OA20783A - Carotenoid compositions and uses thereof - Google Patents

Carotenoid compositions and uses thereof Download PDF

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OA20783A
OA20783A OA1202000394 OA20783A OA 20783 A OA20783 A OA 20783A OA 1202000394 OA1202000394 OA 1202000394 OA 20783 A OA20783 A OA 20783A
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liposome
pharmaceutical composition
disease
carotenoid
ionizable
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OA1202000394
Inventor
Clet Niyikiza
Victor Mandla MOYO
Bolin Geng
Zhenghong Xu
Kaniz KHALIFA
Gwangseong Kim
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L.E.A.F. Holdings Group Llc
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Publication of OA20783A publication Critical patent/OA20783A/en

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Abstract

Provided herein are pharmaceutical compositions comprising carotenoids, including liposomes that encapsulate carotenoids including ionizable carotenoids such as trans-crocetin. The provided compositions have uses in treating diseases, disorders and conditions associated with, but not limited to, infection, endotoxemia, inflammation, sepsis, ischemia, hypoxia, shock, stroke, lung injury, wound healing, traumatic injury, reperfusion injury, cardiovascular disease, kidney disease, liver disease, inflammatory disease, metabolic disease, pulmonary disorders, blood related disorders and hyperproliferative diseases such as cancer. Methods of making, delivering, and using the pharmaceutical compositions are also provided.

Description

CAROTENOID COMPOSITIONS AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001} This application daims priority of U.S. Provisional Application Nos. 62/666,699 filed May 3, 2018, and 62/809,123 filed February 22, 2019, each of which is incorporated herein in its entirety.
BACKGROUND
[0002] Under normal conditions, bacteria and related toxins (endotoxemia) should not routinely be found in the blood of healthy human beings. There is, however, an increasing récognition 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 conséquences of this process known as bacterial translocation are a State of chronic bacteremîa (bacteria) in the blood stream. Bacterial translocation leads to chronic bacteremîa that sheds endotoxins such as bacterial lipopolysaccharides, LPS that gives rise to chronic low grade chronic endotoxemia and the resulting chronic inflammatory state. This chronic low-grade inflammation associated with bacterial translocation has been linked to the pathogenesis of many diseases.
[0003] A more severe form of endotoxemia is associated with sepsîs, a lifethreatening medical condition caused by dysregulated host inflammatory response to infection. Sepsis is a global healthcare problem that strikes an estimated 30 million people worldwide every year. The mortality rate from sepsis is approximately 40% in adults. Sepsis arises when the body’s attempt to fight an infection results in the immune System damaging tissues and organs. This uncontrolled response, normally designed to protect the body, causes widespread inflammation, leaky blood vessels, and abnormal blood clotting resulting in organ damage. In severe cases, blood pressure drops, multiple organ failures ensue, and the patient can die rapidly from septic shock.
[0004] Management of sepsis is a complicated and unmet clinîcal challenge requîring early récognition and management of infection, hémodynamie issues, and other organ dysfunctions. The sepsis underlying infection is treated with antimicrobiais, most commonly broad spectrum anti-bacterial, anti-viral and anti-fungal agents. Current treatment guidelines for management of the hémodynamie issues associated with sepsis and septic shock, recommend use of vasopressors, with norepinephrine as first-line therapy.
[0005] Despite these measures discussed above, sepsis remains a major killer and there remains a great need for new treatments for sepsis and its associated medical conditions. One observation îs that most treatments geared towards sepsis tend to address treating infection and individual failing organ Systems, and not the treatment of the key underlying pathophysiological drivers of sepsis. An alternative approach would be to address the underlying mechanisms of sepsis, in addition to treating the concomitant infection.
[0006] Sepsis, along with many other medical conditions, îs associated with oxygen deprivation (hypoxîa). The major causes of death globally are related to some extent to hypoxia. Examples include, but are not limited to, coronary artery disease, stroke, chronic and acute respiratory diseases. In addition, hypoxia is a common feature of many cancers, and leads to résistance to radiation therapy, chemotherapy and potentially immunotherapy. Preclinically, reversai of hypoxia in cancer has been associated with an improved response to treatment. This suggests that strategies in the clînic to reverse hypoxia could resuit in improved outcomes in cancer.
