation showed negligible washout (change in D/L ratio) over the 6-month evaluation period.

In fig. Figure 3 illustrates the batch reproducibility of liposomal STS. Four batches of liposomal STS were reproducible and stable at 4°C for at least 7 months.

In fig. 4 illustrates the stability of magnesium trans-crocetinate liposome (MTC-LP) at 4°C for 6 months. These MTC-LP test formulations have drug/lipid (D/L) ratios of 80, 60, and 40. Each MTC-LP test formulation showed negligible washout (change in D/L ratio) over the 2-month evaluation period.

In fig. 5 illustrates Survival Study 1 (TP-936): a study of the effectiveness of CTC-LP in the CLP mouse model of sepsis. Survival curve of mice treated with test drugs (a) liposomal CTC (D/L 80) + antibiotic, (b) liposomal CTC (D/L 80) and PGPC + antibiotic, (c) saline + antibiotic and (d) placebo. Test drugs (a) and (b) (in combination with imipenem) showed a trend towards reduced mortality compared with control treated with imipenem (c).

In fig. 6 illustrates Survival Study 2 (TP-967): CLP sepsis study in mice. Survival curve of mice treated with test drugs (a) liposomal PGPC + antibiotic, (b) liposomal (PGPC and CTC) (D/L 80) + antibiotic, (c) liposomal CTC (D/L 80) + antibiotic and (d) saline solution + antibiotic. The test drug (c) showed a trend toward reduced mortality compared with the imipenem-treated control (d).

In fig. 7 illustrates Survival Study 3 (TP-986): CLP sepsis study in mice. Survival curve of mice treated with test drugs: (a) liposomal STS (D/L 80) (1 mg/kg) + antibiotic, (b) liposomal STS (D/L 80) (5 mg/kg) + antibiotic, (c ) liposomal STS (D/L 80) (25 mg/kg) + antibiotic, (d) liposomal STS (D/L 80) (50 mg/kg) + antibiotic, and (e) saline + antibiotic. Each of the tested drugs (a), (c) and (d) showed a trend toward reduced mortality compared with the imipenem-treated control (e). Test drug (b) (liposomal STS (D/L 80) (5 mg/kg) + antibiotic) showed a statistically significant reduction in mortality compared with imipenem-treated control (d) (P=0.0321).

Detailed description of the invention

Applicants have unexpectedly discovered that pharmaceutical compositions, such as liposomes containing polyvalent ionizable carotenoid salts containing polyvalent counterions, significantly improve pharmacokinetics (e.g., half-life, stability and bioavailability) and dramatically increase drug exposure by sustained release of ionizable carotenoids compared to, e.g. , with free carotenoid acids and ionizable carotenoid salts containing monovalent counterions.

Definitions

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the proposed compositions, suitable methods and materials are described herein. Each publication, patent application, patent and other reference mentioned herein is hereby incorporated by reference in its entirety. In the event of any conflict, this description, including definitions, shall control. Moreover, these materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of these compositions and methods will be apparent from the following detailed description, drawings, and claims.

It should be understood that where embodiments of the invention are described herein in the terms containing, similar embodiments described in terms of consisting of and/or consisting essentially of are otherwise also provided. However, when used in the claims as transitional phrases, each of them must

- 4 047345 be interpreted separately and in the relevant legal and factual context (for example, in the claims, the transitional phrase containing is considered a more open phrase, while consisting of is more exclusive and essentially consisting of is a transitional form).

As used herein, singular forms include references to the plural unless the context clearly indicates otherwise, singular forms also include the statistical average of composition, characteristics, or particle size among a plurality of particles (e.g., average liposome diameter, average liposome zeta potential , average number of targeting fragments on a liposome in a liposome solution, average number of encapsulated carotenoids). The average particle size and zeta potential of liposomes in a pharmaceutical composition can generally be measured by methods known in the art, such as dynamic light scattering. The average amount of therapeutic agent in the nanoparticle composition can typically be measured, for example, by absorption spectroscopy (eg, UV spectroscopy).

As used herein, the terms approximately and about, when applied to one or more values of interest, refer to a value that is similar to an established reference value. In certain embodiments of the invention, the term about or about refers to a range of values that fall within the limits of 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6. 5, 4, 3, 2, 1% or less in either direction (more or less) of the specified reference value, unless otherwise stated or otherwise obvious from the context (unless such number would exceed 100% of the possible value ). For example, when used in the context of the amount of a given compound in the lipid component of a nanoparticle composition, about may mean ±10% of the reported value. For example, a nanoparticle composition, including a lipid component, containing about 40% of a given compound may contain 30-50% of the specified compound.

The term and/or, when used in a phrase such as A and/or B, is intended to include both A and B; A or B; A (separately) and B (separately). Likewise, the term and/or when used in a phrase such as A, B and/or C is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (one); Into one); and C (one).

When embodiments herein are described in terms of Markush groups or other grouping of alternatives, the composition or method described includes not only the entire group listed as a whole, but also each member of that group individually and all possible subgroups of the main group, as well as the main group with the missing one or more group members. The described compositions and methods also provide for the explicit exclusion of one or more of any of the group members in the described compositions or methods.

The term liposome refers to a closed vesicle having an internal phase (ie, internal space (internal solution)) surrounded by a lipid bilayer. The liposome may be a small single membrane liposome such as a small unilamellar vesicle (SUV), a large single membrane liposome such as a large unilamellar vesicle (LUV), an even larger single membrane liposome such as a giant unilamellar vesicle (GUV), a multilayer liposome having multiple concentric membranes (eg, 2, 3, 4, 5, 6, 7, 8, 9 or 10), such as a multilayer vesicle (MLV), or a liposome having multiple membranes that are irregular rather than concentric, such as a multivesicular vesicle (MVV). Liposomes and liposome formulations are well known in the art. Lipids that are capable of forming liposomes include all substances that have fatty or fat-like properties. Lipids that can form liposomes include, but are not limited to, glycerides, glycerophospholipids, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids, stearins, sterols, archeolipids, synthetic cationic lipids, and carbohydrate-containing lipids.

A liposome composition is a resulting composition containing a liposome and contents within the liposome, in particular containing lipids that form the liposomal bilayer(s), compounds other than lipids within the liposome bilayer(s), compounds found within and associated with aqueous the internal space(s) of the liposome, and connections attached or associated with the outer layer of the liposome. Thus, in addition to liposome lipids, the liposome composition described herein may suitably contain, but is not limited to, therapeutic agents, immunostimulating agents, vaccine antigens and adjuvants, excipients, carriers and buffering agents. In a preferred embodiment, such compounds are complementary and/or not significantly detrimental to the stability or efficiency of AGP incorporation of said liposome composition.

The terms internal phase, internal space and internal core of a liposome are used interchangeably to refer to the aqueous region contained within (i.e. encapsulated)

- 5 047345 lipid bilayer of liposome. The solution in the internal phase of the liposome is referred to as the internal solution. In contrast, the term outer phase of a liposome refers to the region not covered by the lipid bilayer of the liposome, such as a region other than the inner phase and lipid bilayer when the liposome is dispersed in a liquid.

The term counterion refers to an anionic or cationic counterion.

A cationic counterion is a positively charged atom or group bonded to an anionic atom or group to maintain electrical neutrality. Typical cationic counterions include inorganic cations (eg, metal cations (eg, alkali metal cations, alkaline earth metal cations, and transition metal cations)) and organic cations (eg, ammonium cations, sulfonium cations, phosphonium cations, and pyridinium cations). An anionic counterion is a negatively charged atom or group bonded to a cationic atom or group to maintain electrical neutrality. Typical anionic counterions include halide anions (e.g. F ^- , Cl ^- , Br ^- and I ^- ), NO3 ^- , ClO4 ^- OH ^- , H2PO4 ^-2 , HSO ₄ ^- , sulfonate anions (e.g. methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate , benzenesulfonate, 10-camphorasulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethane-1-sulfonic acid-2-sulfonate, etc.) and carboxylate anions (for example, acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate and glycolate). The counterion may be monovalent or polyvalent (eg, divalent, trivalent, tetravalent, etc.).

The term ionizable refers to a compound containing at least one functional group that (a) carries a positive or negative charge (i.e., is ionized) and is therefore associated with a counterion of opposite charge, or (b) is electrically neutral but ionizes when higher or lower pH. Thus, ionizable compounds include quaternary ammonium salts, as well as uncharged amines and carboxylate moieties, as well as uncharged carboxyl groups.

As used herein, the term carotenoid refers to organic pigments that are structurally composed of a polyene hydrocarbon chain and that may be ring-terminated. Carotenoids are divided into two classes: xanthophylls (which contain oxygen atoms) and carotenes (which do not contain oxygen atoms). Non-limiting examples of carotenoids suitable for use in the present compositions and methods are described in FIG. 1A-1D.

Carotenoids with ionizable functional groups include naturally occurring carotenoid sulfates, carboxylic acids/carboxylates, synthetic phosphates, blue carotenoid oxonium ions, and blue carotenoid proteins.

As used herein, the term polyene carotenoid refers to a carotenoid containing three or more conjugated double bonds and methyl or lower alkyl (C ₂ -C ₃ ) substitutions.

The term naturally occurring refers to a compound or composition that occurs naturally, regardless of whether said compound or composition was isolated from a natural source or chemically synthesized. Examples of naturally occurring mono- and dicarboxylic acid carotenoids include crocetin, norbixin, azaphrine and carotenoid acid.

An apocarotenoid is a carotenoid degradation product in which the normal structure (ie _C40 ) has been reduced by removing fragments from one or both ends. Examples of naturally occurring apocarotenoids include crocetin ( _C20 ), bixin ( _C25 ), vitamin A, abscisic acid, micorradicin and blumenin.

As used herein, the term targeting moiety refers to a molecule that provides increased affinity for a selected target, eg a cell, cell type, tissue, organ, body region, or compartment, eg a compartment of cells, tissues or organs. The targeting fragment can include a wide range of objects. Targeting moieties may include naturally occurring molecules or recombinant or synthetic molecules. In some embodiments, the targeting fragment is an antibody, an antigen binding fragment of an antibody, a bispecific antibody, or another antibody-based molecule or compound. In some embodiments, the targeting moiety is an aptamer, avimer, receptor binding ligand, nucleic acid, biotin-avidin binding pair, peptide, protein, carbohydrate, lipid, vitamin, toxin, microbial component, hormone, receptor ligand, or any derivative thereof. Other targeting moieties are known in the art and are covered by this invention.

The terms specific binding or specifically binds mean that a targeting moiety, such as an antibody or an antigen-binding fragment of an antibody, reacts or binds more frequently, more quickly, for a longer duration, with greater affinity, or some combination of the foregoing, to an epitope, protein, or molecule of the target than to alternative substances, including proteins not associated with the target epitope. Because of the sequence identity between homologous proteins in different species, specific binding in some embodiments may involve a binding agent that recognizes a protein or target in more than one species. Likewise,

- 6 047345 Because of the homology within certain regions of the polypeptide sequences of different proteins, the term specific binding or specifically binds can include a binding agent that recognizes more than one protein or target. It is understood that in some embodiments, a targeting moiety that specifically binds a first target may or may not specifically bind a second target. As such, specific binding does not necessarily require (although may include) exclusive binding, such as binding to a single target. Thus, a targeting moiety may, in certain embodiments, specifically bind more than one target. In certain embodiments of the invention, multiple targets may be linked by the same targeting moiety.

The term epitope refers to that portion of an antigen that can be recognized by and specifically bind to a targeting moiety (ie, binding moiety), such as an antibody. When the antigen is a polypeptide, epitopes can be formed by both contiguous amino acids and non-contiguous amino acids formed by the folding of the tertiary structure of the protein. Epitopes formed from contiguous amino acids tend to be retained upon protein denaturation, whereas epitopes formed from tertiary structure tend to be lost upon protein denaturation. An epitope typically includes at least 3 or more, usually at least 5 or 8-10 amino acids in a unique spatial conformation.

Expressions such as target binding affinity, target binding, and similar expressions known in the art refer to a property of a targeting moiety that can be directly measured by determining affinity constants, e.g., the amount of a targeting moiety that associates and dissociates at a certain antigen concentration. Various techniques can be used to characterize molecular interactions, such as competition analysis, equilibrium analysis, and microcalorimetric analysis, as well as real-time interaction analysis based on surface plasmon resonance interaction (eg, using the BIACORE® instrument). These methods are well known to those skilled in the art and are described, for example, in Neri et al., Tibtech., 14:465-470 (1996) and Jansson et al., J. Biol. Chem. 272:8189–8197 (1997).

As used herein, an effective amount refers to a dosage of an agent sufficient to provide the desired medical result. The effective amount will vary depending on the desired result, the particular condition being treated or prevented, the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent or combination therapy (if any), the particular route of administration, and the like. factors within the knowledge and experience of the practitioner. The effective amount can be determined empirically and in a standard manner relative to the stated purpose. In the case of cancer, an effective amount of the agent can reduce the number of cancer cells; reduce tumor size; inhibit (ie, slow to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (ie, slow to some extent and preferably stop) tumor metastasis; inhibit to some extent tumor growth and/or alleviate to some extent one or more symptoms associated with the disorder. To the extent that a drug can prevent the growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy may, for example, be measured by assessing survival time, progression-free survival time (PFS), efficacy of therapy (ET), duration of response, and/or quality of life.

The terms hyperproliferative disorder, proliferative disease, and proliferative disorder, as used interchangeably herein, refer to unwanted or uncontrolled cellular proliferation of excess or abnormal cells that is undesirable, such as neoplastic or hyperplastic growth, in vitro or in vivo. In some embodiments, said proliferative disease is a cancer or tumor disease (including benign or malignant) and/or any metastasis, regardless of where the cancer, tumor and/or metastasis is located. In some embodiments, said proliferative disease is a benign or malignant tumor. In some embodiments, the proliferative disease is a non-cancerous disease.

In some embodiments, said proliferative disease is a hyperproliferative condition such as hyperplasia, fibrosis (especially pulmonary fibrosis, but also other types of fibrosis such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation in blood vessels, such as stenosis or restenosis after angioplasty. Cancer, tumor, or malignancy are used interchangeably and refer to any of a number of diseases that are characterized by uncontrolled, abnormal cell proliferation, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body ( metastasis), as well as any of a number of characteristics

- 7 047345 ternary structural and/or molecular features.

The term tumor as used herein refers to all growth and proliferation of a tumor cell, whether malignant or benign, and all precancerous and cancerous cells and tissues.

A cancerous tumor or malignant cell is understood as a cell that has specific structural properties, lacks differentiation and is capable of invasion and metastasis. Cancers that can be treated using the pharmaceutical carotenoid composition provided herein include, but are not limited to, non-hematological malignancies such as, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head cancer and neck, stomach cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (eg, osteosarcoma), brain cancer, central nervous system cancer and melanoma; and hematologic malignancies, such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias. Other types of cancer and tumors that can be treated using the trans-crocetin composition are described herein or otherwise known in the art.

The terms cancer, cancerous, cell proliferation disorder, proliferative disorder, and tumor are not mutually exclusive as defined herein.

Terms such as treat, treat, or cure refer to both of (a) therapeutic measures that cure, slow, mitigate, reduce symptoms, and/or stop the progression of a diagnosable condition or disorder and (b) prophylactic or preventative measures that prevent and /or slow down the progression of a disease or condition. Thus, subjects in need of treatment include persons who are already suffering from the specified cancer, disorder or disease; persons at increased risk of a specified cancer or condition and persons in whom a specified infection or condition must be prevented. Subjects are determined to be suffering from or at risk of sepsis, an infectious disease, an immune system disorder, a metabolic disorder (eg, diabetes), a hyperproliferative disease, or another disease or disorder mentioned herein using well known medical and diagnostic techniques. In certain embodiments of the invention, a subject is successfully treated in accordance with the methods provided herein if the subject experiences, for example, complete, partial, or transient improvement or elimination of symptoms associated with the specified disease or condition (for example, cancer and arthritis, such as rheumatoid arthritis). In specific embodiments, the terms treating, treating, or treating refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, not necessarily discernible to the patient. In other embodiments, the terms treating, treating, or treating refer to inhibiting the progression of a proliferative disorder, either physically, such as by stabilizing a discernible symptom, or physiologically, such as by stabilizing a physical parameter, or both. In other embodiments, the terms treating, treating, or treating refer to reducing or stabilizing tumor size, tumor cell proliferation or survival, or the number of cancer cells. Treatment can be carried out with the proposed pharmaceutical composition described herein (eg, liposomal transcrocetinate), alone or in combination with an additional therapeutic agent.

Subject, patient, and animal are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats and mice, and other animals. Animals include all vertebrates, such as mammals and non-mammalian animals such as chickens, amphibians and reptiles.

As used herein, the term mammal refers to any member of the class mammals, including, without limitation, humans and non-human primates such as chimpanzees and other species of great apes and great apes; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs, and other members of the class of mammals known in the art. In a particular embodiment of the invention, the patient is a human.

The term pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, carrier, excipient, stabilizer, diluent or preservative. Pharmaceutically acceptable carriers may include, for example, one or more compatible solid or liquid excipients, diluents, or encapsulating agents that are suitable for administration to a human or other subject.

Therapeutic Agent: In some embodiments, the proposed compositions

- 8 047345 liposome formulations and liposome formulations contain liposomes encapsulating or otherwise associated with one or more therapeutic agents present anywhere in, in, on or around said liposome. For example, the therapeutic agent may be incorporated into the lipid bilayer of the liposome, encapsulated in the internal phase of the liposome or associated with the external side of the liposome. The therapeutic agent or therapeutic agents used in accordance with the described compositions and methods may include any agent intended to treat a condition in a subject. Examples of therapeutic agents that may be suitable for use in accordance with the described methods include vitamin C, thiamine, hydrocortisone or other corticosteroid (for example, a glucocorticoid such as cortisone, etamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone and methylprednisolone; and mineralocorticoids , such as fludrocortisone), astaxanthin, abscisic acid, vitamin A, angiotensin II (eg, GIAPREZA™), tissue plasminogen activator (tPA), antimicrobial agent (eg, antibiotic) and anti-inflammatory agent.

Additional examples of therapeutic agents that may be suitable for use in accordance with the described methods include, but are not limited to, anti-restenotic, pro- or anti-proliferative, anti-tumor, anti-mitotic, anti-platelet, anti-coagulant, anti-fibrin, anti-thrombin, cytostatic, antibiotic and other anti-infective agents, anti-enzymatic , antimetabolic, angiogenic, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and/or cardioprotective agents. In general, any therapeutic agent known in the art can be used, including, without limitation, agents listed in the United States Pharmacopoeia (USP), Goodman and Gilman's The Pharmacological Basis of Therapeutics, ^10th Ed., McGraw Hill, 2001 ; Katzung, Ed., Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange, ^8th ed., Sep. 21, 2000; Physician's Desk Reference (Thomson Publishing; and/or The Merck Manual of Diagnosis and Therapy, ^18th ed., 2006, Beers and Berkow, Eds., Merck Publishing Group; or, in the case of animals, The Merck Veterinary Manual, ^9th ed. ., Kahn Ed., Merck Publishing Group, 2005; each of which is incorporated herein by reference to mean an agent or derivative thereof that can interact with a hyperproliferative cell, such as a cancer cell or an immune cell, thereby reducing the proliferative status of a given cell and/or killing a given cell Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic agents, platinum-based agents (eg, cisplatin, carboplatin, oxaliplatin), taxanes (eg, Taxol), etoposide. , alkylating agents (e.g., cyclophosphamide, ifosamide), metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil, gemcitabine, pemetrexed, or derivatives thereof), antineoplastic antibiotics (e.g., mitomycin, doxorubicin), and herbal antineoplastic agents (e.g., vincristine, vindesine, Taxol). Such agents may further include, but are not limited to, the anticancer agents trimetrexate, TEMOZOLOMIDE™, RALTRITREXED™, 8-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU ) and CAMPTOTHECIN™ or a therapeutic derivative of any of them. Therapeutic agents also refer to salts, acids and free base forms of the above agents.

The term pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, carrier, excipient, stabilizer, diluent or preservative. Pharmaceutically acceptable carriers may include, for example, one or more compatible solid or liquid excipients, diluents, or encapsulating agents that are suitable for administration to a human or other subject.

The term kit refers to a collection of two or more components necessary to implement the methods and compositions provided herein. Kit components may include, but are not limited to, liposome compositions and liposome formulations described herein, reagents, buffers, containers and/or equipment. The phrase stored separately refers to a type of liposome storage that prevents a first population of liposomes from coming into contact with another population of liposomes.

The term radiosensitizing agent means a compound that makes tumor cells more sensitive to radiation therapy. Examples of radiosensitizing agents include misonidazole, metronidazole, tirapazamine and trans-crocetin.

Pharmaceutical compositions.

The pharmaceutical compositions provided can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates using standard synthetic methods and techniques either known to those skilled in the art or that would be apparent to one skilled in the art. field of technology in light of the principles contained herein. Standard synthetic methods and techniques for the preparation of organic molecules, transformation and manipulation of functional groups can be obtained from the relevant scientific literature or from standard

- 9 047345 textbooks in this area. Although not limited to any one or more sources, classic texts such as Smith et al., March's Advanced Organic Chemistry. Reactions, Mechanisms, and Structure, ^5th edition, John Wiley & Sons: New York, 2001; Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, ^3rd edition, John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), incorporated herein by reference, are useful and established textbooks on organic synthesis known to those skilled in the art. The following descriptions of synthetic methods are intended to illustrate, and not to limit, general procedures for preparing the compounds of this invention.

In some embodiments, a new class of polyvalent ionizable carotenoid (eg, trans-carotenoid) salts is provided herein.

In some embodiments, provided herein is a pharmaceutical composition comprising an ionizable carotenoid having the formula

Polyene Carotenoid-Q, wherein said Polyene Carotenoid contains:

(a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10 or more than 9 conjugated double bonds, (b) substitution with methyl or lower alkyl (C ₂ -C ₃ ) and (c) 1, 2, 3 or more 3 ionizable groups;

Q is a polyvalent counterion.

In some embodiments, said Polyene Carotenoid contains all trans-conjugated double bonds. In some embodiments, said Polyene Carotenoid contains 6-9 conjugated double bonds. In specific embodiments of the invention, said Polyene Carotenoid contains 7 conjugated double bonds.

Said Polyene Carotenoid may be naturally occurring or synthetic. In some embodiments, said Polyene Carotenoid is naturally occurring. In other embodiments, said Polyene Carotenoid is synthetic. Said ionizable group(s) may be anionic and/or cationic. In some embodiments, said Polyene Carotenoid-Q contains two or more identical ionizable groups. In some embodiments, said Polyene Carotenoid contains two or more different ionizable groups. In some embodiments, said Polyene Carotenoid contains one or more anionic ionizable groups. In some embodiments, said Polyene Carotenoid contains at least one ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate or phosphate group, and a hydroxamate moiety. In other embodiments, said Polyene Carotenoid contains one or more cationic ionizable groups (eg, a primary, secondary or tertiary amino group, a quaternary ammonium group, a choline group, a guanidine group, or an imidazole group). In specific embodiments of the invention, said Polyene Carotenoid contains one or more cationic ionizable groups and said pharmaceutical composition is substantially free of nucleic acids.

In some embodiments, Q is a multivalent cationic counterion. In some embodiments, Q is a polyvalent metal cation. In further embodiments, Q is a polyvalent transition metal cation. In some embodiments, Q is a divalent counterion. In some embodiments, Q is a divalent cationic counterion. In some embodiments, Q is a divalent metal cation. In additional embodiments, Q is a divalent transition metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In some embodiments, Q is Ca ²⁺ or Mg ²⁺ . In some embodiments, Q is Ca ²⁺ . In additional embodiments, said Polyene Carotenoid-Q is calcium trans-crocetinate (CTC). In some embodiments, Q is Mg2 ⁺ . In additional embodiments, said Polyene Carotenoid-Q is magnesium trans-crocetinate (MTS). In other embodiments, Q is a trivalent cationic counterion such as Fe ³⁺ . In other embodiments, Q is a polyvalent organic counterion. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a divalent organic cation, such as a protonated diamine. Liposomes containing Polyene Carotenoid-Q compositions (eg, liposome compositions) and pharmaceutical compositions containing said liposomes are also provided herein.

- 10 047345 document.

In some embodiments, provided herein is a pharmaceutical composition comprising an ionizable carotenoid having the formula

Q-Rj-Polyene Carotenoid-U^.

where the specified C-Polyene Carotenoid-I2 contains:

(a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10 or more than 9 conjugated double bonds, (b) substitution with methyl or lower alkyl (C2- _C3 ), (c) 1, 2, 3 or more than 3 ionizable groups;

Ri and R2 are ionizable groups;

Q is a polyvalent counterion.