[0007] Carotenoids are a class of natural lipid-soluble pigments found principally in plants where they fonction as accessory pigments and impart protection of tissue through their ability to quench singlet oxygen and free radical species. Carotenoids are known to hâve antioxidant properties and consequently, provide numerous bénéficiai health effects including reducing the potential risks of cardiovascular diseases, cancers, and slowing and/or reversing the degenerative effects of aging on various human physiological activities. However, carotenoids are typically very lipophilie compounds and the clinîcal use of many carotenoids is limited by their instability and low bioavailability.
[0008] Crocetin is a carotenoîd with antîoxidative properties that is sparingly soluble in water. Chemically, crocetin is a 20-carbon apocarotenoid molécule containing seven double bonds and a carboxylic acid group at each end. The administration of trans crocetin (fiee acid), and its sait sodium trans crocetinate in free form (e.g., unencapsulated) phannaceutical formulations has been reported to offer promise in treatment for conditions caused by hypoxia, ischemia, and other medical conditions. However, neither has demonstrated clinical therapeutic efficacy. This is partly due to the fact that formulations of trans crocetin and its sodium sait, sodium trans crocetinate, hâve been to date limited by instability, low bioavailability and short half-lîfe.
[0009] In view of the health benefits conferred by carotenoids and the low bioavailability and instability outlined above, there is a need for providing phannaceutical compositions comprising carotenoids with improved bioavailability and stability. The provided compositions and methods address the shortcomings of carotenoids described above. These compositions and methods will further help overcome the limitations of current therapeutic approaches to disease States linked to endotoxemia and hypoxia as well as other unmet medical needs. The compositions hâve applications as single agents and in combination with other thérapies.
BRIEF SUMMARY
[0010] The dîsclosure provides phannaceutical compositions comprising carotenoids, including liposomes that encapsulate carotenoids such as trans crocetin, trans norbixin, and salts thereof. The provided compositions hâve uses in treating diseases and disorders and conditions associated with, but not limited to, infection, inflammation, sepsis, ischemia, hypoxta, shock, stroke, mjury, cardiovascular disease, rénal dîsease, liver disease, inflammatory disease, metabolic disease, pulmonary disease, neurodegenerative disease, disease of the immune System, and hyperproliferative diseases such as cancer. Methods of making, delîvering, and using the pharmaceuticai compositions are also provided, as are kits containing the compositions.
[0011] The disclosed pharmaceuticai compositions provide for the enhanced delivery of carotenoids including ionizable Polyene Carotenoids such as transcrocetin, with poor pharmacokinetics and biodistribution. The disclosure also provides liposome compositions that display high encapsulation efïiciencies (>98%), high drug-to-lipid ratios, and/or enhanced drug rétention. The provided pharmaceuticai compositions hâve uses in treating diseases and disorders and conditions associated with, but not limited to, infection, inflammation, sepsis, ischemia, hypoxia, anémia, trauma, injury, stroke, shock, diabètes, wound healing, injury (e.g., reperfusion injury, neural injury, rénal injury, livery injury and lung injury), and hyperproliferative diseases such as cancer, as well as conditions associated with the treatment of these diseases and disorders (e.g., anémia, neutropenia and immunosuppression). Methods of making, delîvering, and using the compositions are also provided.
[0012] In some embodiments, the disclosure provides:
[1] a pharmaceuticai composition comprising an ionizable carotenoid sait having the formula: Polyene Carotenoid-Q, wherein, the Polyene Carotenoid comprises (a) 3,4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1, 2, 3, or more than 3, ionizable groups; and Q is a multivalent counterion;
[2] a pharmaceutical composition comprising an ionizable carotenoid sait having the formula:
Q- Ri-Polyene Carotenoid-Rz -Q, wherein, the Polyene Carotenoid comprises (a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1, 2, 3, or more than 3, ionizable groups;
Ri and Rz are ionizable groups e.g,, the same ionizable group or different ionizable groups; and
Q îs a multivalent counterîon;
[3] the pharmaceutical composition of [1] or [2], wherein the Polyene Carotenoid comprises 1, 2, 3, or more than 3, anionic ionizable groups;
[4] the pharmaceutical composition according to [3], wherein the Polyene Carotenoid comprises at least one anionic ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, a phosphate group, and a hydroxamate group;
[5] the pharmaceutical composition of [1] or [2], wherein the Polyene Carotenoid comprises 1, 2, 3, or more than 3, cationic ionizable groups (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, or an imidazole group);
[6] a pharmaceutical composition comprising an ionizable carotenoid sait having the formula: Q-trans-crocetin-Q, wherein, Q is a multivalent cation counterîon;
[7] a pharmaceutical composition comprising an ionizable carotenoid sait having the formula: Q-norbixin-Q, wherein, Q is a multivalent cation counterîon;
[8] the pharmaceutical composition according to any of [l]-[7], wherein the multivalent counterion (Q) is a multivalent cation (e.g., a divalent cation such as a divalent métal cation or a divalent organic cation, or a trivalent cation such as Fe3+);
[9] the pharmaceutical composition of [8], wherein the multivalent cation is at least one divalent cation selected from Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+, a divalent organic cation such as protonated diamine, or a trivalent cation such as Fe3+;
[10] the pharmaceutical composition according to [6], which comprises magnésium trans-crocetinate (MTC) or calcium trans-crocetinate (CTC);
[11] the pharmaceutical composition according to [7], which comprises magnésium trans- norbixinate (MTN) or calcium trans-norbixinate (CTN);
[12] a delivery vehicle comprising the pharmaceutical composition according to any of [1]-[11];
[13] the delivery vehicle according to [12], which is a liposome;
[14] a pharmaceutical composition comprising a liposome encapsulating an ionizabie carotenoîd sait, having the formula:
Polyene Carotenoid-Q, wherein, the Polyene Carotenoid comprises (a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1, 2, 3, or more than 3, ionizabie groups; and
Q is (i) a multivalent counterion or (îî) a monovalent counterion;
[15] a pharmaceutical composition comprising a liposome encapsulating an ionizabie carotenoid sait having the formula:
Q- Ri-Polyene Carotenoid-R2 -Q, wherein
the Polyene Carotenoid comprises (a) 3,4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1, 2, 3, or more than 3, ionizable groups;
Ri and Rs are ionizable groups e.g., the same ionizable group or different ionizable groups; and
Q is (i) a multivalent counterion or (ii) a monovalent counterion;
[16] the pharmaceutical composition according to [14] or [15], wherein the Polyene Carotenoid comprises anionic ionizable groups;
[17] the pharmaceutical composition of [16], wherein the Polyene Carotenoid comprises at least one anionic ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, a phosphate group, and a hydroxamate group;
[18] the pharmaceutical composition a according to [14] or [15], wherein the Polyene Carotenoid comprises cationic ionizable groups (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, or an imidazole group),
[19] a pharmaceutical composition comprising a liposome encapsulatîng an ionizable carotenoid sait having the formula:
Q-trans-crocetin-Q, wherein,
Q is (i) a multivalent cation counterion or (ii) a monovalent cation;
[20] a pharmaceutical composition comprising a liposome encapsulatîng an ionizable carotenoid sait having the formula: Q-trans-norbixin-Q, wherein,
Q is (i) a multivalent cation counterion or (ii) a monovalent cation;