In some embodiments, said QR ₁ -Polyene Carotenoid-E12 contains all trans-conjugated double bonds. In specific embodiments of the invention, said QR ₁ -Polyene Carotenoid-E12 contains 6-9 conjugated double bonds. Said QR ₁ -Polyene Carotenoid-C ₂ may be naturally occurring or synthetic. In some embodiments, said Q-Rj-Polyene Carotenoid-C ₂ is naturally occurring. In other embodiments, said Q-Rj-Polyene Carotenoid-U is synthetic. In some embodiments, R1 and R2 are the same ionizable groups. In other embodiments, R ₁ and R ₂ are different ionizable groups. In some embodiments, R ₁ and R ₂ are the same cationic ionizable groups (eg, a primary, secondary or tertiary amino group, a quaternary ammonium group, a choline group, a guanidine group, and an imidazole group). In other embodiments, R1 and R2 are different cationic groups. In some embodiments, R1 and R2 are the same anionic ionizable groups (eg, a carboxyl group, a sulfonate group, a sulfate group, a phosphonate group, a phosphate group, and a hydroxamate group). In other embodiments, R1 and R2 are different anionic groups. In some embodiments, R ₁ is a cationic ionizable group or an anionic ionizable group and R2 is an anionic ionizable group or a cationic group, respectively. In some embodiments, said Polyene Carotenoid contains at least one ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate or phosphate group, and a hydroxamate moiety. In some embodiments, R1 and/or R2 is at least one ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate or phosphate group, and a hydroxamate moiety. In some embodiments, R2 is at least one ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate or phosphate group, and a hydroxamate moiety. In other embodiments, R1 and/or R2 is a cationic ionizable group (eg, a primary, secondary or tertiary amino group, a quaternary ammonium group, a choline group, a guanidine group, or an imidazole group). In certain embodiments of the invention, R1 is a cationic ionizable group and the pharmaceutical composition is substantially free of nucleic acids.

In some embodiments, Q is a multivalent cationic counterion. In some embodiments, Q is a polyvalent metal cation. In additional embodiments, Q is a polyvalent transition metal counterion. In some embodiments, Q is a divalent counterion. In some embodiments, Q is a divalent cationic counterion. In additional embodiments, Q is a divalent metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In additional embodiments, Q is Ca ²⁺ or Mg2 ⁺ . In additional embodiments, Q is Ca ²⁺ . In some embodiments, Q is Mg2 ⁺ . In other embodiments, Q is a trivalent cationic counterion such as Fe ³⁺ . In other embodiments, Q is a polyvalent organic cation. In additional embodiments, Q is a divalent organic cation, such as a protonated diamine. Liposomes containing _RJ -Polyene Carotenoid^ compositions (eg, liposome compositions) and pharmaceutical compositions containing said liposomes are also provided herein.

- 11 047345

In some embodiments, provided herein is a pharmaceutical composition comprising an ionizable bis-alpha, omega-carotenoid having the formula:

Q-Rj-Polyene Carotenoid-Rj-Q, wherein said Rj-Polyene Carotenoid-Rj-Q contains:

(a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10 or more than 9 conjugated double bonds, (b) substitution with methyl or lower alkyl (C ₂ -C ₃ ) and (c) 1, 2, 3 or more 3 ionizable groups; And

R ₁ represents an ionizable group;

Q is a polyvalent counterion.

In some embodiments, said bis-alpha, omega-carotenoid contains all trans-conjugated double bonds. In some embodiments, said bis-alpha, omega-carotenoid contains 6-9 conjugated double bonds. In specific embodiments, said bis-alpha, omega-carotenoid contains 7 conjugated double bonds. Said bis-alpha, omega-carotenoid may be naturally occurring or synthetic. In some embodiments, said bis-alpha, omega-carotenoid is naturally occurring. In other embodiments, said bis-alpha omega-carotenoid is synthetic. In some embodiments, R ₁ is an anionic ionizable group. In some embodiments, said bisalpha, omega-carotenoid contains an ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate group, a phosphate group, and a hydroxamate moiety. In other embodiments, R1 is a cationic ionizable group (eg, a primary, secondary or tertiary amino group, a quaternary ammonium group, a choline group, a guanidine group, or an imidazole group). In certain embodiments of the invention, R1 is a cationic ionizable group, and the pharmaceutical composition is substantially free of nucleic acids.

In some embodiments, Q is a multivalent cationic counterion. In additional embodiments, Q is a polyvalent metal cation. In some embodiments, Q is a polyvalent transition metal counterion. In some embodiments, Q is a divalent counterion. In some embodiments, Q is a divalent cationic counterion. In additional embodiments, Q is a divalent metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In additional embodiments, Q is Ca ²⁺ or Mg ²⁺ . In some embodiments, Q is Ca ²⁺ . In some embodiments, Q is Mg2 ⁺ . In other embodiments, Q is a trivalent cationic counterion such as Fe ³⁺ . In some embodiments, Q is a polyvalent organic cation. In further embodiments, Q is a divalent organic cation, such as a protonated diamine or a protonated polyamine. Liposomes containing R ₁ -Polyene Carotenoid-B compositions (eg, liposome compositions), and pharmaceutical compositions containing said liposomes are also provided herein.

In some embodiments, provided herein is a pharmaceutical composition comprising an ionizable bis-alpha, omega-carotenoid having the formula

R ₁ -Polyene Carotenoid-Ri.

wherein said bis-alpha, omega-carotenoid contains:

(a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10 or more 9 conjugated double bonds and (b) 1,2,3 or more 3 ionizable groups ; and said bis-alpha, omega-carotenoid is optionally substituted with 1 to n methyl or lower C1-C3 alkyl substitutions, where n = 1 to 4; And

Ri represents a polar group and/or a monocyclic functional group.

In some embodiments, said bis-alpha, omega-carotenoid contains all trans-conjugated double bonds. In some embodiments, said bisalpha, omega-carotenoid contains 6-9 conjugated double bonds. In specific embodiments, said bis-alpha, omega-carotenoid contains 7 conjugated double bonds. Said bis-alpha, omega-carotenoid may be naturally occurring or synthetic. In some embodiments, said bis-alpha, omega-carotenoid is naturally occurring. In other embodiments, said bis-alpha omega-carotenoid is synthetic. In some embodiments, R1 is a polar group. In some embodiments, R1 is a monocyclic functional group. In some embodiments, R ₁ is a polar group and a monocyclic functional group. In some implementations

- 12 047345 version of the invention, said bis-alpha, omega-carotenoid contains a monocyclic and/or polar functional group selected from the functional group present in astaxanthin, lutein, xanthophyll and zeaxanthin. In some embodiments, said bisalpha, omega-carotenoid is selected from astaxanthin, lutein, xanthophyll, and zeaxanthin (for example, as shown below):

Liposomes containing bis-alpha, omega-carotenoid compositions (eg, liposome compositions), and pharmaceutical compositions containing these liposomes are also provided herein.

In some embodiments, said pharmaceutical composition contains trans-crocetin having the formula Q-trans-crocetin-Q:

O

O where Q is a polyvalent cationic counterion.

In some embodiments, Q is a polyvalent metal cation. In further embodiments, Q is a polyvalent transition metal cation. In some embodiments, Q is a divalent cationic counterion. In additional embodiments, Q is a divalent metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In additional embodiments, Q is Ca ²⁺ or Mg ²⁺ . In some embodiments, Q is Ca ²⁺ . In some embodiments, Q is Mg ²⁺ . In other embodiments, Q is a trivalent cationic counterion such as Fe ³⁺ . In some embodiments, Q is a polyvalent organic cation. In additional embodiments, Q is a divalent organic cation, such as a protonated diamine. Liposomes containing trans-crocetin compositions (eg, liposome compositions) and pharmaceutical compositions containing said liposomes are also provided herein.

In some embodiments, provided herein is a pharmaceutical composition comprising calcium trans-crocetinate (CTC). The specified STS can exist in linear and/or cyclic form (shown below):

⁰ II

Ca ⁺⁺⁺⁴ g I° o

Liposomes containing CTC compositions (eg, liposome compositions) and pharmaceutical compositions containing said liposomes are also provided herein.

In some embodiments, provided herein is a pharmaceutical composition comprising magnesium trans-crocetinate (MTS). The specified MTS can exist in linear and/or cyclic form (shown below):

Liposomes containing MTC compositions (eg, liposome compositions) and pharmaceutical compositions containing said liposomes are also provided herein.

In some embodiments, the disclosure provides a pharmaceutical

- 13 047345 composition containing trans-norbixin having the formula Q-Hop6ukCUH-Q

where Q is a polyvalent cationic counterion.

In some embodiments, Q is a multivalent cationic counterion. In some embodiments, Q is a polyvalent metal cation. In further embodiments, Q is a polyvalent transition metal cation. In some embodiments, Q is a divalent cationic counterion. In additional embodiments, Q is a divalent metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In additional embodiments, Q is Ca ²⁺ or Mg ²⁺ . In some embodiments, Q is Ca ²⁺ . In some embodiments, Q is Mg ²⁺ . In other embodiments, Q is a trivalent cationic counterion such as Fe ³⁺ . In some embodiments, Q is a polyvalent organic cation. In additional embodiments, Q is a divalent organic cation, such as a protonated diamine. Liposomes containing trans-norbixin compositions (eg, liposome compositions) and pharmaceutical compositions containing said liposomes are also provided herein.

In some embodiments, provided herein is a pharmaceutical composition comprising calcium trans-norbixinate (CTN). Said CTN may exist in linear and/or cyclic form (shown below):

O

-oS'L b A'ya \ I I ? /

ABOUT

Liposomes containing CTN compositions (eg, liposome compositions) and pharmaceutical compositions containing said liposomes are also provided herein.

In some embodiments, provided herein is a pharmaceutical composition comprising magnesium trans-norbixinate (MTN). Said MTN may exist in linear and/or cyclic form (shown below):

Liposomes containing MTN compositions (eg, liposome compositions) and pharmaceutical compositions containing these liposomes are also provided herein.

Said lipids and other liposome components contained in said liposome compositions may be any lipid, combination of lipids and ratio, or combination of lipids and other liposome components and their respective ratios known in the art. However, one skilled in the art will appreciate that liposome encapsulation of any particular drug, such as, without limitation, the carotenoid compositions discussed herein, may involve considerable routine experimentation to achieve a suitable and functional liposome formulation. In general, the proposed liposomes can have any liposome structure, for example structures having an internal space, protected from the external environment by one or more lipid bilayers, or any microcapsule that has a semi-permeable membrane with a lipophilic central part, where the membrane encloses the internal space. The lipid bilayer can be any arrangement of amphiphilic molecules characterized by a hydrophilic portion (hydrophilic moiety) and a hydrophobic portion (hydrophobic moiety). Typically, amphiphilic molecules in a bilayer are arranged in two-dimensional sheets, in which the hydrophobic moieties are oriented inward of the sheet and the hydrophilic moieties are oriented outward. The amphiphilic molecules forming the proposed liposomes can be any known or later discovered amphiphilic molecules, for example lipids of synthetic or natural origin or biocompatible lipids. These liposomes can also be formed by amphiphilic polymers and surfactants, for example polymerosomes and niosomes. For purposes of this description, without limitation, these liposome-forming materials are also referred to as lipids.

- 14 047345

The liposome composition formulations provided herein may be in liquid or dry form, such as a dry powder or a dry sludge. Said dry powder or dry cake may be subjected to primary drying, for example, under lyophilization conditions, or optionally, said dry cake or dry powder may be subjected to either only primary drying or both primary and secondary drying. In dry form, said powder or precipitate may contain, for example, 1 to 6% moisture, for example in the range of 2 to 5% moisture or 2 to 4% moisture. One example of drying methods is lyophilization (also called freeze drying or freeze drying). Any of the compositions and methods herein may contain liposomes, lyophilized liposomes, or liposomes reconstituted from lyophilized liposomes. In some embodiments, the compositions and methods contain one or more lyoprotectants or cryoprotectants. These protectors are typically polyhydroxy compounds such as sugars (mono-, di- and polysaccharides), polyhydric alcohols and their derivatives, glycerol or polyethylene glycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol or aminoglycosides. In additional embodiments, said lyoprotectants or cryoprotectants contain up to 10 or up to 20% of the solution outside the liposome, inside the liposome, or both outside and inside the liposome.

The properties of liposomes depend on the nature of the lipids used to obtain liposomes. A wide variety of lipids have been used to prepare liposomes. They include cationic, anionic and neutral lipids. In some embodiments, said liposomes containing carotenoid compositions (eg, CTC and MTC) are anionic or neutral. In other embodiments of the invention, the proposed liposomes are cationic. Determination of charge (eg, anionic, neutral, or cationic) can typically be made by measuring the zeta potential of the liposome. The zeta potential of a liposome can be positive, zero or negative. In some embodiments, the zeta potential of said liposome is -150 to 150 mV, or -50 to 50 mV, or any range between these values. In some embodiments, said zeta potential of the liposome is less than or equal to zero. In some embodiments, said zeta potential of the liposome is -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In other embodiments, said zeta potential of the liposome is greater than zero.

In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

In some embodiments, cationic lipids are used to prepare cationic liposomes, which are typically used as gene transfection agents. The positive charge of cationic liposomes allows interaction with the negative charge on the cell surface. Once these cationic liposomes are attached to a cell, said liposome is transported into the cell by endocytosis.

In some preferred embodiments, neutral to anionic liposomes are used. In a preferred embodiment of the invention, an anionic liposome is used. Using a mixture of, for example, neutral lipids such as HSPC and anionic lipids such as PEG-DSPE results in the formation of anionic liposomes that are less likely to bind nonspecifically to normal cells. Specific binding to tumor cells can be achieved by using a tumor-targeting antibody, such as, for example, anti-folate receptor antibodies, including, for example, anti-folate receptor alpha, anti-folate receptor beta and/or anti-folate receptor delta.

By way of example, at least one (or some) of the lipids is/are amphiphilic lipids, defined as having a hydrophilic and a hydrophobic portion (typically a hydrophilic head and a hydrophobic tail). Said hydrophobic portion is typically oriented toward the hydrophobic phase (eg, inside the bilayer), while the hydrophilic portion is typically oriented toward the aqueous phase (eg, outside the bilayer). The hydrophilic portion may contain polar or charged groups such as carbohydrate, phosphate, carboxyl, sulfate, amino, sulfhydryl, nitro, hydroxyl and other similar groups. Said hydrophobic moiety may contain non-polar groups, which include, without limitation, long chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted with one or more aromatic, cycloaliphatic or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids.

Typically, for example, said lipids are phospholipids. Phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphati

- 15 047345 dilinositol, phosphatidylserine and the like. It should be understood that other lipid membrane components may be used, such as cholesterol, sphingomyelin and cardiolipin.

The lipids contained in the liposomes provided herein may be anionic and neutral (including zwitterionic and polar) lipids, including anionic and neutral phospholipids. Neutral lipids exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, dioleylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols. Examples of zwitterionic lipids include, but are not limited to, dioleylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleylphosphatidylserine (DOPS). Anionic lipids are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloylphosphatidylglycerol (POPG) and other anionic modifying groups, attached to neutral lipids.

Collectively, anionic and neutral lipids are referred to herein as non-cationic lipids. Such lipids may contain phosphorus, but are not limited to this. Examples of non-cationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidyl-1-ethanolamine (DSPE), palmitoyloleylphosphatidylethanolamine (POPE palmi) toyloleylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC) , distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleylphosphatidylglycerol (POPG), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans -PE, palmitoyloleylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleylphosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate and cholesterol.

These liposomes can be assembled by any liposome assembly method using liposome components (also called liposomal components) known in the art. Liposome components include, for example, lipids such as DSPE, HSPC, cholesterol and derivatives of these components. Other suitable lipids are commercially available, for example, from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). A partial list of available negatively or neutrally charged lipids suitable for the preparation of anionic liposomes may be, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA-Na, DPPA- Na, DOPA-Na, DMPG-Na, DPPG-Na, DOPG-Na, DMPS-Na, DPPS-Na, DOPS-Na, DOPE-Glutaryl·(Na) ₂ , tetramyristoyl cardiolipin-(No) ₂ , DSPE-mPEG -2000·Na, DSPE-mPEG5000-Na and C3-maleimide PEG-2000-Na.

In some embodiments, these proposed compositions are formulated into a liposome containing a cationic lipid. In one embodiment of the invention, said cationic lipid is selected from, but is not limited to, cationic lipids described in international patent publications No. WO 2012/040184, WO 2011/153120, WO 2011/149733, WO 2011/090965, WO 2011/043913, WO 2011/022460, WO 2012/061259, WO 2012/054365, WO 2012/044638, WO 2010/080724, WO 2010/21865 and WO 2008/103276, US patents No. 7893302, 74049 69 and 8283333, and U.S. Patent Publication No. US 20100036115 and US 20120202871; each of which is incorporated herein by reference in its entirety. In another embodiment of the invention, said cationic lipid may be selected from, but not limited to, Formula A, described in international patent publications No. WO 2012/040184, WO 2011/153120, WO 201/1149733, WO 2011/090965, WO 2011/ 043913, WO 2011/022460, WO 2012/061259, WO 2012/054365 and WO 2012/044638; each of which is incorporated herein by reference in its entirety. In yet another embodiment, said cationic lipid may be selected from, but not limited to, CLI-CLXXIX of WO 2008/103276, CLI-CLXXIX of US Pat. No. 7,893,302, CLI-CLXXXXII of US Pat. No. 7,404,969, and Formula I-VI of US patent applications No. US20100036115; each of which is incorporated herein by reference in its entirety. By way of non-limiting example, said cationic lipid may be selected from the group consisting of (202,232)-H,N-dimethylnonacosa-20,23-dien-10-amine, (172,202)-N,N-dimethylhexacosa-17,20-dien-9-amine . , (142,172)-N,Ndimethyltricosa-14,17-dien-6-amine, (152,182)-N,Ndimethyltetrakosa-15,18-dien7-amine, (182,212)-N,Ndimethylheptakosa-18,21- dien-10-amine, (152,182)-Ni-dimethyltetrakosa-15,18-diene-5-amine, (142.172)-Ni,Ni-dimethyltricosa-14,17-dien-4-amine, (192.222)-Ni-dimethyloctacosa-19 ,22dien-9-amine, (182,212)-N,Ndimethylhexakosa-18,21-dien-8-amine, (172,202)-H,Ndimethylhexakosa- 16 047345

17,20-dien-7-amine, (162,192)-H, Yudimethylpentacosa-16,19-dien-6-amine, (222,252)-N, Yudimethylhentriaconta-22,25-dien-10-amine, (212,242)- H,H-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-gnMeTn.ageptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-ene- 9-amine, (19Z,22Z)-N,N-dimethyloctacosa19,22-dien-7-amine, N,N-dimethylheptacosane-N-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20, 23-dien-10amine, 1-[(11Z, 14Z)-1-nonyleucosa-11,14-dien-1-yl]n-pyrrolidine, (20Z)-N,N-dimethylgentαcos-20-en-10amine, (15Z) -N,N-dimethylheptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,Ndimethylnonacos-17-en-10-amine, ( 24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylgentriacont-22-en-10 -amine, (16Z)-N,N-dimethylpentacos-16en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylgeneicosa-12,15-dien-1-amine, (13Z,16Z) -N,N-dimethyl-3nonyldocosa-13,16-dien-1-amine, K^dimethyl-1-[( 1 S,2R)-2-octylcyclopropyl]heptαdecane-8-amine,

1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N ,N-dimethyl-21-[R ₁ S,2R)-2-octylcyclopropyl]geneicosan-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)- 2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexαdecane-8-amine, K^dimethyl-[(1K^)-2 -undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclononyl]gentyl}dodecane-1amine, 1-[(1K^)-2-heptylcyclopropyl| -SiUdimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]SiUdimethylpentadecane-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopronyl]pentadecane-8- amine, RN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, SN,N-dimethyl1-[(9Z , 12Z)-octαdeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z, 12Z)-octαdeca-9,12dien-1-yloxy- 1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12dien-1-yloxy]-3-[(5Z)-oct -5-en-1-yloxy]propan-2-αmin, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]1-[(octyloxy)methyl]ethyl}azetidine , (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1yloxy]propan-2-amine, (2S)-1-(gentyloxy )-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[ (9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy] -3 - (octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1 [(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)- 1-[(11Z,14Z)-eicosa11,14-dien-1-yloxy]-11Udimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z, 14Z )eicosa-11,14-dien-1-yloxy]-NiUdimethylpropan-2-amine, 1-[(11Z, 14Z)-eicosa-11,14-dien-1-yloxy]N,N-dimethyl-13 -( octyloxy)propan-2-amine, 1-[(13Z, 16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3 (octyloxy)propan-2-amine, (2S)- 1-[(13Z,16Z)-dokosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-dokosa -13 -en-1 -yloxy]-3 -(hexyloxy)-N,N-dimethylyroian-2-αmin,

1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)proian-2-amine, 1-[(9Z)-hexadec-9-en-1yloxy] -YUdimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3 [(9Z,12Z)-octadeca-9,12-diene-1 -yloxy]proian-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3R9Z,12Z)-octadeca-9,12-dien-1-yloxyproian-2 -amine, Yudimethyl-1-(octyloxy)-3-({8-[(^^)-2{[(1R,2R)-2-nentylcyclopronyl]methyl}cyclopronyl]octyl}oxy)pronan-2-amine, Yudimethyl-1-{[-(2octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacosa11,20,2-trien-10-amine, or pharmaceutically acceptable salts or acids thereof, or stereoisomers thereof.

In one embodiment, said lipid may be a cleavable lipid such as those described in International Publication No. WO 2012/170889, which is incorporated herein by reference in its entirety.

Said cationic lipid can generally be synthesized by methods known in the art (see, for example, international publications No. WO 2012/040184, WO 2011/153120, WO 2011/149733, WO 2011/090965, WO 201/1043913, WO 2011 /022460, WO 2012/061259, WO 2012/054365, WO 2012/044638, WO 2010/080724 and WO 2010/21865; each of which is incorporated herein by reference in its entirety.

The lipid derivatives may include, for example, at least a conjugate (preferably by covalent bond) of one or more steric stabilizers and/or functional groups with a liposome component, whereupon said steric stabilizers and/or functional groups should be considered part of the liposome components. Functional groups include groups that can be used to attach a component of a liposome to another part of a molecule, such as a protein. Such functional groups include at least maleimide. These steric stabilizers include at least one from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); polyacrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[U(2-hydroxypropyl)methacrylamide]; amphiphilic poly-juvinylpyrrolidones; L-amino acid based polymer and polyvinyl alcohol.

In some embodiments, the proposed carotenoid compositions are formulated as a lipid-polycation complex. Formation of the lipid-polycation complex can be accomplished by methods known in the art and/or as described in US Patent Application No. 2012/0178702, which is incorporated herein by reference in its entirety. By way of non-limiting example, said polycation may include a cationic

- 17 047345 peptide or polypeptide, such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in international patent application WO 2012/013326; which is incorporated herein by reference in its entirety. In another embodiment of the invention, the carotenoid composition of the invention is formulated in a lipid-polycation complex that further includes a neutral lipid such as, but not limited to, cholesterol or dioleylphosphatidylethanolamine (DOPE).

Since the components of a liposome can include any molecule(s) (i.e., chemical compound/reagent/protein) that is associated therewith, in some embodiments, said components of the proposed liposomes include at least a member selected from DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol maleimide. In some embodiments, said components of the proposed liposomes include DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol maleimide. In a preferred embodiment of the invention, said liposome components that make up said liposome contain DSPE; DSPE-FITC; DSPE-maleimide; cholesterol and HSPC.

In additional embodiments, said liposomes of the liposome compositions provided herein include oxidized phospholipids. In some embodiments, the liposomes contain an oxidized phospholipid selected from phosphatidylserines, phosphatidylinositols, phosphatidylethanolamines, phosphatidylcholines, and 1-palmitoyl-2-arachidono-glycero-phosphate. In some embodiments, said phospholipids have unsaturated bonds. In some embodiments, said phospholipids are arachidonic acid containing phospholipids. In additional embodiments, said phospholipids are sn-2-oxidized. In additional embodiments, said phospholipids are not fragmented.

In some embodiments, said liposomes of the disclosed liposome compositions comprise oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). As used herein, the term oxPAPC refers to lipids generated by the oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), resulting in a mixture of oxidized phospholipids containing fragmented or full-length oxidized sn-2 residues. Well-characterized oxidation-fragmented compounds contain a five-carbon sn-2 residue bearing omega-aldehyde or omega-carboxyl groups. Oxidation of arachidonic acid residues also generates phospholipids containing esterified isoprostanes. oxPARS includes the compounds HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC among other oxidized products present in oxPARS. In additional embodiments, said oxPARS are phospholipids containing epoxyisoprostane. In additional embodiments, said OxPARS is 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2-(epoxycyclopentenone)-sn -glycero-3 phosphorylcholine (PECPC) and/or 1-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, said phospholipids have unsaturated bonds. In some embodiments, said phospholipids are arachidonic acid containing phospholipids. In additional embodiments, said phospholipids are sn-2-oxidized. In additional embodiments, said phospholipids are not fragmented.