[21] a pharmaceutical composition comprising a liposome encapsulatîng an ionizable carotenoid sait having the formula:
Q- Ra-Polyene Carotenoid-R4-Q, wherein,
[22] the Polyene Carotenoid comprises 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, optionally substituted with 1 to n methyl or low C1-C3 alkyl substitutions, wherein η = 1 to 4; and
R3 and/or R4 îs a monocyclic and/or polar group (e.g., the same or different monocyclic and/or polar group); and
Q is (i) a multivalent counterion or (ii) a monovalent counterion;
a pharmaceutical composition comprising a liposome encapsulating a carotenoid with two attached polar groups, which can be the same or different polar group, and havîng the formula:
QA-Polyene Carotenoid-AQ, wherein, the Polyene Carotenoid comprises 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, optionally substituted with 1 to n methyl or low C1-C3 alkyl substitutions, wherein η = 1 to 4; and QA taken together and AQ taken together is a monocyclic functional group (e.g., the same or different monocyclic functional group), optionally selected from a monocyclic functional group présent in astaxanthin, lutein, xanthophyll and zeaxanthin;
[23] the pharmaceutical composition according to any of [14]-[21] or wherein Q is a multivalent counterion (e.g., a multivalent cation such as a divalent métal cation or a divalent organic cation);
[24] the pharmaceutical composition of [23], wherein Q is at least one divalent cation selected from Ca2+, Mg2+, Zn2+, Cu ’, Co2+, and Fe2+, a divalent organic cation such as protonated diamine, or a trîvalent cation such as Fe3+;
[25] the pharmaceutical composition according to any of [14]-[21], wherein Q is a monovalent counterion (e.g., a monovalent métal cation or a monovalent organic cation);
[26] the pharmaceutical composition of [25], wherein Q is at least one monovalent counterion selected from NH4+, Na+, Li+, and K+, or a monovalent organic cation such as protonated amine;
[27] the pharmaceutical composition according to [19], which comprises magnésium trans-crocetinate (MTC) or calcium trans-crocetinate (CTC);
[28] the pharmaceutical composition according to [20], which comprises magnésium trans-norbixinate (MTN) or calcium trans-norbixinate (CTN);
[29] the pharmaceutical composition according to any of [13]-[28], wherein the ionizable carotenoid/lipid ratio is 1 to 1000 g/M, about 10 to 150 g/mol, about 20 to 100 g/mol, or any range therein between;
[30] the pharmaceutical composition according to any of [13]-[29], wherein the liposomes comprise at least 0.1 % to 97% weight by weight (w/w) ionizable carotenoid, or any range therein between;
[31] the pharmaceutical composition according to any of [13]-[3O], wherein the liposome has a diameter of 20 nm to 500 nm, 20 nm to 200 nm, or 80 nm to 120 nm, or any range therein between;
[32] the pharmaceutical composition according to any of [ 13]-[31 ], wherein the liposome is formed from liposomal components;
[33] the pharmaceutical composition according to [32], wherein the liposomal components comprise at least one of an anionic lipid, a cationic lipid and a neutral lipid;
[34] the pharmaceutical composition according to [32] or [33], wherein the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; HSPC; HSPCPEG; cholestérol; cholesterol-PEG; and cholesterol-maleîmide;
[35] the pharmaceutical composition according to any of [13]-[34], wherein the liposome comprises an oxidized phospholipid such as an OxPAPC;
[36] the pharmaceutical composition according to [35], wherein the OxPAPC is an oxidized phospholipid containing fragmented oxygenated sn-2 resîdues, an oxidized phospholipid containing full length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a five-carbon sn-2 residue bearing omega-aldehyde or omega-carboxyl groups;
[37] the pharmaceutical composition of [35], wherein the liposome comprises an OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOAPC and KOOA-PC, l-palmitoyl-2-(5,6-epoxyisoprostane E2)-snglycero-3-phosphocholine (5,6 PEIPC), l-palmîtoyl-2-(epoxy-cyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), 1 -palmitoyl-2(epoxy-îsoprostane E2)-sn-glycero-4-phosphocholîne (PEIPC), 1palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC); 1palmitoyl-2-(9'oxo-nonanoyl)-sn-glycero-3-phosphocholine; 1palmîtoyl-2-arachinodoyl-sn-glycero-3-phosphocholine; 1 -palmitoyl-2myristoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-hexadecyl-snglycero-3-phosphocholine; 1 -palmitoyl-2-azelaoyl-sn-glycero-3phosphocholine; and l-palmitoyl-2-acetoyl-sn-glycero-3-phospho- choline; or the OxPAPC is an epoxyisoprostane-containing phospholipid;