In some embodiments, said liposomes of the described liposome compositions comprise a lipid selected from the group consisting of 1-palmitoyl-2-glutaroyl-sn-glycero-3phosphocholine (PGPC); 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine. In additional embodiments, said liposome contains PGPC.

In some embodiments, at least one component of the lipid bilayer of the liposome is functionalized (or reactive). As used herein, a functionalized moiety is a moiety that contains a reactive moiety that can be used to link reagents and moieties to a lipid. If said lipid is functionalized, any liposome it forms is also functionalized. In some embodiments, said reactive group is one that will react with a crosslinking agent (or other moiety) to form crosslinks. The reactive group in the lipid bilayer of a liposome is located at any point on the lipid that allows contact with the crosslinker and the possibility of crosslinking with another moiety (for example, a steric stabilizer or targeting moiety). In some embodiments, said reactive group is on a head group of a lipid, including, for example, a phospholipid. In some implementations

- 18 047345 of the invention, said reactive group is a maleimide group. Maleimide groups can be crosslinked to each other in the presence of dithiol crosslinkers, including, but not limited to, dithiothreitol (DTT).

It should be understood that the use of other functionalized lipids, other reactive groups and other crosslinkers in addition to those described above is contemplated. In addition to maleimide groups, other examples of contemplated reactive groups include, but are not limited to, other thiol reactive groups, amino groups such as primary and secondary amines, carboxyl groups, hydroxyl groups, aldehyde groups, alkyne groups, azide groups, carbonyls, haloacetyl groups groups (eg iodoacetyl), imidoester groups, N-hydroxysuccinimide esters, sulfhydryl groups and pyridyl disulfide groups.

Functionalized and nonfunctionalized lipids are available from a number of commercial sources, including Avanti Polar Lipids (Alabaster, AL) and Lipoid LLC (Newark, NJ).

In some embodiments, the liposomes contain a steric stabilizer that increases their durability in the bloodstream. One or more steric stabilizers, such as a hydrophilic polymer (polyethylene glycol (PEG)), a glycolipid (monosialoganglioside (GM1)) or others, occupy the space immediately adjacent to the surface of the liposome and exclude other macromolecules from this space. Consequently, the access and binding of blood plasma opsonins to the surface of the liposome is hindered, and thus the interaction of macrophages with such liposomes or any other clearing mechanism is blocked, and the longevity of the liposomes in the bloodstream is increased. In some embodiments, said steric stabilizer of the plurality of steric stabilizers is PEG or a combination containing PEG. In additional embodiments, said steric stabilizer is a PEG or a combination containing PEG with a number average molecular weight (Mn) of 200 to 5000 Da. These PEGs can have any structure, such as linear, branched, star-shaped or comb, and are commercially available.

In some embodiments, the liposomes of the proposed liposome compositions are pegylated (eg, pegylated liposomal CTC and pegylated liposomal MTC). In some embodiments, said PEGylated liposomes are water soluble, i.e. said pegylated liposomes are in the form of an aqueous solution.

The diameter of the proposed liposomes is not particularly limited. In some embodiments, said liposomes have an average diameter of, for example, 20 to 500 nm (nanometers), 20 to 200 nm, or any range in between. In some embodiments, said liposomes have an average diameter of 80 to 120 nm, or any range in between.

In some embodiments, the said pH of solutions containing the liposome composition is a pH from 2 to 8 or any range between these values. In some embodiments, the said pH of solutions containing the liposome composition is a pH of 5 to 8, or 6 to 7, or any range in between. In some embodiments, the said pH of solutions containing the liposome composition is a pH of 6 to 7 or any range between these values. In some embodiments, the said pH of solutions containing the liposome composition is pH 6 to 7.5, 6.5 to 7.5, 6.7 to 7.5, or 6.3 to 7.0 , or any range between these values.

In additional embodiments of the invention, the proposed liposome composition contains a buffer. In additional embodiments, said buffer is selected from HEPES, sodium citrate, or sodium phosphate (eg, monobasic and/or dibasic sodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, said buffer is sodium phosphate (eg, monobasic and/or dibasic sodium phosphate). In some embodiments, said buffer is present at a concentration of 15 to 200 mM, or any range between these values. In yet further embodiments, said buffer is present at a concentration of from 5 to 200 mM, from 15 to 200 mM, from 5 to 100 mM, from 15 to 100 mM, from 5 to 50 mM, from 15 to 50 mM, from 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, or any range in between. In some embodiments, the buffer is HEPES at a concentration of 5 to 200 mM, or any range between these values. In some embodiments, the buffer is citrate at a concentration of 5 to 200 mM, or any range between these values. In some embodiments, the buffer is sodium phosphate at a concentration of 5 to 200 mM, or any range in between.

In additional embodiments of the invention, said liposome composition comprises

- 19 047345 one or more lyoprotectors or cryoprotectors. In some embodiments, said cryoprotectant is mannitol, trehalose, sorbitol, or sucrose. In some embodiments, said lyoprotectant and/or cryoprotectant is present in said composition at a concentration of 1 to 20%, or 5 to 20% by weight, or any range in between.

In additional embodiments of the invention, the proposed liposome composition contains an isotonic agent. In some embodiments, the specified concentration (percent by weight) of the isotonic agent is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range between these values. In some embodiments, the liposome composition contains a sugar (eg, trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.). In additional embodiments, the specified concentration (percent by weight) of sugar is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range in between. values.

In some embodiments of the invention, the proposed liposome composition contains trehalose. In additional embodiments, the specified concentration (percent by weight) of trehalose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, 5-20%, or 1-50%, or any range between these values. In still further embodiments, the said concentration (percent by weight) of trehalose is 1-15% or any range therebetween. In a further embodiment, trehalose is present at a concentration of from about 5 to 20% by weight of trehalose or any combination of one or more lyoprotectants or cryoprotectants in a total concentration of from 5 to 20%. In some embodiments, said pH of the liposome composition is pH 6 to 7.5, 6.5 to 7.5, 6.7 to 7.5, or 6.3 to 7.0, or any range between these values.

In some embodiments, the liposome composition contains dextrose. In some embodiments, the specified concentration (percent by weight) of dextrose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, 5-20%, or 1-50%, or any range between these values. In specific embodiments of the invention, the specified concentration (percent by weight) of dextrose is 1-20% or any range between these values. In a further embodiment, dextrose is present at a concentration of 1 to 20% by weight of dextrose or any combination of one or more lyoprotectants or cryoprotectants in a total concentration of 1 to 20% or 5 to 20%, or any range between these values.

In some embodiments, provided herein is a liposome composition comprising a liposome encapsulating an ionizable carotenoid salt. In some embodiments, the composition comprises a liposome encapsulating a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said liposome is pegylated. In some embodiments, the liposome is targeted. In some embodiments, the liposome is non-pegylated and targeted. In some embodiments, the liposome is non-pegylated and non-targeted. In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 molecules of said ionizable carotenoid. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 molecules of said ionizable carotenoid, or any range between these values. In additional embodiments, said encapsulated ionizable carotenoid is trans-crocetin, trans-norbixin, or the ionizable carotenoid provided in claims [1] to [8] and/or FIG. 1A-1D in this document. In some embodiments, the liposome encapsulates a plurality of carotenoids. In further embodiments, said liposome encapsulates a plurality of ionizable carotenoids (e.g., a combination of trans-crocetin, trans-norbixin, and/or one or more of the ionizable carotenoids provided in claims [1]-[8] and/or FIG. 1A- 1D in this document).

In some embodiments, provided herein is a liposome composition comprising a non-PEGylated, non-targeted liposome encapsulating an ionizable carotenoid salt. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said encapsulated ionizable carotenoid is trans-crocetin. In some embodiments, said encapsulated ionizable carotenoid is trans-norbixin. In some embodiments, said encapsulated ionizable carotenoid is a carotenoid as set forth in claims [1] to [8] and/or FIG. 1A-1D in this document. In some embodiments, said non-PEGylated, non-targeted liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 molecules of said carotenoid. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or

- 20 047345 from 1000 to 5000 molecules of the specified ionizable carotenoid, or any range between these values. In additional embodiments, the liposome encapsulates a plurality of carotenoids. In some embodiments, said liposome encapsulates a plurality of ionizable carotenoids (e.g., a combination of trans-crocetin, trans-norbixin, and/or one or more of the ionizable carotenoids provided in claims [1]-[8] and/or FIGS. 1A-1D this document).

In some embodiments, provided herein is a liposome composition comprising a pegylated liposome encapsulating an ionizable carotenoid salt. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said PEGylated liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 molecules of said ionizable carotenoid. In some embodiments, said PEGylated liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 molecules of said carotenoid, or any range between these values. In some embodiments, said encapsulated ionizable carotenoid is trans-crocetin. In some embodiments, said encapsulated ionizable carotenoid is trans-norbixin. In some embodiments, said encapsulated ionizable carotenoid is a carotenoid as set forth in claims [1] to [8] and/or FIG. 1A-1D in this document. In some embodiments, said pegylated liposome encapsulates a plurality of carotenoids. In further embodiments, said liposome encapsulates a plurality of ionizable carotenoids (e.g., a combination of trans-crocetin, trans-norbixin, and/or one or more of the ionizable carotenoids provided in claims [1]-[8] and/or FIG. 1A- 1D in this document). In some embodiments, said pegylated liposome is targeted. In some embodiments, the pegylated liposome is non-targeted.

In some embodiments, provided herein is a liposome composition comprising a targeted liposome encapsulating an ionizable carotenoid salt. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, the targeted liposome contains a targeting moiety having a specific affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the outer portion of the liposome. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, the targeting moiety binds a surface antigen with a dissociation constant (Kd) ranging from 50x10 ^-12 to 10x10 ^-6 as determined by the BIACORE® assay. In additional embodiments, Kd is determined by surface plasmon resonance in which the antigen containing the epitope is immobilized, the targeting moiety serves as the analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl at 37°C WITH. In some embodiments, the targeting moiety comprises a protein or folate conjugate with a specific affinity for one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ). In some embodiments, said targeted liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. Said targeted liposome may be pegylated or non-pegylated.

In some embodiments, the targeted liposome is pegylated and encapsulates a salt of an ionizable carotenoid. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, the targeted PEGylated liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said targeted PEGylated liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 molecules of said ionizable carotenoid, or any range between these values. In some embodiments, said encapsulated ionizable carotenoid is trans-crocetin. In some embodiments, said encapsulated ionizable carotenoid is trans-norbixin. In some embodiments, said encapsulated ionizable carotenoid is a carotenoid as set forth in claims [1]-[8] and/or FIG. 1A-1D in this document.

In other embodiments, the targeted liposome is non-pegylated and encapsulates a salt of an ionizable carotenoid. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said targeted non-PEGylated liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In further embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 molecules of said ionizable carotenoid, or any range between these values. In some embodiments, said encapsulated ionizable carotenoid is transcrocetin. In some embodiments, said encapsulated ionizable carotenoid is trans-norbixin. In some embodiments, said encapsulated ionizable carotenoid is a carotenoid as set forth in claims [1] to [8] and/or FIG. 1A-1D in this document. In additional embodiments, the targeted non-pegylated liposome encapsulates a plurality of carotenoids. In further embodiments, said liposome encapsulates a plurality of ionizable carotenoids (e.g., a combination of trans-crocetin, trans-norbixin, and/or one or more of the ionizable carotenoids provided in claims [1]-[8] and/or FIG. 1A- 1D in this document).

In some embodiments, provided herein is a PEGylated liposome composition comprising a liposome encapsulating an ionizable carotenoid salt as provided in any one of, and encapsulating the ionizable carotenoid salt. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 molecules of said ionizable carotenoid. In some embodiments, said PEGylated liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 molecules of said ionizable carotenoid, or any range between these values. In additional embodiments, the liposome contains a plurality of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as two or more ionizable carotenoids as set forth in claims [1] to [8] and/or FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a pegylated and targeted liposome encapsulating an ionizable carotenoid salt. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said pegylated and targeted liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said pegylated and targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 molecules of said ionizable carotenoid, or any range between these values. In additional embodiments, said liposome contains a plurality of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as two or more ionizable carotenoids as set forth in claims [1] to [8] and/or FIG. Ία-ID in this document.

In additional embodiments, provided herein is a liposome composition comprising a pegylated and non-targeted liposome encapsulating an ionizable carotenoid salt. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said pegylated and non-targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1,000 to 5,000 molecules of said ionizable carotenoid, or any range between these values. In additional embodiments, the pegylated and non-targeted liposome contains a variety of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more ionizable carotenoids, for example, two or more ionizable carotenoids as provided in claims [1] to [8] and/or FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a non-PEGylated liposome encapsulating an ionizable carotenoid salt. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 molecules of said ionizable carotenoid. In some implementations

- 22 047345 version of the invention, said non-PEGylated liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 molecules of said ionizable carotenoid, or any range between these values. In additional embodiments, the non-pegylated liposome contains a plurality of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as two or more ionizable carotenoids as set forth in claims [1] to [8] and/or FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a non-PEGylated and targeted liposome encapsulating an ionizable carotenoid salt and encapsulating an ionizable carotenoid salt. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said non-PEGylated and targeted liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said non-PEGylated and targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1,000 to 5,000 molecules of said ionizable carotenoid, or any range between these values. In additional embodiments, the non-pegylated and targeted liposome contains a variety of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as two or more ionizable carotenoids as set forth in claims [1] to [8] and/or FIG. Ία-ID in this document.

In additional embodiments, provided herein is a liposome composition comprising a non-PEGylated and non-targeted liposome that encapsulates an ionizable carotenoid salt and encapsulates an ionizable carotenoid salt. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [8]. In some embodiments, said non-PEGylated and non-targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1,000 to 5,000 molecules of said ionizable carotenoid, or any range between these values. In additional embodiments, the non-pegylated and non-targeted liposome contains a variety of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as two or more ionizable carotenoids as set forth in claims [1] to [8] and/or FIG. 1A-1D in this document.

In some embodiments, provided herein is a pegylated liposome composition comprising a liposome encapsulating a trans-crocetin salt. In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 trans-crocetin molecules. In some embodiments, said PEGylated liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 trans-crocetin molecules, or any range between these values. In additional embodiments, said liposome contains a plurality of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the ionizable carotenoids provided in paragraphs [1]-[8] and/or FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a pegylated and targeted liposome encapsulating a transcrocetin salt. In some embodiments, said pegylated and targeted liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said pegylated and targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1,000 to 5,000 trans-crocetin molecules, or any range between these values. In additional embodiments, the pegylated and targeted liposome contains a variety of carotenoids. In further embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the ionizable carotenoids provided in paragraphs [1]-[8] and/or FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a pegylated and non-targeted liposome encapsulating a transcrocetin salt. In some embodiments, said pegylated and non-targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1,000 to 5,000 molecules of transcrocetin, or any range between these values. In additional embodiments, the pegylated and non-targeted liposome contains a variety of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more ionizable carotenoids as set forth in claims [1][8] and/or in FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a non-PEGylated liposome encapsulating a trans-crocetin salt. Somehow

- 23 047345 In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000 or less than 5,000 trans-crocetin molecules. In some embodiments, said non-pegylated liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 trans-crocetin molecules, or any range between these values. In additional embodiments, the non-pegylated liposome contains a plurality of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the ionizable carotenoids provided in paragraphs [1]-[8] and/or FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a non-PEGylated and targeted liposome encapsulating a transcrocetin salt. In some embodiments, said non-PEGylated and targeted liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said non-pegylated and targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1,000 to 5,000 trans-crocetin molecules, or any range between these values. In additional embodiments, the non-pegylated and targeted liposome contains a variety of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the carotenoids proposed in paragraphs [1] to [8] and/or in FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a non-PEGylated and non-targeted liposome encapsulating a trans-crocetin salt. In some embodiments, said non-pegylated and non-targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 molecules of transcrocetin, or any range between these values. In additional embodiments, the non-pegylated and non-targeted liposome contains a variety of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the carotenoids proposed in paragraphs [1] to [8] and/or in FIG. 1A-1D in this document.

In some embodiments, provided herein is a pegylated liposome composition comprising a liposome encapsulating a trans-norbixin salt. In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 trans-norbixin molecules. In some embodiments, said PEGylated liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 trans-norbixin molecules, or any range between these values. In additional embodiments, said liposome contains a plurality of carotenoids. In further embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the ionizable carotenoids provided in paragraphs [1]-[8] and/or FIG. Ία-ID in this document.

In additional embodiments, provided herein is a liposome composition comprising a pegylated and targeted liposome encapsulating a transnorbixin salt. In some embodiments, said pegylated and targeted liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said pegylated and targeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 trans-norbixin molecules, or any range between these values. In additional embodiments, the pegylated and targeted liposome contains a variety of carotenoids. In further embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the ionizable carotenoids provided in paragraphs [1]-[8] and/or FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a pegylated and non-targeted liposome encapsulating a transnorbixin salt. In some embodiments, said pegylated and non-targeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 transnorbixin molecules, or any range between these values. In additional embodiments, the pegylated and non-targeted liposome contains a variety of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the ionizable carotenoids provided in paragraphs [1]-[8] and/or FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a non-PEGylated liposome encapsulating a trans-norbixin salt. In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 trans molecules

- 24 047345 norbixin. In some embodiments, said non-pegylated liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 trans-norbixin molecules, or any range between these values. In additional embodiments, the non-pegylated liposome contains a plurality of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the ionizable carotenoids provided in paragraphs [1]-[8] and/or FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a non-PEGylated and targeted liposome encapsulating a transnorbixin salt. In some embodiments, said non-PEGylated and targeted liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said non-pegylated and targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1,000 to 5,000 trans-norbixin molecules, or any range between these values. In additional embodiments, the non-pegylated and targeted liposome contains a variety of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the carotenoids proposed in paragraphs [1] to [8] and/or in FIG. 1A-1D in this document.

In additional embodiments, provided herein is a liposome composition comprising a non-PEGylated and non-targeted liposome encapsulating a trans-norbixin salt. In some embodiments, said non-pegylated and non-targeted liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 transnorbixin molecules, or any range between these values. In additional embodiments, the non-pegylated and non-targeted liposome contains a variety of carotenoids. In additional embodiments, said liposome contains a plurality of ionizable carotenoids, such as one or more of the carotenoids proposed in paragraphs [1] to [8] and/or in FIG. 1A-1D in this document.

In some embodiments, the proposed liposome compositions comprise a liposome encapsulating one or more ionizable carotenoid salts (e.g., a divalent, trivalent, or tetravalent ionizable carotenoid salt of any one of claims [1]-[8] and/or FIGS. 1A-1D) and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, the liposome composition contains trehalose. In some embodiments, the liposome composition contains from 1 to 50% by weight trehalose. In some embodiments, the liposome composition contains HBS at a concentration of 1 to 200 mM and a pH of 2 to 8. In some embodiments, the liposome composition has a pH of 5-8 or any range therebetween. In some embodiments, said liposome composition has a pH of 6-7 or any range between these values.

In additional embodiments, the provided liposome compositions comprise a liposome encapsulating a trans-crocetin salt and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, said liposome solution contains trehalose. In some embodiments, said liposome solution contains from 1 to 50% by weight trehalose. In some embodiments, said liposome solution contains HBS at a concentration of 1 to 200 mM and a pH of 2 to 8, or any range between these values. In some embodiments, said liposome solution has a pH of 5-8 or any range between these values. In some embodiments, said liposome solution has a pH of 6-7 or any range between these values. In some embodiments, the provided trans-crocetin salt is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said trans-crocetin salt is CTC. In some embodiments, said trans-crocetin salt is MTC.

In further embodiments, the provided liposome compositions comprise a liposome encapsulating a trans-norbixin salt and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, said liposome solution contains trehalose. In some embodiments, said liposome solution contains from 1 to 50% by weight trehalose. In some embodiments, said liposome solution contains HBS at a concentration of 1 to 200 mM and a pH of 2 to 8, or any range between these values. In some embodiments, said liposome solution has a pH of 5-8 or any range between these values. In some embodiments, said liposome solution has a pH of 6-7 or any range between these values. In some embodiments, the provided trans-norbixin salt is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, the iso

- 25 047345 bretenia, said trans-norbixin salt is CTN. In some embodiments, said trans-norbixin salt is MTN.

The proposed liposomes contain an aqueous medium enclosed by at least one lipid bilayer. When lipids that contain a hydrophilic head group are dispersed in water, they can spontaneously form bilayer membranes called lamellae. These lamellae consist of two monolayer sheets of lipid molecules with their non-polar (hydrophobic) surfaces facing each other and their polar (hydrophilic) surfaces facing the aqueous environment. The term liposome includes unilamellar vesicles that are composed of a single lipid bilayer and typically have a diameter ranging from about 20 to about 500 nm, from about 50 to about 300 nm, from about 50 to about 150 nm, from about 30 to about 1000 nm, about 30 to about 175 nm, about 80 to about 400 nm, or about 80 to about 120 nm. Liposomes can also be multilamellar, which typically have a diameter ranging from 0.5 to 10 μm with 2-100 concentric lipid bilayers alternating with aqueous phase layers. In some embodiments, liposomes may contain multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs), and small unilamellar vesicles (SUVs). The lipids of said liposome may be cationic, zwitterionic, neutral, or anionic, or any mixture thereof.

Any suitable combination of lipids can be used to prepare the liposomes and lipid nanoparticles provided herein. These lipid compositions can be tailored to influence characteristics such as leak rate, stability, particle size (eg, liposome diameter), zeta potential, protein binding, in vivo circulation, and/or tissue or organ accumulation. For example, DSPC and/or cholesterol can be used to reduce leakage from liposomes. Negatively or positively charged lipids, such as DSPG and/or DOTAP, can be contained to influence the surface charge of liposomes or lipid nanoparticles. In some embodiments, the lipid compositions may contain about ten or less types of lipids, or about five or less types of lipids, or about three or less types of lipids. In some embodiments, the molar content (mol %) of a particular type of lipid present typically includes from about 0 to about 10%, from about 10 to about 30%, from about 30 to about 50%, from about 50 to about 70%, about 70 to about 90%, about 90 to 100% of the total lipids present in the liposomal or lipid nanoparticle. In some embodiments, the therapeutic liposome contains 40-80 mol% DSPC, 5-50 mol% cholesterol, 0-30 mol% DSPG, and 0-10 mol% DSPE-PEG(2000). In some embodiments, said attack liposome contains 40-70 mol% DPPC, 5-20 mol% cholesterol, 0-20 mol% DOTAP, and 20-40 mol% TPGS.

Depending on the desired application, the particle size (diameter) of the liposome can be adjusted. For example, when the liposome is intended to be delivered to cancerous tissue or inflamed tissue by an enhanced permeability and retention (EPR) effect as an injection product or the like, it is preferable that the diameter of the liposomes be 20-500 nm, 30-175 nm, or 50-150 nm, or any range between these values. In the case where it is intended to transfer the liposome to a macrophage, it is preferable that the diameter of the liposome be from 30 to 1000 nm, or from 80 to 400 nm, or any range between these values. In the case where the liposome composition is to be used as an oral drug or transdermal drug, the particle size of the liposome can be set to several microns. It should be noted that in normal tissue, vessel walls serve as barriers (since the vessel walls are densely composed of vascular endothelial cells), and microparticles such as supermolecules and liposomes of this size cannot be distributed in a given tissue. However, in diseased tissues, the vessel walls are loosely composed (because there are gaps between the endothelial cells of the vessels), increasing vascular permeability, and supermolecules and microparticles can be distributed to the tissue outside such vessels (increased permeability). In addition, the lymphatic system is well developed in normal tissue, but it is known that the lymphatic system is not developed in diseased tissue, and that supermolecules or microparticles, once incorporated, are not processed through the general system, but are retained in diseased tissue (enhanced retention), which forms basis of the EPR effect (Wang et al., Ann. Rev. Med. 63:185-198 (2012); Peer et al., Nat. Nanotech. 2:751-760 (2007); Governor, Exp. Opin. Drug Deliv 8:565-580 (2011); Int. J. Nanomed. 3:2129 (2008); Torchilin Front. Biosci. 16:1388-1412; Baryshnikov Vest. Med. Nauk. 23-31; . Thus, it is possible to control the pharmacokinetics of liposomes by adjusting the liposome particle size (diameter).

The size of said liposomes in the proposed liposome compositions may vary, for example, from 0.5 nm to 10 μm, or from 20 nm to 5 μm, depending on the composition of the phospholipid, the method used for their preparation and the intended therapeutic use of said liposomes. In some embodiments, the said average diameter of the liposomes in the present liposome composition is 20 to 500 nm, 50 to 200 nm, 20 to 200 nm, or any range between these values. In some embodiments, said average diameter of the liposome

- 26 047345 we are from 80 to 120 nm or any range between these values (for example, 85-115 nm, 90-110 nm, 95-110 nm or 95-105 nm). In some embodiments, the average diameter of the liposomes in the present liposome composition is 10-250 nm or any range between (e.g., 10-225 nm, 10-200 nm, 10-175 nm, 10-150 nm, 40-150 nm , 50-150 nm, 60-150 nm, 70-150 nm, 80-150 nm, 90-150 nm, 100-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, 10-50 nm , 50-100 nm, 50-90 nm, 50-80 nm, 50-70 nm, 50-60 nm, 60-100 nm, 60-90 nm, 60-80 nm, 60-70 nm, 70-100 nm , 70-90 nm, 70-80 nm, 80-100 nm, 80-90 nm or 90-100 nm). In some embodiments, the said average diameter of the liposomes in the present liposome composition is 100-250 nm or any range between these values (for example, 100-225 nm, 100-200 nm, 100-175 nm, or 100-150 nm). In other embodiments, the said average diameter of the liposomes in the present liposome composition is 10-100 nm, or any range between these values (for example, about 10-90 nm, 10-80 nm, 10-70 nm, 10-60 nm, or 10- 50 nm). In some embodiments, said average diameter of the liposomes in the present liposome composition is less than about 500, 450, 400, 350, 300, 250, 200, 150, 145, 150, 135, 130, 125, 120, 115, 110, 105 , 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45 or 40 nm. Dynamic laser light scattering is a method used to measure the diameter of liposomes, which is well known to those skilled in the art. Diameter of liposomes (DLP) can typically be determined by any methods and equipment known in the art, including, for example, dynamic laser light scattering (Coulter N4 Particle Size Analyzer), Zetasizer Nano ZSP (Malvern, UK) and ELS-8000 (Otsuka Electronics Co., Ltd.).

In some embodiments, the proposed liposome compositions have a monodisperse size (diameter) distribution. Monodispersity and uniform size distribution are used interchangeably herein and describe a variety of liposomal nanoparticles or microparticles, wherein the particles have the same or nearly the same diameter. As used herein, monodisperse distribution refers to a particle distribution in which 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 86, 88, 89, 90, 91, 92 , 93, 94, 95 or more of the particle distribution lies within 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 or 10% of the mass average diameter.

In some embodiments, said population of liposomes in the proposed liposome composition is relatively homogeneous. In some embodiments, said population of liposomes in the proposed liposome composition is heterogeneous. The polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, for example, the particle size (diameter) distribution of nanoparticle compositions. A low (eg, less than 0.3) polydispersity index usually indicates a narrow particle size distribution. In some embodiments, said liposome population in a liposome composition of the invention has a polydispersity index of 0 to 0.25, or 0.01 to 0.1, or any range in between (e.g., 0.001 to 0.2, 0.005 to 0.1, from 0.005 to 0, from 0.005 to 0.09, from 0.009 to 0.09, from 0.01 to 0.08, from 0.02 to 0.09, or from 0.02 to 0, 07, or any range between these values).

In some embodiments, the liposomes within said population of liposomes in the proposed liposome composition differ in their lipid composition, molar ratio of lipid components, size, charge (zeta potential), targeting ligands, and/or combinations thereof.

The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable since more highly charged particles may interact undesirably with cells, tissues and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition may be from about -10 to about 20 mV, from about -10 to about 15 mV, from about -10 to about 10 mV, from about -10 to 5 mV, from about - 10 to about 0 mV, about -10 to about -5 mV, about -5 to about 20 mV, about -5 to about 15 mV, about -5 to about 10 mV, about -5 to about 5 mV, about -5 to about 0 mV, about 0 to about 20 mV, about 0 to about 15 mV, about 0 to about 10 mV, about 0 to about 5 mV, about 5 to about 20 mV , from about 5 to about 15 mV or from about 5 to 10 mV. The zeta potential of liposomes is typically determined by methods and equipment known in the art, including, for example, dynamic light scattering (Zetasizer Nano ZSP, Malvern, UK) and laser Doppler electrophoresis.

Encapsulation efficiency of a therapeutic and/or prophylactic agent, such as an ionizable carotenoid (eg, trans-crocetin), refers to the amount of therapeutic and/or prophylactic agent encapsulated or otherwise associated with the nanoparticle composition after preparation, relative to its original amount. Said encapsulation efficiency is preferably high (eg about 100%). Encapsulation efficiency can be

- 27 047345 is measured, for example, by comparing the amount of therapeutic and/or prophylactic agent in a solution containing a nanoparticle composition before and after removal of the unencapsulated therapeutic and/or prophylactic agent. For the liposome compositions described herein, the encapsulation efficiency of the ionizable carotenoid (e.g., trans-crocetin) can be at least 50%, e.g., 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96 , 97, 98 or 99%. In some embodiments, said encapsulation efficiency is at least 80%. In certain embodiments of the invention, said encapsulation efficiency is at least 90%. In certain embodiments of the invention, said encapsulation efficiency is at least 95%. In certain embodiments of the invention, said encapsulation efficiency is at least 98%.

In additional embodiments, the proposed liposome compositions comprise liposomes encapsulating a salt of an ionizable carotenoid. In some embodiments, said ionizable carotenoid (eg, trans-crocetin)/lipid ratio of the present liposome composition is from 1 to 1000 g/mol, or any range between these values. In some embodiments, said ionizable carotenoid/lipid ratio of the liposome composition is 10 to 200 g/mol, 10 to 150 g/mol, 10 to 100 g/mol, 20 to 200 g/mol, 20 to 150 g/mol, from 20 to 100 g/mol, from 30 to 200 g/mol, from 30 to 150 g/mol, from 30 to 100 g/mol, from 40 to 200 g/mol, from 40 to 150 g/mol mol, 40 to 100 g/mol, 50 to 200 g/mol, 50 to 150 g/mol, or 50 to 100 g/mol, or any range between these values. In some embodiments, said ionizable carotenoid/lipid ratio is from 30 to 90 g/mol or any range between these values. In some embodiments, said ionizable carotenoid/lipid ratio is 30 to 50 g/mol, 40 to 60 g/mol, 50 to 70 g/mol, 60 to 80 g/mol, or 70 to 90 g /mol, or any range between these values. In some embodiments, said liposome encapsulates a salt of an ionizable carotenoid according to any one of claims [1] to [7]. In some embodiments, said liposome encapsulates the ionizable carotenoid salt provided in any of FIG. 1A-1D.

In some embodiments, the liposome composition comprises liposomes encapsulating a trans-crocetin salt. In some embodiments, said trans-crocetin/lipid ratio in the liposome composition of the invention is from 1 to 1000 g/mol or any range between these values. In some embodiments, said trans-crocetin/lipid ratio of the liposome composition is from 10 to 200 g/mol, from 10 to 150 g/mol, from 10 to 100 g/mol, from 20 to 200 g/mol, from 20 to 150 g/mol, from 20 to 100 g/mol, from 30 to 200 g/mol, from 30 to 150 g/mol, from 30 to 100 g/mol, from 40 to 200 g/mol, from 40 to 150 g /mol, 40 to 100 g/mol, 50 to 200 g/mol, 50 to 150 g/mol, or 50 to 100 g/mol, or any range between these values. In some embodiments, said trans-crocetin/lipid ratio is from 30 to 90 g/mol or any range between these values. In some embodiments, said transcrocetin/lipid ratio is 30 to 50 g/mol, 40 to 60 g/mol, 50 to 70 g/mol, 60 to 80 g/mol, or 70 to 90 g/mol. mole, or any range between these values.

In some embodiments, the liposome composition comprises liposomes encapsulating a trans-norbixin salt. In some embodiments, said trans-norbixin/lipid ratio of the proposed liposome composition is from 1 to 1000 g/mol or any range between these values. In some embodiments, said trans-norbixin/lipid ratio of the liposome composition is from 10 to 200 g/mol, from 10 to 150 g/mol, from 10 to 100 g/mol, from 20 to 200 g/mol, from 20 to 150 g/mol, from 20 to 100 g/mol, from 30 to 200 g/mol, from 30 to 150 g/mol, from 30 to 100 g/mol, from 40 to 200 g/mol, from 40 to 150 g /mol, 40 to 100 g/mol, 50 to 200 g/mol, 50 to 150 g/mol, or 50 to 100 g/mol, or any range between these values. In some embodiments, said trans-norbixin/lipid ratio is from 30 to 90 g/mol or any range between these values. In some embodiments, said transnorbixin/lipid ratio is 30 to 50 g/mol, 40 to 60 g/mol, 50 to 70 g/mol, 60 to 80 g/mol, or 70 to 90 g/mol. mole, or any range between these values.

In some embodiments, the liposome composition is buffered using a zwitterionic buffer. Accordingly, said zwitterionic buffer is an aminoalkanesulfonic acid or a suitable salt. Examples of aminoalkanesulfone buffers include, but are not limited to, HEPES, HEPPS/EPPS, MOPS, MOBS and PIPES. Preferably, said buffer is a pharmaceutically acceptable buffer suitable for use in humans, for example, for use in a commercial injection product. Most preferably, said buffer is HEPES. Said liposome composition may contain AGP.

- 28 047345

In some embodiments, the liposome composition is buffered using HEPES. In some embodiments, the liposome composition is buffered with HEPES at a pH of about 7.

In some embodiments, said pharmaceutical composition is a liposome composition comprising a cationic liposome. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between. In some embodiments, said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, 30 to 175 nm, 50 to 200 nm, 50 to 150 nm, or any range in between. In some embodiments, said cationic liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said liposome composition contains at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or greater than 75 wt.% wt. ionizable carotenoid (for example, trans-crocetin). In some embodiments, during the preparation of the liposome composition, at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 75, 80, 85 90, 95 or 97% of the starting material of the ionizable carotenoid is encapsulated (entrapped) in the liposomes of the liposome composition. In additional embodiments, said ionizable carotenoid (eg, trans-crocetin) encapsulated in a liposome is contained in a HEPES buffer solution within the liposome. In additional embodiments, said liposome contains at least one OxPARS.

In some embodiments of the invention, the proposed pharmaceutical composition is a liposome composition containing an anionic or neutral liposome. In some embodiments, the liposome composition comprises a liposome that has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In some embodiments, said anionic or neutral liposome has a diameter of 20 to 500 nm, 20 to 200 nm, 30 to 175 nm, 50 to 150 nm, or any range in between. In other embodiments, said anionic or neutral liposome has a diameter of 80 to 120 nm, or any range in between. In some embodiments, said anionic liposome has a diameter of 20 to 500 nm, 20 to 200 nm, 30 to 175 nm, 50 to 150 nm, or any range in between. In further embodiments, said anionic liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said neutral liposome has a diameter of 20 to 500 nm, 20 to 200 nm, 30 to 175 nm, 50 to 150 nm, or any range in between. In some embodiments, said neutral liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said pharmaceutical composition contains at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more wt.% wt. ionizable carotenoid (for example, trans-crocetin). In some embodiments, during the preparation of the liposome composition, at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or greater than 75% from the starting material, the ionizable carotenoid (eg trans-crocetin) is encapsulated (entrapped) in liposomes. In some embodiments, said liposome composition contains at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or greater than 75 wt.% wt. said ionizable carotenoid (eg trans-crocetin). In some embodiments, said anionic or neutral liposome composition comprises at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or greater than 75% wt./wt. said ionizable carotenoid (eg trans-crocetin). In some embodiments, the liposome composition contains at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or greater than 75% w/w . said ionizable carotenoid (eg trans-crocetin). In additional embodiments, said ionizable carotenoid (eg, trans-crocetin) is encapsulated in an anionic or neutral liposome and is contained in a HEPES buffer solution within the liposome. In additional embodiments, said liposome contains at least one OxPARS.

In some embodiments, the proposed pharmaceutical composition is a liposome composition comprising a liposome that contains at least one OxPARS. In some embodiments, said OxPARS is oxidized and/or phospholipid-containing fragmented oxidized sn-2 residues. In some embodiments, said OxPARS is an oxidized phospholipid,

- 29 047345 containing a five-carbon sn-2 residue bearing an omega-aldehyde or omega-carboxyl group. In some embodiments, said OxPAPC is an oxidized phospholipid selected from HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC. In some embodiments, said OxPARS is an epoxysoprostane-containing phospholipid. In some embodiments, said OxPAPC is a PGPC. In some embodiments, said liposome contains at least 0.01, 0.1, 1, 5, 10, 15, 20, 25, or at least 30% OxPARS. In some embodiments, said liposome composition has a cationic liposome that contains 0.01-35%, 0.1-30%, 1-25%, 3-20%, or 5-15% OxPARS, or any range in between. values. In some embodiments, the liposome composition comprises a cationic liposome. In some embodiments, the liposome composition comprises a neutral liposome. In some embodiments, the liposome composition comprises an anionic liposome. In additional embodiments, said liposome composition comprises at least one OxPAPC-containing liposome that has a diameter of 20 to 500 nm, 20 to 200 nm, 30 to 175 nm, 50 to 150 nm, or any range in between. . In further embodiments, said liposome composition comprises at least one OxPAPC-containing liposome that has a diameter between 80 and 120 nm, or any range in between.

In some embodiments, the proposed pharmaceutical composition is a liposome composition comprising a cationic liposome that contains at least 0.01, 0.1, 1, 5, 10, 15, 20, 25, or at least 30% OxPARS. In some embodiments, said liposome composition has a cationic liposome that contains 0.01-35, 0.1-30, 1-25, 3-20, or 5-15% OxPARS, or any range between these values. In some embodiments, said liposome contains at least 0.01, 0.1, 1, 5, 10, 15, 20, 25, or at least 30% OxPARS. In some embodiments, the liposome composition comprises a cationic liposome that contains about 10% OxPARS. In some embodiments, the liposome composition has a cationic liposome that contains at least 0.01, 0.1, 1, 5, 10, 15, 20, 25, or at least 30% PGPC. In some embodiments, said liposome contains 0.01-35%, 0.1-30%, 1-25%, 3-20%, or 5-15% PGPC, or any range between these values. In some embodiments, the liposome composition comprises a cationic liposome that contains about 10% PGPC.

In some embodiments, said pharmaceutical composition is a liposome composition comprising an anionic or neutral liposome that contains at least 0.01, 0.1, 1, 5, 10, 15, 20, 25, or at least 30% OxPARS . In some embodiments, said liposome composition has an anionic or neutral liposome that contains 0.01-35%, 0.1-30%, 1-25%, 3-20%, or 5-15% OxPARS, or any range between these values. In some embodiments, said liposome contains at least 0.01, 0.1, 1, 5, 10, 15, 20, 25, or at least 30% OxPARS. In some embodiments, the liposome composition comprises an anionic or neutral liposome that contains about 10% OxPARS. In some embodiments, the liposome composition has an anionic or neutral liposome that contains at least 0.01, 0.1, 1, 5, 10, 15, 20, 25, or at least 30% PGPC. In some embodiments, said liposome contains 0.01-35%, 0.1-30%, 1-25%, 3-20%, or 5-15% PGPC, or any range between these values. In some embodiments, the liposome composition comprises an anionic or neutral liposome that contains about 10% PGPC.

In some embodiments, the pharmaceutical composition is a liposome composition comprising a neutral liposome that contains at least 0.01, 0.1, 1, 5, 10, 15, 20, 25, or at least 30% OxPARS. In some embodiments, said OxPARS-containing neutral liposome composition contains 0.01-35%, 0.1-30%, 1-25%, 3-20%, or 5-15% OxPARS, or any range between these values. . In some embodiments, the composition of neutral liposomes containing OxPARS contains about 10% OxPARS. In some embodiments, said neutral OxPAPC-containing liposome composition contains at least 0.01, 0.1, 1, 5, 10, 15, 20, 25, or at least 30% PGPC. In some embodiments, said composition of neutral liposomes containing PGPC contains 0.01-35%, 0.1-30%, 1-25%, 3-20%, or 5-15% PGPC, or any range in between. values. In some embodiments, the OxPAPC-containing neutral liposome composition contains about 10% PGPC.

In further embodiments, said liposome in the liposome composition is pegylated.

In some embodiments of the invention, the proposed pharmaceutical composition is a non-targeted composition of liposomes, i.e. said liposomes in the composition

- 30 047345 liposomes do not have a specific affinity for an epitope (eg, a surface antigen epitope) expressed on the surface of the target cell of interest. In additional embodiments, said non-targeted liposome composition is pegylated.

In some cases, accumulation of liposomes at the target site may be due to the increased permeability and retention characteristics of certain tissues, such as cancer tissues. Accumulation in this manner is often dependent on the size of the liposomes and may not require special targeting functionality. In other embodiments of the invention, the proposed liposomes contain a targeting agent. In general, targeting agents can bind to any target of interest, such as a target associated with an organ, tissue, cell, extracellular matrix, or intracellular region. In certain embodiments of the invention, the target may be associated with a specific disease condition, such as a cancer condition. In some embodiments, said targeting component may be specific for a single target, such as a receptor. Suitable targets may include, but are not limited to, nucleic acids such as DNA, RNA, or modified derivatives thereof. Suitable targets may also include, but are not limited to, a protein such as an extracellular protein, receptor, cell surface receptor, tumor marker, transmembrane protein, enzyme or antibody. Suitable targets may include a carbohydrate, such as a monosaccharide, disaccharide or polysaccharide, which may, for example, be present on the surface of a cell.

In certain embodiments, the targeting agent may include a targeting ligand (eg, an RGD-containing peptide), a small molecule or targeting ligand mimetic (eg, a peptidomimetic ligand), or an antibody or antibody fragment specific for a particular target. In some embodiments, the targeting agent may further include folic acid derivatives, B-12 derivatives, integrin RGD peptides, NGR derivatives, somatostatin derivatives, or peptides that bind to the somatostatin receptor, such as octreotide and octreotate, and the like. In some embodiments, said targeting agents include an aptamer. Aptamers can be designed to bind to a target of interest. Aptamers may include, for example, DNA, RNA and/or peptides, and certain aspects of aptamers are known in the art. (See, for example, Klussman, Ed., The Aptamer Handbook, Wiley-VCH (2006); Nissenbaum, Trends in Biotech. 26(8): 442-449 (2008)).

In other embodiments, said liposome composition comprises a targeted liposome, i.e. said liposome contains a targeting moiety having specific affinity for an epitope (eg, surface antigen or other molecule) on the target cell of interest. In some embodiments, said targeting moiety of said liposome is not covalently attached to said liposome. In other embodiments, said targeting moiety of said liposome is attached to one or both of the PEG and the outer portion of the liposome. In additional embodiments, the targeted liposome is pegylated. The functions of the targeting moiety of said targeted liposome may include, but are not limited to, targeting the liposome to a cell of interest in vivo or in vitro; interaction with a surface antigen for which the targeting fragment has a specific affinity, as well as delivery of the liposome payload (for example, trans-crocetin) to the desired area or cell.

Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen binding fragments of antibodies, scaffold proteins, polypeptides and peptides. In some embodiments, the targeting moiety is a polypeptide. In additional embodiments, said targeting moiety is a polypeptide that contains at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues. In some embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In additional embodiments, the targeting fragment includes one or more of the group consisting of: an antibody, a humanized antibody, an antigen binding fragment of an antibody, a single chain antibody, a single domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody. In some embodiments, the targeting moiety has a specific affinity for an epitope that is preferentially expressed on a target cell, such as a tumor cell, over normal or non-tumor cells. In some embodiments, the targeting moiety has a specific affinity for an epitope on a tumor cell surface antigen that is present on the tumor cell but is absent or inaccessible on a normal or non-tumor cell. In some embodiments, the targeting moiety binds to the epitope of interest with a dissociation constant (Kd) ranging from 50x10 ^-12 to 10x10 ^-6 as determined by the BIACORE® assay. In additional embodiments, Kd is determined by surface plasmon resonance, in which anti

- 31 047345 the gene containing the epitope is immobilized, the targeting fragment serves as the analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate and 150 mM NaCl at 37°C.

In specific embodiments, the targeting moiety comprises a polypeptide that specifically binds to the folate receptor. In some embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, the targeting moiety is folate conjugated to the surface of the liposome (eg, folate-PEG conjugate) or a folic acid derivative. In some embodiments, said folate receptor bound by said targeting moiety is one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-α, FOLR|). folate receptor beta (FR-β, FOLR2) and folate receptor delta (FR-δ, FOLR4). In some embodiments, said folate receptor bound by said targeting moiety is folate receptor alpha (FR-α). In some embodiments, said folate receptor bound by said targeting moiety is folate receptor beta (FR-β). In some embodiments, the targeting moiety specifically binds to FR-α and FR-β.

In additional embodiments, said liposome composition comprises one or more of an immunostimulating agent, a detectable marker, and a maleimide located on at least one of the PEGs and the outer surface of the liposome. In some embodiments, said liposome of the liposome composition is cationic. In other embodiments, the liposome of the liposome composition is anionic or neutral. In additional embodiments, the liposome of said liposome composition has a diameter between 20 nm and 500 nm, or any range in between. In further embodiments, the liposome of said liposome composition has a diameter between 80 and 120 nm, or any range in between. In some embodiments, said liposome of the liposome composition is pegylated. In some embodiments, said liposome of the liposome composition is targeted. In additional embodiments, said liposome of the liposome composition is pegylated and targeted.

In some embodiments, provided herein is a pharmaceutical composition comprising an ionizable carotenoid having the formula

A polyene carotenoid-Q encapsulated in a liposome, wherein said polyene carotenoid contains:

(a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10 or more than 9 conjugated double bonds, (b) substitution with methyl or lower alkyl (C ₂ -C ₃ ), (c) 1, 2, 3 or more 3 ionizable groups; And

Q is (a) a polyvalent counterion or (b) a monovalent cation.

In some embodiments, said Polyene Carotenoid contains all trans-conjugated double bonds. In some embodiments, said Polyene Carotenoid contains 6-9 conjugated double bonds. In specific embodiments of the invention, said Polyene Carotenoid contains 7 conjugated double bonds. Said Polyene Carotenoid may be naturally occurring or synthetic. In some embodiments, said Polyene Carotenoid is naturally occurring. In other embodiments, said Polyene Carotenoid is synthetic. Said ionizable group(s) may be anionic and/or cationic. In some embodiments, said Polyene Carotenoid-Q contains two or more identical ionizable groups. In some embodiments, said Polyene Carotenoid contains all trans conjugated double bonds. In some embodiments, said Polyene Carotenoid contains 6-9 conjugated double bonds. In some embodiments, said Polyene Carotenoid contains two or more different ionizable groups. In some embodiments, said Polyene Carotenoid-Q contains one or more anionic ionizable groups. In some embodiments, said Polyene Carotenoid contains at least one ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate or phosphate group, and a hydroxamate moiety. In other embodiments, said Polyene Carotenoid-Q contains one or more cationic ionizable groups (eg, a primary, secondary or tertiary amino group, a quaternary ammonium group, a choline group, a guanidine group, or an imidazole group). In specific embodiments of the invention, said Polyene Carotenoid contains at least one cationic ionizable group, and said pharmaceutical composition is substantially free of nucleic acids.

- 32 047345

In some embodiments, Q is a multivalent counterion. In some embodiments, Q is a multivalent cationic counterion. In some embodiments, Q is a polyvalent metal cation. In some embodiments, Q is a polyvalent transition metal cation. In some embodiments, Q is a divalent counterion. In some embodiments, Q is a divalent cationic counterion. In some embodiments, Q is a divalent metal cation. In some embodiments, Q is a divalent transition metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In some embodiments, Q is Ca ²⁺ or Mg ²⁺ . In some embodiments, Q is Ca ²⁺ . In additional embodiments, said Polyene Carotenoid-Q is calcium trans-crocetinate (CTC). In some embodiments, Q is Mg ²⁴ . In additional embodiments, said Polyene Carotenoid-Q is magnesium transcrocetinate (MTS). In other embodiments, Q is a trivalent cationic counterion such as Fe ³⁴ . In some embodiments, Q is a polyvalent organic counterion. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a divalent organic cation, such as a protonated diamine.

In additional embodiments, Q is a monovalent counterion. In some embodiments, Q is a monovalent cationic counterion. In some embodiments, Q is a monovalent metal cation. In some embodiments, Q is at least one member selected from Na ⁴ , Li ⁴ or K ⁴ . In some embodiments, Q is an organic cation. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a monovalent organic cation, such as a protonated amine (eg, a protonated diamine or a protonated polyamine). In some embodiments, Q is an organic cation, such as NH ₄ ⁴ , a protonated diamine, or a protonated polyamine.