[38] the pharmaceutical composition according of [37], wherein the liposome comprises PGPC;
[39] the pharmaceutical composition according to any of [13]-[38], wherein the liposome comprises 0% to 100%, 0.1% to 30%, 1% to 25%, 5% to 20%, or 7% to 15% OxPAPC (e.g., about 10% OxPAPC), or any range therein between;
[40] the pharmaceutical composition according to any of [13]-[39], wherein the liposome comprises HSPE, cholestérol, PEG-DSPE-2000, and OxPAPC at a molar ratio of 2 to 5: 1 to 4: 0.01 to 0.3: 0.05 to 1.5;
[41 ] the pharmaceutical composition according to any of [ 13 ] -[40], wherein the liposome is pegylated;
[42] the pharmaceutical composition according to any of [13]-[41], wherein one or more liposomal components further comprises a steric stabilizer;
[43] the pharmaceutical composition according to [42], wherein the steric stabilizer is at least one selected from consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic poIy-Nvinylpyrrolîdones; L-amino-acid-based polymer; oligoglycerol, copolymer containing polyethylene glycol and polypropylene oxide, Poloxamer 188, and polyvinyl alcohol;
[44] the pharmaceutical composition according to [43], wherein the steric stabilizer is PEG and the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons;
[45] the pharmaceutical composition according to any of [ 13]-[44], wherein the liposome is anionic or neutral;
[46] the pharmaceutical composition according to any of [13]-[44], wherein the liposome has a zêta potential of -150 to 150 mV, or -50 to 50 mV, or any range therein between;
[47] the pharmaceutical composition according to any of [13]-[45], wherein the liposome has a zêta potential that is less than or equal to zéro (e.g., -150 to 0, or -50 to 0 mV, or any range therein between);
[48] the pharmaceutical composition according to any of [13]-[47], wherein the liposome has a zêta potential greater than 0 (e.g., 0.2 to 150 mV, or 1 to 50 mV, or any range therein between);
[49] the pharmaceutical composition according to any of [13]-[45], or [48], wherein the liposome is cationic;
[50] the pharmaceutical composition according to any of [l]-[49], which further comprises a pharmaceutically acceptable carrier;
[51] the pharmaceutical composition according to any of [1] to [50], which comprises a tonicity agent such as dextrose, mannitol, glycerin, potassium chloride, or sodium chloride, optionally at a concentration of greater than 0.1%, or a concentration of 0.3% to 2.5%, or any range therein between;
[52] the pharmaceutical composition of [51], which comprises trehalose or dextrose;
[53] the pharmaceutical composition of [52], which contains 1% to 50% trehalose;
[54] the pharmaceutical composition of [51], which contains dextrose, optionally 1% to 50% dextrose;
[55] the pharmaceutical composition according to any of [l]-[54], which contains 5% dextrose in a HEPES buffered solution;
[56] the pharmaceutical composition according to any of [l]-[55], which comprises a buffer such as HEPES Buffered Saline (HBS) or similar, at a concentration of 1 to 200 mM and a pH of 2 to 8, or any ranges therein between;
[57] the pharmaceutical composition according to any of [l]-[56], wherein the pharmaceutîcally acceptable carrier comprises a total concentration of multivalent métal acetate salts such as magnésium acetate or calcium acetate of 0.1 mM to 2000 mM, or 50 mM to 500 mM, or any range therein between;
[58] the pharmaceutical composition according to any of [l]-[57], which has a pH of 5-8, or a pH of 6-7, or any range therein between;
[59] the pharmaceutical composition according to any of [13]-[58], wherein the liposome comprises 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, molécules of ionizable carotenoid;
[60] the pharmaceutical composition according to any of [13]-[59], wherein the liposome comprises 10 to 100,000, 100 to 10,000, or 500 to 5,000, molécules of the ionizable carotenoid, or any range therein between;
[61 ] the pharmaceutical composition according to any of [ 13 ] - [60], wherein the liposome further comprises a targeting moiety and wherein the targeting moiety has a spécifie affinity for a surface antigen on a target cell of interest;