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 molecules of ionizable carotenoid. In some embodiments, said liposome contains from 10 to 100,000 molecules of ionizable carotenoid, or any range between these values. In some embodiments, said ionizable carotenoid/lipid ratio in said liposome composition is from 1 to 1000 g/mol or any range between these values. In some embodiments, said ionizable carotenoid/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g/mol, or any range between these values. In some embodiments, said liposome contains at least 0.1 to 97% trans-crocetin. In some embodiments, said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, 80 to 120 nm, or any range in between. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEGmaleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG and cholesterol-maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-TOr-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In further embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups. In some embodiments, said liposome comprises OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, said liposome contains OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)^n-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2 -(epoxycyclopentenoneIp-glycero-3-phosphocholine (PECPC), 1-palmitoyl-2-(epoxyisoprostane E2)^p-glycero-4-phosphocholine (PEIPC), 1-palmitoyl-2-glutaroyl^p-glycero-3-phosphocholine

- 33 047345 (PGPC); Gpalmitoyl-2-(9'-oxononanoyl-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-glycero-3-phosphocholine; 1-palmitoyl- 2-hexadecyl-n-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-n-glycero-3-phosphocholine and 1-palmitoyl-2-acetoyl-n-glycero-3-phosphocholine. In some embodiments, the liposome contains. PGPC. In some embodiments, said OxPAPC within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range therebetween. In some embodiments, said liposome contains a targeting moiety having a specific affinity for a surface antigen or other molecules. on the target cell of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the outer portion of the liposome, optionally the targeting moiety is attached to one or both of the PEG and the outer portion of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (ie, said liposome is pegylated). In some embodiments, said PEG has a number average molecular weight (MW) of 200 to 5000 Daltons. In additional embodiments, said liposome is anionic or neutral. In some embodiments, said liposome has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In other embodiments, said liposome is cationic. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

In some embodiments, provided herein is a pharmaceutical composition comprising an ionizable carotenoid having the formula

Q-Ri-Polyene Carotenoid-K^X encapsulated in a liposome, wherein said Polyene Carotenoid contains:

(a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10 or more than 9 conjugated double bonds, (b) substitution with methyl or lower alkyl (C ₂ -C ₃ ), (c) 1, 2, 3 or more 3 ionizable groups;

R ₁ and R 2 represent ionizable groups;

Q is (a) a polyvalent counterion or (b) a monovalent cation.

In some embodiments, said Polyene Carotenoid contains all trans-conjugated double bonds. In specific embodiments of the invention, said Polyene Carotenoid contains 6-9 conjugated double bonds. Said Polyene Carotenoid may be naturally occurring or synthetic. In some embodiments, said Polyene Carotenoid is naturally occurring. In other embodiments, said Polyene Carotenoid is synthetic. In some embodiments, R ₁ and R 2 are the same ionizable groups. In other embodiments, R ₁ and R 2 are different ionizable groups. In some embodiments, R ₁ and R ₂ are the same cationic ionizable groups. In other embodiments, R ₁ and R 2 are different cationic groups. In some embodiments, R ₁ and R ₂ are the same anionic ionizable group. In other embodiments, R ₁ and R 2 are different anionic groups. In some embodiments, R ₁ is a cationic ionizable group or an anionic ionizable group, and R2 is an anionic ionizable group or a cationic group, respectively. In some embodiments, said Polyene Carotenoid contains at least one anionic ionizable group. In some embodiments, said Polyene Carotenoid contains at least one ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate or phosphate group, and a hydroxamate moiety. In neko

- 34 047345 In other embodiments of the invention, R ₁ represents at least one ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate or phosphate group, and a hydroxamate moiety. In some embodiments, R2 is at least one ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate or phosphate group, and a hydroxamate moiety. In other embodiments, said Polyene Carotenoid-Q contains one or more cationic ionizable groups (eg, a primary, secondary or tertiary amino group, a quaternary ammonium group, a choline group, a guanidine group, or an imidazole group). In specific embodiments of the invention, said Polyene Carotenoid contains at least one cationic ionizable group, and said pharmaceutical composition is substantially free of nucleic acids.

In some embodiments, Q is a multivalent counterion. In some embodiments, Q is a multivalent cationic counterion. In additional embodiments, Q is a polyvalent metal cation. In some embodiments, Q is a polyvalent transition metal counterion. In some embodiments, Q is a divalent counterion. In some embodiments, Q is a divalent cationic counterion. In additional embodiments, Q is a divalent metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In additional embodiments, Q is Ca ²⁺ or Mg2 ⁺ . In additional embodiments, Q is Ca ²⁺ . In some embodiments, Q is Mg2 ⁺ . In other embodiments, Q is a trivalent cationic counterion such as Fe ³⁺ . In other embodiments, Q is a polyvalent organic counterion. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a divalent organic cation, such as a protonated diamine.

In some embodiments, Q is a monovalent cationic counterion. In further embodiments, Q is at least one member selected from Na ⁺ , Li ⁺ or K ⁺ . In some embodiments, Q is an organic counterion. In some embodiments, Q is a polyvalent organic cation. In further embodiments, Q is a divalent organic cation, such as a protonated diamine or a protonated polyamine. In other embodiments, Q is a monovalent organic cation, such as NH ₄ ⁺ , a protonated diamine, or a protonated polyamine.

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000 molecules of ionizable carotenoid. In some embodiments, said liposome contains from 10 to 100,000 molecules of ionizable carotenoid, or any range between these values. In some embodiments, said ionizable carotenoid/lipid ratio in said liposome composition is from 1 to 1000 g/mol or any range between these values. In some embodiments, said ionizable carotenoid/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g/mol, or any the range between these values. In some embodiments, said liposome contains at least 0.1 to 97% ionizable carotenoid. In some embodiments, said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, 80 to 120 nm, or any range in between. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEGmaleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG and cholesterol-maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In additional embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups. In some implementations

- 35 047345 of the invention, said liposome contains OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, said liposome comprises OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2 -(epoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), 1-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), 1-palmitoyl-2-glutaroyl-sn-glucero- 3-phosphocholine (PGPC); 1 -palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glucero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-n-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-glycero-3-phosphocholine; and 1-palmitoyl-2 acetoyl-sn-glycero-3-phosphocholine. In some embodiments, said liposome contains PGPC. In some embodiments, said OxPARS within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range between these values. In some embodiments, the liposome contains a targeting moiety having a specific affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the liposome exterior, and optionally, the targeting moiety is covalently attached to one or both of the PEG and the liposome exterior. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (ie, said liposome is pegylated). In some embodiments, said PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In some embodiments, the zeta potential of said liposome is in the range of -150 to 150 mV or -50 to 50 mV, or any range between these values. In some embodiments, said liposome is cationic. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between. In some embodiments, said liposome is anionic or neutral. In some embodiments, the liposome composition comprises a liposome that has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between.

In some embodiments, provided herein is a pharmaceutical composition comprising an ionizable bis-alpha, omega-carotenoid having the formula Q-Ri-Polyene Carotenoid-Ri-Q encapsulated in a liposome, wherein said Polyene Carotenoid contains:

(a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10 or more than 9 conjugated double bonds, (b) substitution with methyl or lower alkyl (C ₂ -C ₃ ), (c) 1, 2, 3 or more 3 ionizable groups; and R1 represents an ionizable group;

Q is (a) a polyvalent counterion or (b) a monovalent cation.

In some embodiments, the R ₁ -Polyene Carotenoid X contains all trans-conjugated double bonds. In some embodiments, said K1-Polyene Carotenoid-Sh contains 6-9 trans-conjugated double bonds. In specific embodiments of the invention, said K1-Polyene Carotenoid-I1 contains 7 trans-conjugated double bonds. Said N-Polyene Carotenoid-I1 may be naturally occurring or synthetic. In some embodiments, said N-Polyene Carotenoid R1 is naturally occurring. In other embodiments, said R1Polyene Carotenoid^ is synthetic. In some embodiments, R1 is an anionic ionizable group. In some embodiments, said R ₁ -Polyene Carotenoid X contains an ionizable group selected from a carboxyl group, a sulfonate group, a sulfate group, a phosphonate group, a phosphate group, and a hydrox group.

- 36 047345 matte fragment. In other embodiments, R ₁ is a cationic ionizable group (eg, a primary, secondary or tertiary amino group, a quaternary ammonium group, a choline group, a guanidine group, or an imidazole group). In specific embodiments, R ₁ is a cationic ionizable group, and the pharmaceutical composition is substantially free of nucleic acids.

In some embodiments, Q is a multivalent counterion. In some embodiments, Q is a multivalent cationic counterion. In additional embodiments, Q is a polyvalent metal cation. In some embodiments, Q is a polyvalent transition metal counterion. In some embodiments, Q is a divalent counterion. In some embodiments, Q is a divalent cationic counterion. In additional embodiments, Q is a divalent metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In additional embodiments, Q is Ca ²⁺ or Mg ² '. In additional embodiments, Q is Ca ²⁺ . In some embodiments, Q is Mg ² '. In other embodiments, Q is a trivalent cationic counterion such as Fe ³⁺ . In other embodiments, Q is a polyvalent organic counterion. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a divalent organic cation, such as a protonated diamine.

In some embodiments, Q is a monovalent cationic counterion. In additional embodiments, Q is at least one member selected from Na ⁺ , Li ⁺ or K ⁺ . In some embodiments, Q is an organic counterion. In some embodiments, Q is a polyvalent organic cation. In further embodiments, Q is a divalent organic cation, such as a protonated diamine or a protonated polyamine. In other embodiments, Q is a monovalent organic cation, such as NH ₄ ⁺ , a protonated diamine, or a protonated polyamine.

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, or less than 10,000 molecules of ionizable carotenoid. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 molecules of ionizable carotenoid, or any range between these values. In some embodiments, said ionizable carotenoid/lipid ratio in said liposome composition is from 1 to 1000 g/mol or any range between these values. In some embodiments, said ionizable carotenoid/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g/mol, or any range between these values. In some embodiments, said liposome contains at least 0.1 to 97% ionizable carotenoid. In some embodiments, said liposomes have an average diameter, for example, from 20 to 500 nm, from 20 to 200 nm, or any range between these values. In some embodiments, said liposomes have an average diameter of 80 to 120 nm, or any range between these values. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEGmaleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG and cholesterol-maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-TOr-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In further embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups. In some embodiments, said liposome comprises OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, said liposome contains OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)^n-glycero-3-phosphocholine (5,6-PEIPC),

- 37 047345

-palmitoyl-2-(epoxycyclopentenone)^p-glycero-3-phosphorylcholine (PECPC), 1-palmitoyl-2-(epoxyisoprostane E2)^p-glycero-4-phosphocholine (PEIPC), 1-palmitoyl-2-glutaroyl^ p-glycero-3-phosphocholine (PGPC); 1-palmitoyl-2-(9'-oxononanoyl)^n-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-n-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-n-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-n-glycero-3-phosphocholine and 1-palmitoyl-2-acetoyl-n-glycero-3-phosphocholine. In some embodiments, said liposome contains PGPC. In some embodiments, said OxPARS within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range in between. In some embodiments, the liposome contains a targeting moiety having a specific affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the liposome exterior, and optionally, the targeting moiety is covalently attached to one or both of the PEG and the liposome exterior. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, said targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (ie, said liposome is pegylated). In some embodiments, said PEG has a number average molecular weight (MW) of 200 to 5000 Da. In additional embodiments, said liposome is anionic or neutral. In some embodiments, said liposome has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV? or -8 to 2 mV, or any range between these values. In other embodiments, said liposome is cationic.

In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

In some embodiments, provided herein is a pharmaceutical composition comprising a bis-alpha, omega-carotenoid having the formula

Rj -Polyene Carotenoid-Rj, encapsulated in a liposome, wherein said bis-alpha, omega-carotenoid contains:

(a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10 or more 9 conjugated double bonds, (b) 1, 2, 3 or more 3 ionizable groups ; and said Polyene Carotenoid is optionally substituted with 1 to n methyl or lower C ₁ -C3 alkyl substitutions, where n = 1-4; And

R ₁ represents a polar group and/or a monocyclic functional group.

In some embodiments, said bis-alpha, omega-carotenoid contains all trans-conjugated double bonds. In some embodiments, said bisalpha, omega-carotenoid contains 6-9 conjugated double bonds. In specific embodiments, said bis-alpha, omega-carotenoid contains 7 conjugated double bonds. Said bis-alpha, omega-carotenoid may be naturally occurring or synthetic. In some embodiments, said bis-alpha, omega-carotenoid is naturally occurring. In other embodiments, said bis-alpha omega-carotenoid is synthetic. In some embodiments, R ₁ is a polar group. In some embodiments, R ₁ is a monocyclic functional group. In some embodiments, R ₁ is a polar group and a monocyclic functional group. In some embodiments, said bis-alpha, omega-carotenoid contains a monocyclic and/or polar functional group selected from the functional group present in astaxanthin, lutein, xanthophyll and zeaxanthin. In some embodiments, said bisalpha, omega-carotenoid is selected from astaxanthin, lutein, xanthophyll, and zeaxanthin (eg, as shown below).

- 38 047345

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, or less than 10,000 bis-alpha omega-carotenoid molecules. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 bis-alpha, omega-carotenoid molecules, or any range between these values. In some embodiments, said bis-alpha, omega-carotenoid/lipid ratio in said liposome composition is from 1 to 1000 g/mol or any range between these values. In some embodiments, said bis-alpha, omega-carotenoid/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g /mol, or any range between these values. In some embodiments, said liposome contains at least 0.1 to 97% bis-alpha, omega carotenoid. In some embodiments, said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, or any range in between. In some embodiments, said liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In further embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups. In some embodiments, said liposome comprises OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, said liposome comprises OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2 (epoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), 1-palmitoyl-2-(epoxyisoprostane E2)sn-glycero-4-phosphocholine (PEIPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3- phosphocholine (PGPC); 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-sn-glycero3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-snglycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and 1-palmitoyl-2-acetoylsn-glycero-3-phosphocholine. In some embodiments, said liposome contains PGPC. In some embodiments, said OxPARS within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range between these values. In some embodiments, the liposome contains a targeting moiety having a specific affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the liposome exterior, and optionally, the targeting moiety is covalently attached to one or both of the PEG and the liposome exterior. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100

- 39 047345 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (ie, said liposome is pegylated). In some embodiments, said PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In additional embodiments, said liposome is anionic or neutral. In some embodiments, said liposome has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In other embodiments, said liposome is cationic. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

In some embodiments, said pharmaceutical composition contains a trans-crocetin salt having the formula Q-trans-crocetin-Q

encapsulated in a liposome, where Q is (a) a polyvalent counterion or (b) a monovalent cation.

In some embodiments, Q is a multivalent cationic counterion. In some embodiments, Q is a polyvalent metal cation. In further embodiments, Q is a polyvalent transition metal cation. In some embodiments, Q is a divalent cationic counterion. In additional embodiments, Q is a divalent metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In additional embodiments, Q is Ca ²⁺ or Mg ²⁺ . In some embodiments, Q is Ca ²⁺ . In some embodiments, Q is Mg ²⁺ . In some embodiments, Q is a divalent organic counterion. In other embodiments, Q is a trivalent cationic counterion such as Fe ³⁺ . In other embodiments, Q is a polyvalent organic counterion. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a divalent organic cation, such as a protonated diamine.

In additional embodiments, Q is a monovalent cationic counterion. In some embodiments, Q is a monovalent metal cation. In some embodiments, Q is at least one member selected from Na, Li, or K. In some embodiments, Q is an organic cation. In some embodiments, Q is a monovalent organic cation, such as a protonated amine (eg, a protonated diamine or a protonated polyamine). In some embodiments, Q is an organic cation, such as NH4 ⁺ , a protonated diamine, or a protonated polyamine.

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, or less than 10,000 trans-crocetin molecules. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 1,000 to 5,000 trans-crocetin molecules, or any range between these values. In some embodiments, said trans-crocetin/lipid ratio in said liposome composition is from 1 g/mol to 1000 g/mol, or any range between these values. In some embodiments, said trans-crocetin/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g/mol, or any range between these values. In some embodiments, said liposome contains at least 0.1 to 97% trans-crocetin. In some embodiments, said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, or any range

- 40 047345 between these values. In some embodiments, said liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEGmaleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In additional embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups. In some embodiments, said liposome comprises OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid.

In some embodiments, said liposome comprises OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2 -(epoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), 1-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), 1-palmitoyl-2-glutaroylsn-glycero-3 - phosphocholine (PGPC); 1 -palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine;

-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3 phosphocholine; 1 -palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and 1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine. In some embodiments, said liposome contains PGPC. In some embodiments, said OxPARS within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range between these values. In some embodiments, the liposome contains a targeting moiety having a specific affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the liposome exterior, and optionally, the targeting moiety is covalently attached to one or both of the PEG and the liposome exterior. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (ie, said liposome is pegylated). In some embodiments, said PEG has a number average molecular weight (Mn) of 200 to 5000 Da. In additional embodiments, said liposome is anionic or neutral. In some embodiments, said liposome has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In other embodiments, said liposome is cationic. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

In some embodiments, provided herein is a pharmaceutical composition comprising calcium trans-crocetinate (CTC) encapsulated in a liposome. The specified STS can exist in linear and/or cyclic form (shown below):

- 41 047345

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, or less than 10,000 trans-crocetin molecules. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 trans-crocetin molecules, or any range between these values. In some embodiments, said trans-crocetin/lipid ratio in said liposome composition is from 1 to 1000 g/mol or any range between these values. In some embodiments, said trans-crocetin/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g/mol, or any range between these values. In some embodiments, said liposome contains at least 0.1 to 97% trans-crocetin. In some embodiments, said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, or any range in between. In some embodiments, said liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In further embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups.

In some embodiments, said liposome comprises OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, said liposome comprises OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2-( epoxycyclopentenone)sn-glycero-3-phosphorylcholine (PECPC), 1-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4phosphocholine (PEIPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC ); 1-palmitoyl-2-(9'oxononanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-sn-glycero-3phosphocholine; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and 1-palmitoyl-2-acetoyl-sn-glycero3-phosphocholine. In some embodiments, said liposome contains PGPC. In some embodiments, said OxPARS within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range between these values. In some embodiments, the liposome contains a targeting moiety having a specific affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the liposome exterior, and optionally, the targeting moiety is covalently attached to one or both of the PEG and the liposome exterior. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome contains less than 500,000 or less than 200,000 trans-crocetin molecules. In some embodiments, said liposome contains from 10 to 100,000 molecules of trans-crocetin, or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (i.e., said liposome

- 42 047345 is pegylated). In some embodiments, said PEG has a number average molecular weight (Mn) of 200 to 5000 Da. In additional embodiments, said liposome is anionic or neutral. In some embodiments, said liposome has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In other embodiments, said liposome is cationic. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

In some embodiments, provided herein is a pharmaceutical composition comprising magnesium trans-crocetinate (MTC) encapsulated in a liposome. The said MTS can exist in linear and/or cyclic form (shown below):

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, or less than 10,000 trans-crocetin molecules. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 500 to 5,000 molecules of trans-crocetin, or any range between these values. In some embodiments, said trans-crocetin/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g/mol, or any range between these values. In some embodiments, said liposome contains at least 0.1 to 97% trans-crocetin. In some embodiments, said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, or any range in between. In some embodiments, said liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In additional embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups. In some embodiments, said liposome comprises OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, said liposome comprises OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2 -(epoxycyclopentenone)-sn-glycero-3-phosphocholine (PECPC), 1 -palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), 1 -palmitoyl-2-glutaroyl-sn-glycero- 3-phosphocholine (PGPC); 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and 1-palmitoyl-2acetoyl-sn-glycero-3-phosphocholine. In some embodiments, said liposome contains PGPC. In some embodiments, said OxPARS within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range between these values. In some embodiments, the liposome contains a targeting moiety having a specific affinity for a surface antigen on a target cell of interest. In some embodiments of the invention, said targeting fragment

- 43 047345 ment is attached to one or both of the PEG and the outer portion of the liposome, optionally said targeting moiety is attached to one or both of the PEG and the outer portion of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (ie, said liposome is pegylated). In some embodiments, said PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In additional embodiments, said liposome is anionic or neutral. In some embodiments, said liposome has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In other embodiments, said liposome is cationic. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

In some embodiments, provided herein is a pharmaceutical composition comprising trans-norbixin having the formula Q-hop6ukcuh-Q

encapsulated in a liposome, where Q is a polyvalent cationic counterion.

In some embodiments, Q is a multivalent cationic counterion. In some embodiments, Q is a polyvalent metal cation. In further embodiments, Q is a polyvalent transition metal cation. In some embodiments, Q is a divalent cationic counterion. In additional embodiments, Q is a divalent metal cation. In some embodiments, Q is at least one member selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ and Fe ²⁺ . In additional embodiments, Q is Ca ²⁺ or Mg ²⁺ . In some embodiments, Q is Ca ²⁺ . In some embodiments, Q is Mg ²⁺ . In some embodiments, Q is a divalent organic counterion.

In some embodiments, Q is a monovalent cationic counterion. In some embodiments, Q is a monovalent metal cation. In some embodiments, Q is at least one member selected from Na ⁺ , Li ⁺ or K ⁺ . In some embodiments, Q is an organic cation. In some embodiments, Q is a monovalent organic cation, such as a protonated amine (eg, a protonated diamine or a protonated polyamine). In some embodiments, Q is an organic cation, such as NH ₄ ⁺ , a protonated diamine, or a protonated polyamine.

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, or less than 10,000 trans-norbixin molecules. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 trans-norbixin molecules, or any range between these values. In some embodiments, said trans-norbixin/lipid ratio in said liposome composition is from 1 to 1000 g/mol or any range between these values. In some embodiments, said trans-norbixin/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g/mol, or any range between these values. In some embodiments, the liposome contains at least 0.1 to 97% Q-norbixin-Q. In some implementations

- 44 047345 of the invention, said liposome has a diameter of from 20 to 500 nm, from 20 to 200 nm, or any range between these values. In some embodiments, said liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEGmaleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In additional embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups. In some embodiments, said liposome comprises OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, said liposome comprises OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2 -(epoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), 1-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), 1-palmitoyl-2-glutaroyl-sn-glycero- 3-phosphocholine (PGPC); 1 -palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine; 1 -palmitoyl-2-arachidonoyl-snglycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-n-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-glycero-3-phosphocholine; and 1-palmitoyl-2 acetoyl-sn-glycero-3-phosphocholine. In some embodiments, said liposome contains PGPC. In some embodiments, said OxPARS within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range between these values. In some embodiments, the liposome contains a targeting moiety having a specific affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the liposome exterior, and optionally, the targeting moiety is covalently attached to one or both of the PEG and the liposome exterior. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (ie, said liposome is pegylated). In some embodiments, said PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In additional embodiments, said liposome is anionic or neutral. In some embodiments, said liposome has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In other embodiments, said liposome is cationic. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

In some embodiments, provided herein is a pharmaceutical composition comprising calcium trans-norbixinate (CTN) encapsulated in a liposome. Said CTN may exist in linear and/or cyclic form (shown below):

- 45 047345

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, or less than 10,000 trans-norbixin molecules. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 trans-norbixin molecules, or any range between these values. In some embodiments, said trans-norbixin/lipid ratio in said liposome composition is from 1 to 1000 g/mol or any range between these values. In some embodiments, said trans-norbixin/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g/mol, or any range between these values. In some embodiments, said liposome contains at least 0.1 to 97% trans-norbixin. In some embodiments, said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, or any range in between. In some embodiments, said liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEGmaleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-TOF-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In additional embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups. In some embodiments, said liposome comprises OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, said liposome comprises OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6-epoxyisoprostane E2Si-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2-(epoxycyclopentenone Sup -glycero-3-phosphorylcholine (PECPC), 1 -palmitoyl-2-(epoxyisoprostane E2Si-glycero-4-phosphocholine (PEIPC), 1 -palmitoyl-2-glutaroyl-glycero-3-phosphocholine (PGPC); 1 - palmitoyl-2-(9'-oxononanoyl 1-glycero-3-phosphocholine; 1 -palmitoyl-2-arachidonoyl-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-glycero-3-phosphocholine; 1 -palmitoyl- 2-hexadecyl-p-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-p-glycero-3-phosphocholine; and 1-palmitoyl-2-acetoyl-p-glycero-3-phosphocholine; in some embodiments, said liposome. contains PGPC. In some embodiments, the specified OxPARS within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range between these values. In some embodiments, the specified liposome contains a targeting moiety having a specific affinity for a surface antigen on the cell. target of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the liposome exterior, and optionally, the targeting moiety is covalently attached to one or both of the PEG and the liposome exterior. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (ie, said liposome is pegylated). In some embodiments, said PEG has a number average molecular weight (Mn) of 200 to 5000 Da. In additional embodiments, said liposome is anionic or neutral. In some embodiments, said liposome has a zeta potential of less than or equal to zero. In some countries

- 46 047345 in the implementation of the invention, the said liposome has a zeta potential from -150 to 0 mV, from -50 to 0 mV, from -40 to 0 mV, from -30 to 0 mV, from -25 to 0 mV, from -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In other embodiments, said liposome is cationic. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

In some embodiments, provided herein is a pharmaceutical composition comprising magnesium trans-norbixinate (MTN) encapsulated in a liposome. Said MTN may exist in linear and/or cyclic form (shown below):

In some embodiments, said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, or less than 10,000 trans-norbixin molecules. In some embodiments, said liposome contains from 10 to 100,000, from 100 to 10,000, or from 1000 to 5000 trans-norbixin molecules, or any range between these values. In some embodiments, said trans-norbixin/lipid ratio in said liposome composition is from 1 to 1000 g/mol or any range between these values. In some embodiments, said trans-norbixin/lipid ratio is 10-150 g/mol, 10-100 g/mol, 30-200 g/mol, 40-200 g/mol, or 50-200 g/mol, or any range between these values. In some embodiments, said liposome contains at least 0.1 to 97% trans-norbixin. In some embodiments, said liposome has a diameter of 20 to 500 nm, 20 to 200 nm, or any range in between. In some embodiments, said liposome has a diameter between 80 and 120 nm, or any range between these values. In some embodiments, said liposome is formed from liposome components. In further embodiments, said liposome components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEGmaleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol maleimide. In further embodiments, said liposome components comprise at least one selected from DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC. In additional embodiments, said liposome further comprises an oxidized phospholipid such as OxPARS. In some embodiments, said liposome comprises OxPARS, which is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing full-length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega -aldehyde or omega-carboxyl groups. In some embodiments, said liposome comprises OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC, or said OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, said liposome comprises OxPAPC selected from the group consisting of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2 -(epoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), 1-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), 1-palmitoyl-2-glutaroyl-sn-glycero- 3-phosphocholine (PGPC); 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and 1-palmitoyl-2acetoyl-sn-glycero-3-phosphocholine. In some embodiments, said liposome contains PGPC. In some embodiments, said OxPARS within the lipid bilayer of the liposome is 0-100% of the total lipids, or any range between these values. In some embodiments, the liposome contains a targeting moiety having a specific affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of the PEG and the liposome exterior, optionally the targeting moiety is attached to one or both of the PEG and the liposome exterior

- 47 047345 covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting fragment is an antibody or an antigen-binding fragment of an antibody. In some embodiments, said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500, or from 30 to 200 targeting fragments, or any range between these values. In some embodiments, said liposome contains less than 500,000 or less than 200,000 trans-norbixin molecules. In some embodiments, said liposome contains from 10 to 100,000 trans-norbixin molecules or any range between these values. In some embodiments, said liposome further comprises an immunostimulating agent (eg, 1,6-beta-glucan). In some embodiments, said liposome contains a steric stabilizer. In some embodiments, said steric stabilizer is polyethylene glycol (ie, said liposome is pegylated). In some embodiments, said PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In additional embodiments, said liposome is anionic or neutral. In some embodiments, said liposome has a zeta potential of less than or equal to zero. In some embodiments, said liposome has a zeta potential of -150 to 0 mV, -50 to 0 mV, -40 to 0 mV, -30 to 0 mV, -25 to 0 mV, -20 to 0 mV, -10 to 0 mV, -9 to 0 mV, -8 to 0 mV, -7 to 0 mV, -6 to 0 mV, -5 to 0 mV, -4 to 0 mV, -3 to 0 mV, -2 to 0 mV, -1 to 0 mV, or -8 to 2 mV, or any range in between. In other embodiments, said liposome is cationic. In some embodiments, the liposome composition comprises a liposome that has a zeta potential greater than zero. In some embodiments, said liposome zeta potential is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, 1 to 30 mV, 1 to 25 mV, 1 to 20 mV, 1 to 15 mV, 1 to 10 mV, 1 to 5 mV, 2 to 10 mV, 3 to 10 mV, 4 to 10 mV, or 5 to 10 mV, or any range in between.

Compilation and introduction.

The proposed compositions can be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may contain one or more nanoparticle compositions. For example, a pharmaceutical composition may contain one or more nanoparticle compositions containing one or more different therapeutic or prophylactic agents. The pharmaceutical compositions may further contain one or more pharmaceutically acceptable excipients or additional ingredients, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21 ^st Edition, AR Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and additional ingredients may be used in any pharmaceutical composition, except that any conventional excipient or additional ingredient may be incompatible with one or more components of the nanoparticle composition. An excipient or additional ingredient may be incompatible with a component of a nanoparticle composition if its combination with that component would result in any undesirable biological effect or otherwise harmful effect.

In some embodiments, one or more excipients or additional ingredients may constitute more than 50% of the total weight or volume of the pharmaceutical composition containing the nanoparticle composition. For example, one or more excipients or additional ingredients may constitute 50, 60, 70, 80, 90% or more of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99, or 100% pure. In some embodiments, the excipient is approved for use in humans and for veterinary use. In some embodiments, the excipient is approved by the US Food and Drug Administration. In some embodiments, the excipient is a pharmaceutical grade excipient. In some embodiments, the excipient complies with the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia and/or International Pharmacopoeia.

Standard methods for preparing liposomes include, but are not limited to, those described in Liposomes: A Practical Approach, V.P. Torchilin, Volkmar Weissig Oxford University Press, 2003, and are well known in the art.

In some embodiments, provided herein is a liposome composition and a physiologically (ie, pharmaceutically) acceptable carrier. As used herein, the term carrier generally refers to an inert substance used as a diluent or carrier medium for a drug, such as a therapeutic agent. This term is also

- 48 047345 generally covers an inert substance which imparts cohesive properties to the composition. Typically, physiologically acceptable carriers are present in liquid form. Examples of liquid carriers include saline, phosphate buffer, buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins for enhanced stability (eg, albumin, lipoprotein , globulin, etc.) etc. Since physiologically acceptable carriers are determined in part by the particular composition administered, as well as the particular method used to administer the composition, there is a wide range of suitable pharmaceutical composition formulations provided herein (see, for example, Remington's Pharmaceutical Sciences, 17 ^th ed., 1989) .

The present compositions can be sterilized by conventional known sterilization methods or can be prepared under sterile conditions. Aqueous solutions may be packaged for use or aseptically filtered and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. Said compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting agents and buffering agents, tonicity adjusting agents, wetting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride , calcium chloride, sorbitan monolaurate and triethanolamine oleate. Sugars may also be included to stabilize the compositions, such as a stabilizer for lyophilized liposome compositions. In some embodiments, the pharmaceutical composition contains an isotonic agent at a concentration greater than 0.1%, or at a concentration of 0.3 to 2.5%, 0.5 to 2.0%, or 0.5 to 1.5% , from 0.5 to 1.5%, from 0.6 to 1.1%, or any range in between. In some embodiments, the pharmaceutical composition contains an isotonic agent such as dextrose, mannitol, glycerin, potassium chloride or sodium chloride. In additional embodiments of the invention, said pharmaceutical composition contains dextrose, mannitol, glycerin, potassium chloride or sodium chloride in a concentration of more than 0.1% or in a concentration of 0.3 to 2.5%, 0.5 to 2.0%, 0.5 to 1.5%, 0.5 to 1.5%, 0.6 to 1.1%, or any range in between.

Formulations suitable for parenteral administration, for example, intra-articular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal and subcutaneous routes include aqueous or non-aqueous, isotonic sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic and solutes, which render the composition isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives. Injectable solutions and suspensions can also be prepared from sterile powders, granules and tablets. In some embodiments, the proposed liposome compositions are administered, for example, by intravenous infusion, topically, intraperitoneally, intravesically, or intrathecally. In specific embodiments of the invention, these liposome compositions are administered parenterally or intravenously. Preferably, the pharmaceutical liposome compositions are administered parenterally, i.e. intra-articularly, intravenously, subcutaneously or intramuscularly. In other embodiments, the pharmaceutical preparation may be administered topically.

In some embodiments, the provided pharmaceutical compositions (e.g., liposome compositions) are presented in unit dosages or in sealed multi-dose containers, such as ampoules and vials.

In some embodiments, the pharmaceutical preparations are administered in a unit dosage form. In this form, the preparation is divided into unit doses containing appropriate amounts of the active component, for example a liposome composition. Said unit dosage form may be a packaged preparation, a package containing discrete quantities of the drug. If desired, the composition may also contain other compatible therapeutic agents (eg, as described herein).

In some embodiments, these liposome compositions containing a therapeutic and/or diagnostic agent used in the pharmaceutical compositions provided herein can be administered at an initial dosage of from about 0.001 to about 1000 mg/kg per day. The range of daily dosage that can be used is from about 0.01 to about 500 mg/kg, or from about 0.1 to about 200 mg/kg, or from about 1 to about 100 mg/kg, or from about 10 up to about 50 mg/kg. Dosages, however, may vary depending on the requirements of the patient, the severity of the condition being treated and the liposome composition used. For example, dosages may be empirically determined based on the type and stage of disease, disorder, or condition diagnosed in a particular patient. The dosage administered to a patient in the context of the proposed pharmaceutical compositions (eg, liposome compositions) should be sufficient to influence a beneficial therapeutic response in the patient over time. The dose size will also be determined by the presence, nature and extent of any adverse side effects that accompany the administration of a particular liposome composition in a particular patient. Determining the correct

- 49 047345 dosage for a specific situation is within the competence of the practitioner. Typically, treatment begins with lower doses that are less than the optimal dose of the liposome formulation. Thereafter, the dosage is increased in small increments until the optimal effect is achieved under the circumstances. For convenience, the total daily dosage can be divided and administered in portions throughout the day, if desired.

Loading into liposomes.

The proposed carotenoid compositions can be loaded into liposomes using active or passive techniques.

In some embodiments, this description provides a method for preparing a liposome composition containing ionizable crocetin (for example, according to claims [1]-[23]), this method includes the following steps:

(a) forming a mixture containing: liposome components in solution;

(b) homogenizing the mixture to form liposomes in solution;

(c) treating the mixture to form liposomes containing an ionizable carotenoid.

In some embodiments, the processing step includes one or more of the following: thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw technique, reverse phase evaporation, high pressure dynamic microfluidization, microfluidic mixing, dual emulsification, double lyophilization emulsification, 3D printing, membrane contactor technique and mixing. In some embodiments, said processing step includes one or more steps of modifying the size of the liposomes by one or more of extrusion, high pressure microfluidization, and/or sonication steps.

In some embodiments, this disclosure provides an active loading method for producing a carotenoid salt within a liposome formulation using a concentration gradient of soluble metal acetate salts (calcium acetate or magnesium acetate).

Polyvalent counterions used in accordance with this description can be encapsulated in liposomes in accordance with the methods described herein or otherwise known in the art. This includes passive encapsulation techniques described below or otherwise known in the art.

In some embodiments, this disclosure provides a method for preparing a pharmaceutical composition containing:

(a) preparing a solution of liposomes containing liposomes in a solution of a weak acid polyvalent metal salt;

(b) adding an ionizable carotenoid to said liposome solution;

(c) maintaining the ionizable carotenoid in the liposome solution for a sufficient time for the carotenoid to penetrate into the liposomes.

In some embodiments, said ionizable carotenoid is an ionizable carotenoid in the compositions of any one of claims [1] to [8] (eg, trans-crocetin and trans-norbixin). In some embodiments, said carotenoid is a carotenoid described in any of FIGS. 1A-1D. In some embodiments, said weak acid is selected from acetic acid, gluconic acid, tartaric acid, glutamic acid, citric acid, formic acid and glycic acid. In some embodiments, said polyvalent metal weak acid salt is used at a concentration of 0 to 2000 mM, or 50 to 500 mM, or any range in between. In some embodiments, said polyvalent metal is selected from Ca ²⁺ , Mg 2 ⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ , Fe ²⁺ and Fe ³⁺ . In some embodiments, said weak acid is acetic acid and said polyvalent metal is Ca ²⁺ (ie, said weak acid salt of the polyvalent metal is calcium acetate). In some embodiments, said weak acid is acetic acid and said polyvalent metal is Mg2 ⁺ (ie, said polyvalent metal weak acid salt is magnesium acetate). Pharmaceutical compositions prepared in accordance with these proposed methods are also covered by this invention. Said liposome solution is preferably a buffered solution. However, it is stated that any suitable solvent may be used for the preparation and use of the present compositions. A preferred liposome solution has a pH around physiological pH and contains a buffer that has a buffering range that includes physiological pH. A non-limiting example of a suitable liposome solution buffer is HEPES (eg, 5 mM HEPES buffered saline pH 6.5). Pharmaceutical compositions obtained in accordance with this method are also covered by this invention.

Polyvalent metals used in accordance with the proposed methods can be encapsulated in liposomes in accordance with conventional methods known in the art. These methods include, for example, the passive encapsulation methods described in this document.

- 50 047345 document or otherwise known in the art. Loading of an ionizable carotenoid, such as trans-crocetin, can be accomplished by keeping the ionizable carotenoid in the liposome solution for a suitable amount of time at a suitable temperature. Depending on the composition of the liposomes and the temperature, pH and chemical nature of the ionizable carotenoid, loading of the ionizable carotenoid can occur over a period of minutes or hours. In some embodiments, the loading is carried out at a temperature, for example, from 0 to 95°C, or from 20 to 75°C, or any range between these values, preferably from about 40 to about 80°C, or any range between these values.

In some embodiments, this specification further provides step (d) removing the non-encapsulated ionizable carotenoid from the liposome preparation prepared in accordance with (c). In some embodiment, said removal is accomplished by passing the liposome preparation through a gel filtration column equilibrated with a second aqueous buffer solution, centrifugation, or dialysis, or related methods. After removal of the non-encapsulated ionizable carotenoid, the degree of ionizable carotenoid loading can be determined by measuring the levels of ionizable carotenoid and lipids in accordance with conventional methods. Lipid and drug concentrations can be determined using any suitable method known in the art, such as scintillation counting, spectrophotometric assays and high performance liquid chromatography. Replacing the liposome preparation solution to remove unencapsulated carotenoid and counterion such as sodium acetate can be achieved using any of various methods known in the art, including, but not limited to, chromatography of the liposome preparation through a long gel filtration column equilibrated with a second aqueous buffered solution, centrifugation, extensive or repeated dialysis, exchange of the liposome preparation, treatment of the liposome preparation with chelating agents, or related methods. Pharmaceutical compositions prepared in accordance with these proposed methods are also covered by this invention.

In some embodiments, this disclosure provides a method for preparing a pharmaceutical composition comprising:

(a) preparing a solution of liposomes containing liposomes in a solution of a weak acid polyvalent metal salt;

(b) adding trans-crocetin to said liposome solution;

(c) maintaining the ionizable carotenoid in the liposome solution for a sufficient time for the carotenoid to penetrate into the liposomes.

In some embodiments, said weak acid is selected from acetic acid, gluconic acid, tartaric acid, glutamic acid, citric acid, formic acid and glycic acid. In some embodiments, said polyvalent metal weak acid salt is used at a concentration of 0 to 2000 mM, or 50 to 500 mM, or any range in between. In some embodiments, said polyvalent metal is selected from Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Co ²⁺ , Fe ²⁺ and Fe ³⁺ . In some embodiments, said weak acid is acetic acid and said polyvalent metal is Ca ²⁺ (ie, said weak acid salt of the polyvalent metal is calcium acetate). In some embodiments, said weak acid is acetic acid and said polyvalent metal is Mg ²⁺ (ie, said polyvalent metal weak acid salt is magnesium acetate). Pharmaceutical compositions obtained in accordance with this method are also covered by this invention. Said liposome solution is preferably a buffered solution. However, it is stated that any suitable solvent may be used to operate the present compositions or methods. A preferred liposome solution has a pH around physiological pH and contains a buffer that has a buffering range that includes physiological pH. A non-limiting example of suitable liposome solution buffers is a 5 mM HEPES buffered saline solution at pH 6.5. Pharmaceutical compositions obtained in accordance with this method are also covered by this invention.

Loading of trans-crocetin can be accomplished by keeping trans-crocetin in the liposome solution for a suitable amount of time at a suitable temperature. Depending on the composition of the liposomes and the temperature, pH and chemical nature of trans-crocetin, loading of trans-crocetin can occur over a period of minutes or hours. In some embodiments, the loading is carried out at a temperature, for example, from 0 to 95°C, or from 20 to 75°C, or any range between these values, preferably from about 40 to about 80°C.

In some embodiments, this specification further provides step (d) of removing unencapsulated trans-crocetin from the liposome preparation prepared in accordance with (c). In some embodiment of the invention, said removal is accomplished by

- 51 047345 passing a given liposome preparation through a gel filtration column equilibrated with a second aqueous buffered solution, centrifugation or dialysis, or related methods. After removal of unencapsulated trans-crocetin, the extent of trans-crocetin loading can be determined by measuring trans-crocetin and lipid levels according to conventional methods. Lipid and drug concentrations can be determined using any suitable method known in the art, such as scintillation counting, spectrophotometric assays and high performance liquid chromatography. Replacing the liposome preparation solution to remove unencapsulated trans-crocetin and a counterion such as sodium acetate can be achieved using any of various methods known in the art, including, but not limited to, chromatography of the liposome preparation through a long gel filtration column , equilibrated with a second aqueous buffered solution, centrifugation, extensive or repeated dialysis, exchange of the liposome preparation, treatment of the liposome preparation with chelating agents, or related methods. Pharmaceutical compositions prepared in accordance with these proposed methods are also covered by this invention.

Pharmaceutical compositions containing an ionizable carotenoid salt prepared in accordance with these proposed methods are also covered by this invention. In some embodiments, said ionizable carotenoid is an ionizable carotenoid in the compositions of any one of claims [1] to [8] (eg, trans-crocetin and trans-norbixin). In some embodiments, said ionizable carotenoid is a carotenoid described in any of FIGS. 1A-1D. In some embodiments, this disclosure provides a pharmaceutical composition comprising a liposome encapsulating an ionizable carotenoid, wherein said ionizable carotenoid is loaded into the liposomes in the presence of intraliposomal multivalent counterions (e.g., Ca ²⁺ , Mg ² ', Zn ²⁺ , Cu ²⁺ , Co ²⁺ , Fe ²⁺ and Fe ³⁺ ). In some embodiments, said multivalent counterions include Ca ^2' . In some embodiments, said multivalent counterions include Mg ^2' . In some embodiments, said multivalent counterions include Fe ^3' .

In some embodiments, this disclosure provides a pharmaceutical composition comprising a liposome encapsulating a trans-crocetin salt, wherein trans-crocetin is loaded into the liposomes in the presence of intraliposomal polyvalent counterions (e.g., Ca ^2' , Mg ^2' , Zn ^2' , Cu ^{2 '} , Co ^2' , Fe ^2' and Fe ^3' ). In some embodiments, said multivalent counterions include Ca ^2' . In some embodiments, said multivalent counterions include Mg ^2' . In some embodiments, said multivalent counterions include Fe ^3' .

Methods of treatment and application.

The proposed pharmaceutical compositions, such as liposome compositions, have applications that provide advantages over prior methods of treating diseases and disorders, which include, but are not limited to, infection and infectious diseases such as HIV/AIDS: human immunodeficiency virus-1 (HIV -1), tuberculosis, malaria and its complications such as cerebral malaria, severe anemia, acidosis, acute renal failure and ARDS, sepsis, inflammation (eg, chronic inflammatory diseases), ischemia (including an ischemic condition such as ischemic stroke, disease coronary arteries, peripheral vascular diseases, cerebrovascular diseases, renal pathologies associated with ischemia and ischemia associated with wounds); shock (eg, hemorrhagic shock), stroke, cardiovascular diseases, renal pathologies, wound healing, metabolic diseases, hyperproliferative diseases such as cancer and disorders of the immune system, cardiovascular system, digestive, nervous, respiratory and endocrine systems. In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any one of claims[ 1]-[24]). The use of a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any one of claims [1] to [24]) in the manufacture of a medicament for treating a disease, disorder or condition in a subject is also provided herein, as is, pharmaceutical compositions according to any one of claims [1] to [24] for use in a medicinal product.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with endotoxemia in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any of paragraphs [1]-[24]).

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with sepsis in a subject in need of

- 52 047345 in such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (eg, a pharmaceutical composition according to any one of claims [1] to [24]). In some embodiments, the subject suffers from a mild endotoxemic disease.

In some embodiments, provided herein is a method of treating or preventing the risk of sepsis in a subject, comprising administering to said subject a pharmaceutical composition provided herein (eg, a pharmaceutical composition according to any one of claims [1] to [24]). In some embodiments, the subject is immunodeficient or immunosuppressed. In some embodiments, the subject is critically ill. In some embodiments, the subject is elderly or neonatal. In some embodiments, the subject suffers from febrile neutropenia. In some embodiments, the subject is suffering from an infection.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with a burn injury in a subject who is a burn victim, comprising administering to the subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any one of pp. [1]-[24]).