[62] the pharmaceutical composition of [61], wherein the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond;
[63] the pharmaceutical composition of [61] or [62], wherein the targeting moiety is a polypeptide;
[64] the pharmaceutical composition according to any of [61]-[63], wherein the targeting moiety is an antibody or an antigen binding fragment of an antibody;
[65] the pharmaceutical composition according to any of [61]-[64], wherein the targeting moiety binds the surface antigen with an equilibnum dissociation constant (Kd) in a range of 50 x 1012 to 10 x I0'6 as determined using BIACORE® analysis;
[66] the pharmaceutical composition according to any of [61 ]-[65], wherein the targeting moiety specifically binds one or more folate receptors selected from: folate receptor alpha (FR-α). folate receptor beta (FR-β), and folate receptor delta (FR-δ);
[67] the pharmaceutical composition according to any of [61]-[66], wherein the targeting moiety comprises one or more selected from: an antibody, a humanized antibody, an antigen binding fragment of an antibody, a single chain antibody, a single-domain antibody, a bi-specific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody;
[68] the pharmaceutical composition according to any of [62]-[67], wherein each pegylated liposome comprises 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between;
[69] the pharmaceutical composition according to any of [13]-[67], further comprising one or more of an FABP, an immunostimulatory agent, an immunosuppressing agent, a détectable marker and a maleimide, wherein the FABP, the immunostimulatory agent, the immunosuppressing agent, the détectable marker or the maleimide is attached to said PEG or the exterior of the liposome;
[70] the pharmaceutical composition according to [69], which comprises at least one immunostimulatîng agent selected from: a protein immunostimulating agent; a nucleic acid immunostimulating agent; a Chemical immunostimulating agent; a hapten; and an adjuvant;
[71] the pharmaceutical composition of [69] or [60], wherein the immunostimulating agent is at least one selected from: a fluorescein; a fluorescein isothiocyanate (FITC); a DNP; a beta glucan; a beta-1,3 glucan; a beta-l,6-glucan; a resolvin (e.g., a resolvin D such as Dn6DPA or Dn-3DPA, a Resolvin E, or a T sériés resolvin); and a Tolllike receptor (TLR) modulating agent such as, an oxidized low-density lipoprotein (e.g., OXPAC, PGPC), or an eritoran-like lipid (e.g., E5564);
[72] the pharmaceutical composition according to any of [69]-[71], which comprises FABP;
[73] the pharmaceutical composition according to any of [69]-[72], which further comprises a hapten;
[74] the pharmaceutical composition of [73], wherein the hapten comprises one or more of fluorescein or Beta 1,6-glucan;
[75] the pharmaceutical composition according to any of [l]-[74], which further comprises at least one cryoprotectant selected from consisting of mannitol; trehalose; sorbitol; and sucrose;
[76] a targeted composition comprising the pharmaceutical composition according to any of [l]-[75];
[77] a non-targeted liposome composition comprising the pharmaceutical composition according to any of [13]-[60] or [69]-[75];
[78] the pharmaceutical composition according to any of [1 ]-[77], for use in the treatment of disease or a condition in a subject;
[79] use of the pharmaceutical composition according to any of [l]-[78], in the manufacture of a médicament for the treatment of disease in a subject;
[80] a method for treatîng or preventing a disease in a subject needing such treatment or prévention, the method comprising administering the pharmaceutical composition of any of [l]-[79] to the subject;
[81] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is associated with endotoxemia;
[82] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is sepsis;
[83] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the subject is a bum victim;
[84] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is an infection (e.g., a bacterial infection such as an P. aeruginosa infection, an 5. aureus infection (e.g., MRSA) or a condition associated therewith, or an enterococcal infection (e.g., VRE), a fungal infection (e.g., a candidiasîs infection (e.g., invasive candidiasîs) or a condition associated therewith, or a parasitic infection or a condition associated therewith such as malaria (or an associated condition such as cérébral malaria, severe anémia, acidosis, acute kidney faîlure and ARDS), Schistosomiasis, and human Afirican trypanosomiasis, and conditions associated therewith; a viral infection or a condition assicated therewith such as Ebola, Dengue and Marburg (or an associated condition such as influenza, measles, and a viral hémorrhagie fever)