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with an infection in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any of paragraphs [1]-[24]). In some embodiments, the infection is a bacterial infection (eg, P. aeruginosa infection, S. aureus infection (eg, MRSA), mycobacterium tuberculosis infection, enterococcal infection (eg, VRE), or a condition related thereto. In some embodiments In embodiments, said infection is a fungal infection (eg, a candidal infection, such as invasive candidiasis) or a condition related thereto. In some embodiments, said infection is a parasitic infection (eg, schistosomiasis and human African trypanosomiasis) or a condition related thereto. with it. In some embodiments, the infection is malaria or a condition associated therewith, such as cerebral malaria, severe anemia, acidosis, acute renal failure, and ARDS. In some embodiments, the infection is a viral infection (eg, Ebola, Dengue and Marburg fever) or related conditions such as influenza, measles and viral hemorrhagic fever.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with ischemia or hypoxia in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical compositions according to any one of paragraphs [1]-[24]). In some embodiments, the ischemia- or hypoxia-related disease or condition is associated with a surgical or traumatic injury. In some embodiments, the disease or condition is ischemic reperfusion injury, transient cerebral ischemia, cerebral ischemia-reperfusion, ischemic stroke, hemorrhagic stroke, traumatic brain injury, migraine (eg, chronic migraine or severe migraine), gastrointestinal ischemia , kidney disease, pulmonary embolism, acute respiratory failure, neonatal respiratory distress syndrome, obstetric emergencies to reduce perinatal comorbidity (such as pre/eclampsia and conditions that lead to cerebral palsy), myocardial infarction, acute limb or mesenteric ischemia , cirrhosis of the heart, chronic peripheral vascular disease, congestive heart failure, atherosclerotic stenosis, anemia, thrombosis, embolism, macular degeneration, neurodegenerative disease (eg, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS)), sleep apnea, as well as surgical or traumatic injury. In some embodiments, the disease or condition associated with ischemia or hypoxia is myocardial infarction or congestive heart failure with or without cirrhosis. In some embodiments, the disease or condition is pulmonary embolism, acute respiratory failure, chronic peripheral vascular disease, atherosclerotic stenosis, anemia, thrombosis, or embolism. In some embodiments, said ischemia- or hypoxia-related disease or condition is macular degeneration or hypoxia-related cancer. In some embodiments, the disease or condition is a kidney disease. In some embodiments, the disease or condition is lipopolysaccharide drug or toxin-induced acute kidney injury (AKI) or

- 53 047345 end-stage kidney disease.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with shock in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any of paragraphs [1]-[24]). In some embodiments, the disease or condition is associated with cardiogenic shock. In some embodiments, the disease or condition is associated with hypovolemic shock. In some embodiments, the disease or condition is associated with septic shock or other forms of distributive shock. In some embodiments, the disease or condition is associated with neurogenic shock. In some embodiments, the disease or condition is associated with anaphylactic shock.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with nitric oxide deficiency in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical compositions according to any one of claims [1]-[24]). In some embodiments, the disease or disorder is sickle cell disease, paroxysmal nocturnal hemoglobinuria (PNH), hemolytic anemia, thalassemia, another red blood cell disorder, or a condition related thereto. In some embodiments, the disease or disorder is purpura, such as thrombotic platelet purpura (TTP), hemolytic uremic syndrome (HUS), idiopathic thrombocytopenia (ITP), and another platelet disorder or condition related thereto. In some embodiment, the disease or disorder is a coagulation abnormality such as disseminated intravascular coagulopathy (DIC), purpura fulminans, heparin-induced thrombocytopenia (HIT), hyperleukocytosis and hyperviscosity syndrome, or a condition associated therewith.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with inflammation in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any of paragraphs [1]-[24]). In some embodiments, the inflammatory disease or condition is a low-grade inflammation. In some embodiments, said inflammatory disease or condition is systemic inflammation. In some embodiments, said inflammatory disease or condition is an acute inflammatory disease or a chronic inflammatory disease.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with a cardiovascular disease or condition in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein ( for example, a pharmaceutical composition according to any one of claims [1] to [24]). In some embodiments, said cardiovascular disease or condition is coronary artery disease. In some embodiments, said cardiovascular disease or condition is myocardial infarction, sudden cardiac death, cardiac and respiratory arrest, hypertension, pulmonary arterial hypertension, atherosclerosis, occlusive arterial disease, Raynaud's disease, peripheral vascular disease, other vasculopathies, such as Buerger's disease, Takayasu's arthritis and post-cardiac arrest syndrome (PACS), chronic venous insufficiency, heart disease, congestive heart failure or chronic skin ulcer.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with a liver disease or condition in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., pharmaceutical composition according to any one of claims [1] to [24]). In some embodiments, said liver disease or condition is cirrhosis. In some embodiments, the liver disease or condition is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). In some embodiments, said liver disease or condition is alcoholic liver disease. In some embodiments, said liver disease or condition is acute liver injury.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with a lung disease or condition in a subject in need of such treatment or prevention, comprising administering said

- 54 047345 to the subject of the pharmaceutical composition proposed herein (for example, the pharmaceutical composition according to any one of claims [1] to [24]). In some embodiments, said lung disease or condition is acute respiratory distress syndrome (ARDS). In some embodiments, said lung disease or condition is chronic obstructive pulmonary disease. In some embodiments, said lung disease or condition is pulmonary fibrosis. In some embodiments, said lung disease or condition is pulmonary hemorrhage. In some embodiments, said lung disease or condition is asthma. In some embodiments, said lung disease or condition is lung damage. In some embodiments, said lung disease or condition is lung cancer. In some embodiments, the condition is cystic fibrosis.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with a kidney disease or condition in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (eg, pharmaceutical composition according to any one of claims [1] to [24]). In some embodiments, said kidney disease or condition is lipopolysaccharide drug-induced acute kidney injury (AKI). In some embodiments, said kidney disease or condition is chronic renal failure with or without end-stage renal disease.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with a vascular disease in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any of paragraphs [1]-[24]). In some embodiments, said disease or condition is coronary artery disease. In some embodiments, the disease or condition is hypertension. In some embodiments, said disease or condition is atherosclerosis. In some embodiments, the disease or condition is post-cardiac arrest syndrome (PACS). In some embodiments, the disease or condition is arterial occlusive disease, peripheral vascular disease, chronic venous insufficiency, chronic skin ulcers, or Raynaud's disease. In some embodiments, said disease, disorder, or condition associated with vascular disease is a heart disease. In further embodiments, said disease, disorder or condition is congestive heart failure. In some embodiments, said disease, disorder, or condition associated with vascular disease is an ischemic bowel disease.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with a heart attack or stroke in a subject in need of such treatment or prevention and/or at risk for a heart attack or stroke, comprising administering to said subject a pharmaceutical composition provided herein (eg, a pharmaceutical composition according to any one of claims [1] to [24]). In some embodiments, the disease, disorder or condition is ischemic stroke. In some embodiments, the disease, disorder, or condition is a hemorrhagic stroke.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with the nervous system in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any of paragraphs [1]-[24]). In some embodiments, said disease or condition is pain (eg, chronic pain). In some embodiments, the disease or condition is a neurodegenerative disease (eg, Alzheimer's disease or Parkinson's disease). In some embodiments, said disease, disorder, or condition associated with the nervous system is nerve damage.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with inflammatory bowel disease in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical compositions according to any one of claims [1]-[24]). In some embodiments of the invention

- 55 047345 The specified disease, disorder or condition is Crohn's disease. In some embodiments, said disease, disorder, or condition is ulcerative colitis.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with type 2 diabetes or a predisposition to diabetes in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (for example, a pharmaceutical composition according to any one of claims [1] to [24]). In some embodiments, said disease, disorder, or condition is a metabolic disease. In some embodiments, said disease, disorder, or condition is insulin resistance. In some embodiments, the disease, disorder, or condition is a diabetic vascular disease (eg, a microvascular disease such as retinopathy and nephropathy). In some embodiments, the disease, disorder, or condition is diabetic neuropathy. In some embodiments, the disease, disorder, or condition is ulcers, diabetic necrosis, or gangrene.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with an autoimmune disorder in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any of paragraphs [1]-[24]). In some embodiments, said autoimmune disorder is psoriasis. In some embodiments, said autoimmune disorder is cystic fibrosis. In some embodiments, said autoimmune disorder is rheumatoid arthritis.

In some embodiments, provided herein is a method of treating or preventing a disease, disorder, or condition associated with sclerosis in a subject in need of such treatment or prevention, comprising administering to said subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any of paragraphs [1]-[24]). In some embodiments, said disease, disorder, or condition associated with sclerosis is systemic sclerosis.

In some embodiments, provided herein is a method of treating endotoxemia in a subject in need of such treatment, comprising administering to said subject a pharmaceutical composition provided herein (eg, a pharmaceutical composition according to any one of claims [1] to [24]). In some embodiments, said endotoxemia is associated with a condition such as periodontal disease (eg, periodontitis or gum disease), chronic alcoholism, chronic smoking, transplantation, neonatal necrotizing enterocolitis, or neonatal ear infection.

In some embodiments, provided herein is a method of reducing systemic levels of LPS, endotoxin, and/or other systemic inflammatory trigger in a subject in need thereof, comprising administering to the subject a pharmaceutical composition provided herein (e.g., a pharmaceutical composition according to any one of claims. [1]-[24]).

Combination therapy.

The compositions provided herein may be administered alone or in combination therapy with one or more additional therapeutic agents. In some embodiments, the composition is administered in combination therapy with another therapeutic agent. The combinations can be administered either simultaneously, for example, combined in the same delivery vehicle (eg, liposome), in mixture with each other, separately but simultaneously; or sequentially. This includes proposals in which the combined therapeutic agents are administered together as a therapeutic mixture, as well as techniques in which the combined agents are administered separately but simultaneously, such as through separate intravenous lines to the same individual. Administration in combination further includes separate administration of one of the therapeutic agents prior to administration of the second. Methods of treatment using the specified combination therapy are also proposed.

In additional embodiments, the composition provided herein is administered in combination with another therapeutic agent. In some embodiments, the composition of any one of claims [1] to [8] is administered in combination with another therapeutic agent. In some embodiments, a composition comprising a carotenoid salt as provided in any of FIGS. 1A-1D herein are administered in combination therapy with another therapeutic agent. In some embodiments, a composition comprising a polyvalent salt (eg, a divalent salt or a trivalent salt) of a carotenoid as provided in any of FIGS. 1A-1D herein are administered in combination therapy with another therapeutic agent. In specific embodiments of the invention, a composition containing a polyvalent

- 56 047345 new salt of trans-crocetin (for example, CTC or MTS), administered in combination therapy with another therapeutic agent. In other specific embodiments, a composition containing a polyvalent trans-norbixin salt (eg, CTN or MTN) is administered in combination therapy with another therapeutic agent.

In some embodiments, a pharmaceutical composition containing a salt of one or more ionizable carotenoids is administered in combination therapy with a carotenoid containing at least one polar group or monocyclic group. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, a salt containing a divalent, trivalent, or tetravalent counterion). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In one embodiment of the invention, said carotenoid containing at least one polar group or monocyclic polar group is symmetrical. In another embodiment of the invention, the divalent salt composition of an ionizable carotenoid is administered in combination therapy with at least one carotenoid selected from: zeaxanthin, astaxanthin, lutein and xanthophyll. In another embodiment of the invention, said divalent salt of an ionizable carotenoid composition is administered in combination therapy with astaxanthin. In another embodiment of the invention, said carotenoid containing at least one polar group or monocyclic polar group is asymmetric. In another embodiment, the ionizable carotenoid divalent salt composition described herein is administered in combination with abscisic acid (ABA).

In some embodiments, a pharmaceutical composition containing an ionizable carotenoid salt provided herein is administered in combination therapy with a standard of care for the disease, disorder, or condition being treated. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with an antimicrobial agent. In some embodiments, the antimicrobial agent is an antibacterial agent. In some embodiments, said antibacterial agent is selected from, but is not limited to, ertapenem, piperacillin-tazobactam, cefepime, aztreonam, metronidazole, meropenem, ceftriaxone, ciprofloxacin, vancomycin, linezolid, tobramycin, levofloxacin, azithromycin, cefazolin, and mpicillin. In some embodiments, the antibacterial agent is selected from, but is not limited to, ceftriaxone, levofloxacin, ciprofloxacin, cefazolin, piperacillinatazobactam, meropenem, metronidazole, vancomycin, and ampicillin. In other embodiments, said antimicrobial agent is an antifungal agent. In additional embodiments, said antifungal agent is caspofungin or another antifungal drug. In other embodiments, said antimicrobial agent is an antimalarial agent. In additional embodiments, said antimalarial agent is selected from, but is not limited to, artemisinin and analogs thereof, chloroquine and analogs thereof, atovaquone, a quinine derivative, proguanil, or other antimalarial drug. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with activated protein C (eg, rhAPC) or drotrecogin alfa (activated) (DAA). In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments of the invention,

- 57 047345 The ionizable carotenoid present is trans-crocetin (eg CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition containing an ionizable carotenoid salt provided herein is administered in combination therapy with a corticosteroid (eg, a glucocorticoid or mineralocorticoid, such as fludrocortisone). In some embodiments, said corticosteroid is a glucocorticoid. In additional embodiments, said glucocorticoid is selected from cortisone, etamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone, and methylprednisolone. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with an intravenous vitamin. In some embodiments, said vitamin is vitamin C (ascorbic acid). In some embodiments, said vitamin is vitamin A. In some embodiments, the provided ionizable carotenoid salt is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with a glucocorticoid and vitamin C (eg, intravenous vitamin C). In some embodiments, said glucocorticoid is selected from cortisone, etamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone, and methylprednisolone. In additional embodiments, said glucocorticoid is hydrocortisone. In additional embodiments, at least one ionizable carotenoid composition provided herein (e.g., a divalent salt composition containing the ionizable carotenoid described in FIGS. 1A-1C and/or 1D) is administered in combination therapy with a glucocorticoid, vitamin With and thiamine. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is transcrocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with a vasopressor agent. In some embodiments, said vasopressor therapeutic agent is norepinephrine or similar drugs, or angiotensin II (eg, GIAPREZA™). In some embodiments, said vasopressor therapeutic agent is epinephrine, phenylnephrine, dopamine, or vasopressin. In some embodiments, said vasopressor therapeutic agent is ephedrine, milrinone, isoproterenol, dobutamine, isoproterenol, or dopamine.

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with a thrombolytic therapeutic agent. In some embodiments, said thrombolytic therapeutic agent is tissue plasminogen activator (tPA). In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

- 58 047345

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with a therapeutic agent. In some embodiments, the pharmaceutical composition of any one of claims [1] to [28] is administered in combination with a therapeutic agent. In some embodiments, a pharmaceutical composition comprising a polyvalent carotenoid salt as provided in any of FIG. 1A-1D herein are administered in combination therapy with a therapeutic agent. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with a therapeutic agent selected from: heparin, vasopressin, antidiuretic hormone (ADH), and a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin). In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1][8] and/or in FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with an anti-inflammatory therapeutic agent. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with oxygen and/or intravenous fluids to maintain/increase blood oxygen levels and/or blood pressure, or with hyperbaric therapy. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with a chemotherapeutic agent (eg, to improve the effect of chemotherapy on cancer cells and mitigate the effects of myelosuppression and anemia caused by chemotherapy). In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is transnorbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid salt provided herein is administered in combination therapy with immunotherapy. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (e.g.

- 59 047345

STS and MTS). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

In some embodiments, a pharmaceutical composition containing an ionizable carotenoid salt provided herein is administered in combination therapy with radiation therapy. In some embodiments, the ionizable carotenoid salt provided is a multivalent salt (eg, divalent, trivalent, or tetravalent). In some embodiments, said ionizable carotenoid is a carotenoid according to any one of claims [1] to [8] and/or FIG. 1A-1D. In specific embodiments, said ionizable carotenoid is trans-crocetin (eg, CTC and MTC). In other specific embodiments, said ionizable carotenoid is trans-norbixin (eg, CTN and MTN).

Kits for the administration of active agents.

In other embodiments, provided herein is a kit for administering a disclosed ionizable carotenoid composition to a subject to treat a disease, disorder, or condition. In some embodiments, provided herein is a kit for delivering a therapeutic agent to a subject, comprising (a) a first composition comprising a disclosed ionizable carotenoid composition (eg, a liposome containing a polyvalent trans-crocetin salt); and (b) a second composition containing, for example, reagents, buffers, excipients or other therapeutic agent, which is stored separately until administration to the subject. Such kits typically contain two or more components necessary to treat a disease condition, such as hypoxia or an inflammatory disease. In some embodiments, these kits contain, for example, the proposed lipid compositions, reagents, buffers, containers and/or equipment. These compositions and liposome formulations may be in lyophilized form and then reconstituted prior to administration. In some embodiments, the kits comprise a packaging assembly that contains one or more components used to treat a disease condition in a patient. For example, said packaging assembly may contain separate containers that hold therapeutic liposomes and other excipients or therapeutic agents that may be mixed with said compositions prior to administration to a patient. In some embodiments, a physician may select and match certain components and/or packaging assemblies depending on the treatment or diagnosis needed for a particular patient.

Examples

Example 1. Preparation of calcium trans-crocetinate liposomes.

Two different variants of trans-crocetin have been used to prepare trans-crocetin liposomes, namely trans-crocetin free acid (TC) and its sodium salt, sodium trans-crocetinate (STC). trans-Crocetin was encapsulated in liposomes using the following methods.

Preparation of multi-bilayer (multilamellar) vesicles (MLV).

First, the lipid components of the liposome lipid membrane were weighed and combined as a concentrated solution in ethanol at a temperature of approximately 65°C. In one formulation, the lipids used were hydrogenated soybean phosphatidylcholine, cholesterol, and DSPE-PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]).

The molar ratio of HSPC:cholesterol:PEG-DSPE was about 3:2:0.15. In another formulation, the lipids used were HSPC, cholesterol, PEG-DSPE-2000 and 1-palmitoyl-2-glutarylsn-glycero-3-phosphocholine (PGPC). The molar ratio of HSPC:cholesterol:PEG-DSPE:PGPC was about 2.7:2:0.15:0.3. Calcium acetate was then dissolved in aqueous buffer at a concentration of 125 or 250 mM, pH 7.0. The calcium acetate solution was heated to 65°C.

The ethanol solution of the lipid was added to the calcium acetate solution using a pipette. At this stage, the solution was mixed well with a magnetic stirrer. Mixing was carried out at elevated temperature (63-72°C) to ensure that the lipids were in a liquid crystalline state (as opposed to the gel state, which they assume at a temperature below the lipid transition temperature (Tm=51-54°Q). As a result, the indicated the lipids were hydrated and formed multi-lamellar vesicles (MLVs) containing calcium acetate in the inner space.

Reduction of MLV size by filter extrusion.

MLVs were fragmented into unilamellar vesicles (one bilayer) of the desired size by high-pressure extrusion using two passes through stacked (polycarbonate) membranes. The stacked membranes had two layers with a pore size of 200 nm and six layers with a pore size of 100 nm. During extrusion, the temperature was maintained above Tm to ensure the plasticity of the lipid membranes. As a result of extrusion, large and heterogeneous in size and lamellarity MLVs were converted into small homogeneous (100–120 nm) unilamellar vesicles (ULVs), which sequestered calcium acetate into their interior. A Malvern Zetasizer Nano ZS instrument (Southboro, MA) with a scattering detector (90°) was used to measure the hydrodynamic size (diameter) of vesicles at 25°C in a plastic microcuvette. Samples were diluted 50 times in a matrix composition

- 60 047345 va before analysis.

After ULVs containing calcium acetate were obtained, extraliposomal calcium acetate was removed by gel filtration chromatography (SEC, with PD-10 columns) or TFF (tangential flow diafiltration). An isotonic agent was added to the liposomes to balance osmolality (final concentration: 5% dextrose for 125 mM calcium acetate liposome and 10% dextrose for 250 mM calcium acetate liposome). Once a calcium acetate concentration gradient had formed, the trans-crocetin loading procedure was preferably carried out for 24 hours. The lipid content of the resulting liposome solution was determined by phosphate analysis.

A solution of 1 mg/ml trans-crocetin was prepared in 10% dextrose (for liposomes 250 mM calcium acetate) and the pH was adjusted to 8. The trans-crocetin solution was mixed with a solution of calcium acetate liposomes at various drug/lipid ratios (100, 80, 60 or 40 g/mM). The mixture was then thoroughly stirred and heated to 65°C for 30 minutes and then quickly cooled to room temperature using an ice bath. This step can be replaced by stirring the mixture at room temperature overnight.

The movement of the trans-crocetin molecule (uncharged, neutral form) through the lipid bilayer of the liposome was achieved using a gradient generated by calcium acetate (in other words, acetic acid diffused outward, trans-crocetin diffused inward). Transcrocetin was then retained within the liposomes by ionization and then precipitating with calcium (as a calcium salt form (calcium trans-crocetinate, CTC)).

Purification of liposomes.

Extraliposomal trans-crocetin was removed by gel filtration chromatography (PD-10 columns) or TFF. In this example, the buffer used in SEC was HBS (HEPES buffered saline, pH 6.5). Upon completion of cleaning, filter sterilization was performed using a 0.22 μm filter. A Malvern Zetasizer Nano ZS instrument (Southboro, MA) with a scattering detector (90°) was used to measure the hydrodynamic size (diameter) of vesicles at 25°C in a plastic microcuvette. Samples were diluted before analysis.

Table 1

Physical characteristics of typical nanoparticles loaded with HTS

Initial concentration of IH The effect of encapsulation Final concentration Drug/lipide ratio Diameter tr PDI Zetapotential STS LP 1 mg/ml transcrocet 96.9% 0.24 mg/ml 78.6 g/mM lipids 105.7 nm 0.05 6 -2.88 mV

in disodium STS LP 0.75 mg/ml transcrocet in disodium 98.32% 3.92 mg/ml 68.23 g/mM lipids 103.8 nm 0.04 1 -2.71 mV STS LP 0.75 mg/ml transcrocet in disodium 99.47% 3.90 mg/ml 66.23 g/mM lipids 100.8 nm 0.03 1 -3.67 mV STS LP 0.75 mg/ml transcrocet in disodium 92.59% 2.49 mg/ml 34.74 g/mM lipids 101.9 nm 0.03 8 -3.83 Mv STS LP 0.75 mg/ml transcrocet in disodium 98.30% 5.34 mg/ml 85.74 g/mM lipids 95.9 nm 0.04 -3.66 mV

Example 2. Preparation of calcium acetate liposomes with Nanoassemblr®.

Liposomes loaded with calcium acetate were prepared by the following procedure. First, the lipid components of the liposome lipid membrane were weighed and combined as a concentrated solution in ethanol at a temperature of approximately 65°C. In one example, the lipids used were hydrogenated soybean phosphatidylcholine, cholesterol, and DSPE-PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-I-[methoxy(polyethylene glycol)-2000]).

The molar ratio of HSPC:cholesterol:PEG-DSPE was about 3:2:0.15. In another example, the lipids used were HSPC, cholesterol, PEG-DSPE^OOO and 1-palmitoyl-2-glutarylsn-glycero-3-phosphocholine (PGPC). The molar ratio of HSPC:cholesterol:PEG-DSPE:PGPC was

- 61 047345 about 2.7:2:0.15:0.3.

Calcium acetate was then dissolved in aqueous buffer at a concentration of 125 or 250 mM, pH 7.0. The calcium acetate solution was heated to 65°C. The lipid solution in ethanol and the calcium acetate solution were separately transferred into syringes. The two solutions were introduced into the microfluidic channel and mixed by passing through it with a NanoAssemblr® device from Precision NanoSystems. Mixing was carried out at elevated temperature (63-72°C) to ensure that these lipids were in a liquid crystalline state (as opposed to the gel state, which they assume at a temperature below the lipid transition temperature (Tm=51-54°C)). The size of the liposome can be controlled by the ratio between the lipid solution and the aqueous solution, as well as the mixing flow rate. Example 3- Preparation of MTC Liposomes and its Characteristics Preparation of Trans-crocetin Liposomes by Magnesium Acetate Gradient: To obtain trans-magnesium crocetinate liposomes, two different variants of this molecule were used, namely: trans-crocetin free acid (TC) and its sodium salt, sodium trans -crocetinate (STC).

Liposome with magnesium acetate was prepared by the following procedure. First, the lipid components of the liposome membrane were weighed and combined as a concentrated solution in ethanol at a temperature of about 65°C. In one example, the lipids used were hydrogenated soybean phosphatidylcholine, cholesterol, and DSPE-PEG-2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000]). The molar ratio of HSPC:cholesterol:PEG-DSPE was about 3:2:0.15. In another example, the lipids used were HSPC, cholesterol, PEG-DSPE-2000 and 1-palmitoyl-2-glutaryl-glycero-3-phosphocholine (PGPC). The molar ratio of HSPC:cholesterol:PEG-DSPE:PGPC was about 2.7:2:0.15:0.3. Magnesium acetate was then dissolved in aqueous buffer at a concentration of 125 or 250 mM, pH 7.0. The magnesium acetate solution was heated to 65°C. The lipid solution in ethanol was added to the magnesium acetate solution using a pipette. At this stage, the solution was mixed well with a magnetic stirrer. Mixing was carried out at elevated temperature (63-72°C) to ensure that the lipids were in a liquid crystalline state (as opposed to the gel state, which they take on below the lipid transition temperature (Tm=51-54°C)). As a result, these lipids were hydrated and formed multilamellar vesicles (MLVs) containing magnesium acetate in their internal space (internal solution). MLV size reduction by filter extrusion: MLVs were fragmented into unilamellar vesicles (one bilayer) of the desired size by high pressure extrusion using two passes through stacked (polycarbonate) membranes. The stacked membranes had two layers with a pore size of 200 nm and six layers with a pore size of 100 nm. During extrusion, the temperature was maintained above Tm. As a result of extrusion, large and heterogeneous in size and lamellarity MLVs were converted into small homogeneous (100–120 nm) unilamellar vesicles (ULVs), which sequestered calcium acetate into their interior. A Malvern Zetasizer Nano ZS instrument (Southboro, MA) with a scattering detector (90°) was used to measure the hydrodynamic size (diameter) of vesicles at 25°C in a plastic microcuvette. Samples were diluted 50-fold in the formulation matrix prior to analysis.

Generating a gradient.

After ULVs containing magnesium acetate were obtained, extraliposomal magnesium acetate was removed by gel filtration chromatography (SEC, with PD-10 columns) or TFF (tangential flow diafiltration). Isotonic agent solutions (such as 50% dextrose) were added to the liposomes to balance osmolality (final concentration: 5% dextrose for 125 mM magnesium acetate liposome and 10% dextrose for 250 mM magnesium acetate liposome). The lipid content of the resulting liposome solution was determined by phosphate analysis.

Loading of trans-crocetin into magnesium acetate liposomes.

A solution of 1 mg/ml trans-crocetin or sodium trans-crocetinate was prepared in 10% dextrose (for liposomes, 250 mM magnesium acetate) and the pH was adjusted to 8-8.5 with sodium hydroxide. A solution of sodium transcrocetinate was mixed with a solution of magnesium acetate liposomes at various drug/lipid ratios (100, 80, 60 or 40 g/mol). The mixture was then thoroughly stirred and heated to 65°C for 30 minutes and then quickly cooled to room temperature using an ice bath. This step can be replaced by stirring the mixture at room temperature overnight.

Purification of liposomes.

Extraliposomal trans-crocetin was removed by gel filtration chromatography (PD-10 columns) or TFF. In this example, the buffer used in SEC was HBS (HEPES buffered saline, pH 6.5). Upon completion of cleaning, filter sterilization was performed using a 0.2-0.22 μm filter. A Malvern Zetasizer Nano ZS instrument (Southboro, MA) with a scatter detector (90°) was used to measure hydrodynamic size (diameter) at 25°C in a plastic microcuvette. Samples were diluted before analysis.