[85] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is bacteremîa;
[86] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is a liver disease or condition (e.g., cirrhosis, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH); alcoholic liver disease, acute liver injury, and cirrhosis of the liver);
[87] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is a lung disease or condition (e.g., acute respiratory distress syndrome (ARDS), pulmonary fibrosis, pulmonary hemorrhage, lung injury, lung cancer, chronic obstructive pulmonary disease (COPD) and other respiratory disorders);
[88] the phannaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is kidney disease (e.g., lipopolysaccharide médication or toxin induced acute kidney injury (AKI) and end stage kidney disease);
[89] the phannaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is an autoimmune dîsorder (e.g., psoriasis, cystic fibrosis, and rheumatoid arthritis);
[90] the phannaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is sclerosis (e.g., systemic sclerosis);
[91] the phannaceutical composition of [78], use of [79], or method of [80], wherein the subject is a critically ill patient;
[92] the phannaceutical composition of [78], use of [79], or method of [80], wherein the subject is at risk of developing sepsis;
[93] the phannaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is a low grade endotoxemic disease;
[94] the phannaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is inflammation (e.g., systemic inflammation, low-grade inflammation, acute inflammation, and chronic inflammatory disease);
[95] the phannaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is inflammatory bowel disease (e.g., Crohn’s disease and ulcerative colitis);
[96] the phannaceutical composition of [78], use of [79], or method of [80], wherein the subject is immunocompromised;
[97] the phannaceutical composition of [78], use of [79], or method of [80], wherein the subjects receives chemotherapy and/or îs immunesuppressed (e.g., febrile neutropénie patients);
[98] the phannaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is a metabolic disease;
[99] the pharmaceuticai composition of [78], use of [79], or method of [80], wherein the disease or condition is insulin résistance;
[100] the pharmaceuticai composition of [78], use of [79], or method of [80], wherein the disease or condition is diabètes or an associated conditions such as gangrené, diabetic necrosis, diabetic neuropathy, diabetic vascular disease (e.g., microvascular disease such as retinopathy and nephropathy, and diabetic ulcers);
[101] the pharmaceuticai composition of [78], use of [79], or method of [80], wherein the disease or condition is type 2 diabètes;
[102] the pharmaceuticai composition of [78], use of [79], or method of [80], wherein the disease or condition is a cardiovascular disease (e.g., coronary artery disease such as myocardîal infarction, sudden cardiac death, cardiorespiratory arrest, hypertension, pulmonary arterial hypertension, atherosclerosîs, occlusive arterial disease, Raynaud’s disease, peripheral vascular disease, other vasculopathies such as Buerger’s disease, Takayasu’s arthritis, and post-cardiac arrest syndrome (PCAS), chronic venons Însufficiency, heart disease, congestive heart failure, chronic skin ulcers);
[103] the pharmaceuticai composition of [78], use of [79], or method of [80], wherein the disease or condition is characterized by ischemia or hypoxîa (e.g., ischemic-reperfusion injury, transient cérébral ischemia, cérébral ischemia-reperfusion, ischémie stroke, hémorrhagie stroke, traumatic brain injury, migraine (e.g., a chronic migraine or severe migraine disorder), gastrointestinal ischemia, kidney disease, pulmonary embolism, acute respiratory failure, néonatal respiratory distress syndrome, obstetric emergencies to reduce périnatal comorbidity (such as, pre/eclampsia and conditions that lead to cérébral palsy), myocardîal infarction, acute limb or mesenteric ischemia, cardiac cirrhosis, chronic peripheral vascular disease, congestive heart faîlure, atherosclerotic stenosis, anémia, thrombosis, embolism, macular degeneration, a neurodegenerative disease (such as Alzheimer’s disease, Parkinson’s disease, or Amyotrophie Latéral Sclerosis (ALS)), sleep apnea, and surgery or traumatic injury);