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table 2

Physical characteristics of typical MTS-loaded nanoparticles

Initial concentration The effect of encapsulation Final concentration Ratio tion a/lipid preparation Diameter PDI Zetapotential MTS LP (D/L80) 0.75 mg/ml transcrocetin disodium 99.98% 5.03 mg/ml 77.22 g/mM lipids 102.1 nm 0.046 -2.32 mV MTS LP (D/L80) 0.75 mg/ml transcrocetin disodium 98.82% 4.00 mg/ml 58.83 g/mM lipids 103.4 nm 0.034 -3.23 mV MTS LP (D/L80) 0.75 mg/ml transcrocetin disodium 98.90% 2.25 mg/ml 35.13 g/mM lipids 103.7 nm 0.039 -3.23 mV

Table 3

Summary of PK results of liposomal STS and MTS

Product under test T1/2 (h) AUC (mg/ml *h) Stach (mg/ml) Exposure in plasma (Increase by times compared to free drug STC) NCA analysis Free drug STC 0.35 0.21 0.36 ND Free drug STC 0.47 0.26 ND ND CTC-LP-80 5.12 8.36 1.26 40 CTC-LP-60 4.52 6.4 1.1 35 CTC-LP-40 5.8 10.75 1.44 56 MTC-LP-80 2.88 5.29 1.29 25 MTC-LP-60 2.9 6.01 1.44 29 MTC-LP-40 2.67 5.25 1.37 25 Liposome Labeled with Fluorescent Dye 12.2 nd ND nd

Balb/c mice (3 mice/group) were treated with a single dose of free STC, CTC/MTC-LP (D/L ratio 80, 60, 40) and fluorescent dye-labeled liposomes via a slow intravenous bolus to collect serial blood samples in various time points within 24 hours (usually 5 minutes, 1, 2, 4, 8 and 24 hours).

μL of each plasma sample was mixed with 395 μL of methanol containing 1% formic acid. Sample mixtures were well mixed by vortex. Samples were incubated at −20°C for 1 h and then equilibrated at room temperature for 15 min. The samples were mixed by vortex and then centrifuged at 10,000 rpm for 10 min at room temperature. 200 µl

- 63 047345 supernatant was removed from each sample without disturbing the precipitate and analyzed by HPLC. If the amount of plasma allowed, this analysis was duplicated.

The concentration of STC in plasma samples was determined using a standard curve constructed by analyzing plasma samples containing a known amount of STC. PK profiles were analyzed.

Table 4

CTC liposome stability

Testing my product Date of analysis Size P particles c (nm) PDI Zeta potential (mV) Conc. lipid (mM) Conc. crocetin (mg/ml) Resulting D/L CTC-LP- 80 1st Come on 101.1 0.039 -3.09 63.88 5.01 78.47 CTC-LP- 80 1st 1 month 100.3 0.046 -1.56 1.64 0.13 77.73

CTC-LP- 80 1st 2 months 99.33 0.049 -3.44 1.25 0.10 76.46 CTC-LP- 80 1st 5 months 99.56 0.046 -2.49 2.03 0.16 78.37 CTC-LP- 80 1st 6 months 103.3 0.06 -2.21 1.73 0.14 78.66 CTC-LP- 80 2nd Come on 97.3 0.049 -3.44 71.09 5.44 76.57 CTC-LP- 80 2nd 3 months 99.6 0.037 -2.21 2.22 0.17 78.83 CTC-LP- 80 2nd 4 months 99.8 0.038 -3.27 2.14 0.17 79.91 CTC-LP- 80 2nd 5 months 99.7 0.05 -4.55 1.31 0.10 78.73 CTC-LP- 80 3rd Come on 102.3 0.042 -0.80 70.57 5.48 77.64 CTC-LP- 80 3rd 2 months 102.9 0.038 -2D1 2.04 0.16 77.72 CTC-LP- 80 3rd 3 months 102.3 0.042 -2.74 2.01 0.15 76.22 CTC-LP- 80 3rd 4 months 104.2 0.090 -2.17 1.12 0.09 77.10 CTC-LP- 80 4th Come on 99.6 0.037 -2.21 70.57 5.58 79.11 CTC-LP- 80 4th 1 month 101.6 0.054 -2.00 2.52 0.20 80.76 CTC-LP- 80 4th 2 months 100.8 0.042 -3.22 2.05 0.16 77.11 CTC-LP- 3 102.8 0.073 -5.01 1.36 oh and 79.06

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80 4th months CTC-LP- 60 Get out and I 100.8 0.0 -3.7 58.91 3.90 66.23 CTC-LP- 60 4 months 104.0 oh oh -1.9 2.12 0.14 68.12

CTC-LP- 60 month in 103.2 0.037 -2.63 1.70 0.12 68.89 CTC-LP- 40 Get out and I 101.9 oh oh -3.8 71.54 2.49 34.74 CTC-LP- 40 4 months 106.1 oh oh -2.1 2.54 0.09 36.58 CTC-LP- 40 5 months 103.1 0.038 -2.28 2.21 0.08 36.41

The stability of CTC liposomes was further assessed by characterizing the liposome solution after the liposomes had been cleared of potential eluted drug by a gel filtration column after a specified storage period (up to 6 months). Characterization methods were the same as previously described.

These CTC liposomes showed almost the same drug/lipid ratio within the error. Consequently, negligible leaching of the drug was confirmed over a 6-month storage period (4°C).

Table 5

Assessing the reproducibility and stability of liposome batches

D/L batch samples Mar Air May Jun Jul Auto Sep Oct CTC-LP-80 (1st) 76.57 76.37 75.81 75.36 78.10 CTC-LP-80 (2nd) 76.57 76.41 82.12 77.12 CTC-LP-80 (3rd) 77.64 78.13 77.81 76.54 CTC-LP-80 (4th) 79.11 82.93 78.92 80.77

The reproducibility and stability of liposome batches were assessed by D/L characteristic.

CTC liposomes showed negligible change in this assessment. Thus, CTC liposomes showed stability for at least 6 months.

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Table 6

Stability of MTS liposome

Testable product Date of analysis Particle size (nm) PDI Zeta potential (mV) Conc. lipid (mm) Conc. drug (mg/ml) D/L (g/mol) MTC-LP-80 Original 102.1 0.04 6 -2.32 65.2 5.03 77.22 MTC-LP-80 1 month 104.4 0.03 8 -2.84 19.70 1.53 77.72 MTC-LP-80 2 months 105.5 0.05 1 -4.78 18.29 1.41 77.22 MTC-LP-60 Original 103.4 0.03 4 -3.23 57.96 3.27 56.39 MTC-LP-60 10 days 105.2 0.05 -2.46 23.14 1.38 59.67 MTC-LP-60 1 month 105.4 0.05 6 -4.45 23.31 1.39 59.85 MTC-LP-40 Original 103.7 0.03 9 -3.23 64.04 2.25 35.13 MTC-LP-40 10 days 104 0.03 -2 24.39 0.87 35.76 MTC-LP-40 1 month 106.5 0.05 8 -5.74 23.21 0.84 36.27

Determination methods were the same as previously described.

These MTC liposomes showed almost the same drug/lipid ratio within the error. Therefore, the stability of the liposome for at least 2 months under storage conditions (4°C) was confirmed.

Example 4. Efficacy study protocol and results of liposomal STS.

Animals and livestock.

Male and/or female C57BL/6 mice purchased from Envigo Laboratories or Jackson Lab (Bar Harbor, ME) were acclimated to housing conditions and handled in accordance with Animal Handling Protocol (AUP) number TP-05. Animals were acclimatized for approximately 1 week before the start of the study. Only animals considered healthy were included in this study. Animals were fed irradiated Teklad Global Rodent Diet 2918 and water ad libitum. Mice were housed in groups of 5/cage in static cages with Teklad 1/8 7902 irradiated corncob bedding in bioBubble® cleanrooms by introducing H.E.P.A filtered air into a closed environment at 100 complete air changes per hour. The environment was controlled to a temperature range of 74±5°F and a humidity range of 30-70%. Treatment groups were determined by cell map. Individual mice were identified by an inedible marker at the base of the tail. All procedures performed in this experiment were conducted in accordance with laws, regulations, and guidelines of the National Institutes of Health and Institutional Animal Care Committee approval for laboratory research. Scale Ligature Puncture and Postoperative Procedure: On day 1, male and/or female mice were anesthetized with isoflurane and transferred to the surgical table. The lower quadrants of the abdomen were shaved with an electric trimmer. On day 0, mice were anesthetized with isoflurane and transferred to the surgical table. The shaved surface was disinfected with three alternating scrubs of chlorhexidine surgical scrub and 70% isopropanol. An abdominal longitudinal midline skin incision was made with iridectomy scissors without penetrating the abdominal cavity. After the initial incision, small scissors were used to widen the incision 1.5–2 cm to gain entry into the abdominal cavity. The median white fascia of the abdominal musculature was identified and cut for intermuscular incision and incision of the fascia and peritoneal layers. The cecum was removed from the surface of the body using blunt anatomical forceps, leaving the remainder of the small and large intestines within the abdominal cavity and avoiding disruption or damage to the mesenteric blood vessels. The cecum was ligated with a sterile 9.5 mm stainless steel surgical clamp below the ileocecal valve in the designated position (about 70% of the cecum was ligated). Care was taken not to block the intestines. Before cecal perforation, the cecal contents were gently pushed toward the distal cecum. The cecum was then perforated with a 16-gauge needle for severe sepsis. Conducted one end-to-end

- 66 047345 puncture in the middle between the ligature and the tip of the cecum in the direction from the mesenteric to the antimesenteric edge. After removing the needle, the cecum was moved into the abdominal cavity without distributing feces from the cecum onto the wound of the abdominal wall, but a small drop of feces was squeezed out from both the mesenteric and antimesenteric puncture holes. The droplet size was as reproducible as possible. The peritoneum, fascia, and abdominal musculature were closed using simple running sutures (4-0 PDS or chrome catgut surgical sutures) and the skin incision was sealed with 9 mm autoclips or surgical glue. Immediately after surgery, mice received a subcutaneous (SC) injection of 0.5 ml of 0.9% saline at room temperature. Animals were then allowed to recover from surgery in a clean cage placed on a warm, recirculating heating pad with free access to water and food pellets on the floor. The heating pad occupied half the cage so that the animals had the opportunity to move to a cooler part of the cage if desired. The mice remained conscious and able to move in this environment. The heating pad was removed once the animal was stable.

Animals were monitored continuously after surgery at least every 2-3 min for about 30 min until the animals recovered and were able to move independently. After this, the animals were observed every hour for at least 6 hours after surgery. Animals were also closely monitored (every hour from 7:00 to 18:00 each day) throughout the study, with additional observations at 10 pm and 2 am on days 1-3. Postoperative rodent recordings were made (1 recording per animal) during the study. Abnormal clinical signs were recorded during follow-up. Any animal showing signs of impending death was humanely euthanized. If the animal was euthanized, the time and date were recorded in the postoperative note.

Composition and dosage.

Mice were administered the test article by IP injection starting 2 hours after surgery and continued once daily until day 4 inclusive (a total of 5 days of dosing). Mice in groups 1-3 were dosed with a volume of ~10 μl of test article per gram of mouse body weight (as shown in Table 7; these administered doses represent a dose of 50 mg/kg per mouse per day for 5 days). Mice in group 4 were administered 0.9% saline once daily by IP injection in a volume of 0.3 ml on days 0-4. Mice were weighed daily, and dose volumes were administered according to the table. 7.

Table 7

Dose values

Groups 1, 2 and 3 (dose 10 µl/g) Body weight range Dose volume 20-23.9 g 0.2 ml 24-26.9 g 0.25 ml 27-30.9 g 0.3 ml

Endpoint analysis.

The effectiveness of the test products was assessed by listing the tested mortality of animals within 5 days after the CLP operation. Animals that remained alive on day 5 were humanely euthanized by exposure to CO ₂ .

Research results.

In table 8 describes four CLP studies used to test different formulations of liposomal CTC. Studies 1 and 2 examined a male mouse model of CLP. Studies 3 and 4 examined a female mouse model of CLP. The products tested and the results of each study are described below.

Table 8

Typical CLP studies on the effectiveness of liposomal STS

Study 1-TR-936 Study 2-TR-967 Study 3-TR-983 Type Efficiency Efficiency Efficiency Gender Mouse Male Female Female Size Research thirty 40 50 Groups 3 4 5

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Results of Study 1 (TP-936).

All surgical and dosing procedures were performed as detailed in the study protocol (above). Placebo animals demonstrated 100% survival. Mice that underwent CLP and were treated with saline and imipenem showed a 50% mortality rate. Animals receiving test article 1 and imipenem or test article 2 and imipenem showed 30 and 10% mortality, respectively. Five of the nine deaths during the study were the result of euthanasia due to dehydration and side decubitus (Figure 5).

Together, these data demonstrate that caecal ligation and puncture using a 16-gauge needle causes mortality in C57BL/6J mice. However, both drugs tested (in combination with imipenem) showed a trend toward reduced mortality compared with the imipenem-treated control group.

Results of Study 2 (TP-967).

Animals treated with saline and imipenem (Group 4) showed 70% mortality (Fig. 6). One in seven deaths was the result of euthanasia. Mice treated with PGPC-LP and imipenem (Group 1) showed a 60% mortality rate, with one in six deaths resulting from euthanasia. Group 2, which was treated with CTC-LP-80 and imipenem, showed 30% mortality (Fig. 6). Two of the three deaths were the result of euthanasia. Mice treated with PGPC-CTC-LP-80 and imipenem (Group 3) showed 70% mortality (Fig. 6). Two of the seven deaths were the result of euthanasia. Neither treatment group showed a statistically significant difference in mortality compared with the vehicle control group, but there was a strong trend toward improved survival.

Results of Study 3 (TP-986).

Mice treated with CTC-LP-80 (50 mg/kg) and imipenem showed 70% mortality (Fig. 8). Three of the seven deaths were the result of euthanasia. Mice treated with CTC-LP-80 (25 mg/kg) and imipenem showed 40% mortality (Fig. 7). Two of the four deaths were the result of euthanasia. Mice treated with CTC-LP-80 (5 mg/kg) and imipenem showed 20% mortality (Fig. 7). None of the deaths were the result of euthanasia. This treatment demonstrated a statistically significant reduction in mortality compared to the vehicle control group (P=0.0321). Mice treated with CTC-LP-80 (1 mg/kg) and imipenem showed 60% mortality (Fig. 7). None of the deaths were the result of euthanasia, Fig. 7.

Together, these data demonstrate that caecal ligation and puncture using a 16-gauge needle causes mortality in C57Bl/6 mice.

Treatment with test article CTC-LP-80 (5 mg/kg) and imipenem demonstrated a statistically significant reduction in mortality compared with the saline control group.

Example 5. Preparation of STC liposomes by passive loading.

Passive loading of sodium trans-crocetinate by extruder: Sodium trans-crocetinate was dissolved in the aqueous phase at maximum solubility in a given aqueous medium, for example, 0.7 mg/ml in 5% dextrose. A lipid solution in ethanol containing HSPC, cholesterol, PEG-DSPE, with/without PGPC was pipetted into the aqueous solution. At this stage, the solution was mixed well with a magnetic stirrer. Mixing was carried out at elevated temperature (63-72°C) to ensure that these lipids were in a liquid crystalline state (as opposed to the gel state, which they assume at a temperature below the lipid transition temperature (Tm=51-54°C)). As a result, these lipids were hydrated and formed multi-lamellar vesicles (MLVs) containing sodium trans-crocetinate in the aqueous core. The MLV was then reduced in size by extrusion as previously described.

Passive loading of trans-crocetin using Nanoassemblr®: Sodium trans-crocetinate was dissolved in the aqueous phase at maximum solubility in a given aqueous medium, for example, 0.7 mg/ml in 5% dextrose. A lipid solution in ethanol containing HSPC, cholesterol, PEG-DSPE, with/without PGPC and aqueous sodium trans-crocetinate was separately transferred into syringes. The two solutions were introduced into the microfluidic channel and mixed by passing through it with a NanoAssemblr® device from Precision NanoSystems. Mixing was carried out at elevated temperature (63-72°C) to ensure that the lipids were in a liquid crystalline state (as opposed to the gel state, which they take on below the lipid transition temperature (Tm=51-54°C)). The size of the liposome can be controlled by changing the ratio between lipid solution and aqueous solution, as well as the mixing flow rate. Passive loading of trans-crocetin by ethanol injection: trans-crocetin (free acid) was dissolved in a mixture of lipids in ethanol at maximum solubility. The lipid mixture in ethanol containing trans-crocetin was then either mixed with an aqueous solution (eg, a solution of buffers, buffered saline or dextrose) and reduced in size by extrusion, or mixed with the aqueous solution through the microfluidic channel of the NanoAssemblr® device.

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Claims (26)

Passive loading of trans-crocetin by thin film rehydration method. trans-Crocetin (free acid) was dissolved in a volatile organic solvent (eg ethanol, methanol, chloroform, dichloromethane, etc.) along with other lipids: HSPC, cholesterol, PEG-DSPE, with/without PGPC. The organic solvent in the trans-crocetin-lipid mixture was completely dried using a rotary evaporator at elevated temperature (eg, 65°C) in a water bath and vacuum. During drying, the flask was rotated, and a dried thin film of trans-crocetin lipid was formed on the wall of the round-bottomed flask. An aqueous solution was added to the thin film and rotated/stirred at elevated temperature (eg, 65°C). Rehydration of the thin film in aqueous solution forms multi-lamellar vesicles (MLVs) containing transcrocetin in the MLV lipid bilayer. The MLVs were then reduced in size by extrusion to the desired small unilamellar vesicles (SUVs). Example 6. Preparation of targeted trans-crocetin liposomes: After loading. The antibody or its fragments, such as Fab or scFv, which contain a cysteine residue at the C-terminus, were conjugated and incorporated into the trans-crocetin liposome by the post-loading method. Thiol-reactive lipopolymer micelles (eg, DSPE-PEG-maleimide) were prepared by dissolving in an aqueous solution at 10 mg/mL. The cysteine-terminated antibody (or fragment thereof) was dissolved and reduced with 10-20 mM of a reducing reagent (eg, 2-mercaptoethylamine, cysteine, or dithioerythritol) at pH <7. Excess reducing reagent was carefully removed using SEC (gel filtration chromatography) or dialysis. The purified and reconstituted antibody (or fragment thereof) was then incubated with thiol-reactive lipopolymer micelles at a molar ratio of 1:4. At the end of the reaction, excess maleimide groups were quenched with a small amount of cysteine (1 mM) or mercaptoethanol. Unconjugated antibody (or fragment thereof) was removed using SEC. The purified conjugated micelles were then incubated with liposomes at 37°C or elevated temperature at various antibody/lipid ratios (this ratio depends on the antibody). Although the described methods have been described in connection with what are currently considered the most practical and preferred embodiments of the invention, it should be understood that the methods covered by this description are not intended to be limited to the described embodiments, but rather are intended to cover various modifications and equivalents included in the spirit and scope of the appended claims. All publications, patents, patent applications, Internet sites and accession numbers/database sequences, including both polynucleotide and polypeptide sequences, cited herein are incorporated herein by reference in their entirety for all purposes to the same extent , as if each individual publication, patent, patent application, Internet site, or accession number/database sequence were specifically and individually designated for inclusion by reference. CLAIM 1. A pharmaceutical composition containing a liposome encapsulating an ionizable carotenoid salt having the formula Q-trans-crocetin-Q or Q-trans-norbixin-Q, where Q is a polyvalent cationic counterion. 2. The pharmaceutical composition according to claim 1, wherein Q is a divalent metal cation, a divalent organic cation or a trivalent cation such as Fe ³⁺ . 3. The pharmaceutical composition according to claim 2, wherein Q is a divalent metal cation selected from Ca ²⁺ , Mg ²⁺ , Zn ² '' Cu ²⁺ , Co ²⁺ and Fe ²⁺ . 4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the liposome encapsulates an ionizable carotenoid salt having the formula Q-trans-norbixin-Q. 5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the liposome encapsulates an ionizable carotenoid salt having the formula Q-trans-crocetin-Q. 6. Pharmaceutical composition according to any one of claims 1-3 and 5, in which the liposome encapsulates magnesium trans-crocetinate (MTS). 7. Pharmaceutical composition according to any one of claims 1-3 and 5, in which the liposome encapsulates calcium trans-crocetinate (CTC). 8. Pharmaceutical composition according to any one of claims 1 to 4, wherein the liposome encapsulates magnesium trans-norbixinate (MTN) or calcium trans-norbixinate (CTN). 9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the ionizable carotenoid/lipid ratio is from about 1 to 1000 g/mol lipid, from about 10 to about 150 g/mol lipid, or from about 20 to about 100 g/mol lipid. mole of lipid or in which said liposomes contain at least - 69 047345 from 0.1 to 97% by weight (w/w) of ionizable carotenoid. 10. Pharmaceutical composition according to any one of claims 1 to 9, wherein said liposome has a diameter of 20 to 500 nm, 20 to 200 nm or 80 to 120 nm. 11. The pharmaceutical composition according to any one of claims 1 to 10, wherein said liposome is formed from liposome components, wherein the liposome components include: at least one of a cationic lipid, an anionic lipid and a neutral lipid, or at least one selected from the following: distearoylphosphatidyl-1-ethanolamine (DSPE); DSPE-polyethylene glycol (PEG); DSPE-PEG-maleimide; hydrogenated soy phosphatidylcholine (HSPC); HSPC-PEG; cholesterol; cholesterol-PEG and cholesterol-maleimide. 12. The pharmaceutical composition according to any one of claims 1 to 11, wherein said liposome contains an oxidized phospholipid, such as oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3phosphorylcholine (OxPAPC). 13. The pharmaceutical composition according to claim 11, wherein the one or more liposome components further includes at least one steric stabilizer selected from poly-P-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly|N-(2hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidones; polymer based on L-amino acid; oligoglycerin, a copolymer containing polyethylene glycol and polypropylene oxide, Poloxamer 188 and polyvinyl alcohol. 14. Pharmaceutical composition according to any one of claims 1 to 13, wherein said liposome is anionic, neutral or cationic. 15. The pharmaceutical composition according to any one of claims 1 to 14, wherein said liposome has a zeta potential of -150 to 150 mV or -50 to 50 mV. 16. The pharmaceutical composition according to any one of claims 1 to 14, wherein said liposome has a zeta potential of -150 to 0 mV or -50 to 0 mV. 17. The pharmaceutical composition according to any one of claims 1 to 16, further comprising a pharmaceutically acceptable carrier which includes an isotonic agent such as dextrose, mannitol, glycerin, potassium chloride or sodium chloride. 18. The pharmaceutical composition according to any one of claims 1 to 17, in which said liposome contains less than 6 million, less than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000 or less than 5,000 molecules, or from 10 to 100,000, from 100 to 10,000 or 500 to 5,000 molecules of ionizable carotenoid. 19. The pharmaceutical composition according to any one of claims 1 to 18, wherein said liposome further comprises a targeting moiety, and wherein said targeting moiety has a specific affinity for a surface antigen on a target cell of interest. 20. The pharmaceutical composition according to claim 19, wherein said targeting fragment is a polypeptide, antibody, humanized antibody, antigen binding fragment of an antibody, single chain antibody, single domain antibody, bispecific antibody, synthetic antibody, pegylated antibody or multimeric antibody. 21. The pharmaceutical composition according to claim 19 or 20, in which said liposome contains from 1 to 1000, from 50 to 750, from 100 to 500 or from 30 to 200 targeting fragments. 22. The pharmaceutical composition according to any one of claims 1 to 21, further containing one or more of FABP, an immunostimulating agent, an immunosuppressive agent, a detectable marker and a maleimide, wherein said FABP, an immunostimulating agent, an immunosuppressive agent, a detectable marker and maleimide is attached to the specified PEG or the outer part of the liposome. 23. The pharmaceutical composition according to any one of claims 1 to 22, further containing at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol and sucrose. 24. The pharmaceutical composition according to any one of claims 1 to 23, intended for use in treating a disease or condition in a subject. 25. The pharmaceutical composition according to claim 24, wherein said disease or condition is a disease or condition associated with endotoxemia; sepsis; infection; bacteremia; liver disease or condition; or a lung disease or condition such as acute respiratory distress syndrome (ARDS), pulmonary fibrosis, pulmonary hemorrhage, lung injury, lung cancer or chronic obstructive pulmonary disease (COPD); kidney disease; autoimmune disorder; sclerosis; inflammation; inflammatory bowel disease; metabolic disease; insulin resistance; diabetes, such as type 2 diabetes, or a related condition; cardiovascular disease; disease or condition characterized by ischemia or hypoxia; heart attack or stroke; shock; or a condition associated with nitric oxide deficiency. 26. A method for producing a pharmaceutical composition according to any one of claims 1 to 23, including: (a) preparing a liposome solution containing the liposomes in a solution of a weak acid salt of a polyvalent metal or polyvalent organic cations such as a protonated amine; (b) adding an ionizable carotenoid to said liposome solution, wherein the ionizable carotenoid -