[104] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is heart attack or stroke (e.g., ischémie and hémorrhagie stroke);
[105] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is shock (e.g., cardiogenic shock, hypovolémie shock, septic shock, neurogenic shock, and anaphylactic shock);
[106] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is associated with nitric oxide deftciency (e.g., sîckle cell disease, paroxysmal noctumal hemoglobînuria (PNH), a hemolytic anémia, a thalassemia, another red blood cell disorder, a purpura such as thrombotic thrombocytic purpura (TTP), hemolytic urémie syndrome (HUS), idiopathic thrombocytopenia (ITP), another platelet disorder, a coagulation abnormality such as disseminated intravascular coagulopathy (DIC), purpura fulmînans, heparin induced thrombocytopenia (HIT), hyperleukocytosis, and hyper viscosity syndrome, or a condition associated therewith;
[107] the pharmaceutical composition of [78], use of [79], or method of [80], wherein the disease or condition is endotoxemîa, such as the endotoxemia associated with conditions like periodontal disease (e.g., periodontitis or inflammation of the gums), chronic alcoholism, chronic smoking, transplantation, néonatal necrotizing enterocolitis, or néonatal ear infection;
[108] a method of reducing systemic levels of LPS, endotoxin and/or another trigger of systemic inflammation in a subject in need thereof, the method comprising administering the phannaceutical composition of any of ΠΗ78] to the subject;
[109] the method according to any of [80]-[108], wherein the phannaceutical composition is administered in combination therapy with another therapeutic agent;
[110] a method ofpreparing a liposomal composition of any of [13]-[77], the method comprising: forming a mixture comprising: liposomal components in solution; homogenizing the mixture to form liposomes in the solution; and processing the mixture to form liposomes containing an ionizable carotenoid;
[111] the method according to [110], wherein the processing step includes one or more steps of: thin film hydration, extrusion, in-Iine mixing, éthanol injection technique, freezing-and-thawing technique, reversephase évaporation, dynamic high pressure microfluidization, microfluidic mixing, double émulsion, freeze-dried double émulsion, 3D printîng, membrane contacter method, and stirring;
[112] the method according to [110] or [111], wherein said processing step includes one or more steps of modifying the size of the liposomes by one or more of steps of extrusion, high-pressure microfluidization, and/or sonication;
[113] a method of preparing a phannaceutical composition comprising:
(a) preparing a liposomal solution containing liposomes in a weak acid sait of a multivalent métal;
(b) adding an ionizable carotenoid according to any of [l]-[7], [10], or [11], to the liposomal solution; and (c) maintaining the ionizable carotenoid in the liposomal solution for sufficient time to load the carotenoid into liposomes;
[114] the method of [113], wherein the weak acid is an organic acid (e.g., an organic acid selected from acetic acid, gluconic acid, tartane acid, glutamic acid, citric acid, formic acid, and glycînic acid);
[115] the method of [113] or [114], wherein the multivalent métal is selected is a divalent métal (e.g., a divalent métal selected from Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+), or a trivalent métal such as Fe3+;
[116] the method according to any of [113]-[115], wherein the weak acid is acetic acid and the multivalent métal is Ca2+ or Mg2+ (i.e., the weak acid sait of the multivalent métal is calcium acetate or magnésium acetate, respectively);
[117] a pharmaceutical composition prepared according to the method of any of [109]-[l 15];
[118] a method of preparing pharmaceutical composition comprising a liposome encapsulating trans-crocetin, the method comprising:
(a) preparing a liposomal solution comprising liposomes and a solution containing a weak acid sait of a multivalent métal;
(b) adding trans-crocetin to the liposomal solution; and (c) maintaining the trans-crocetin in the liposomal solution for sufficient time to load trans-crocetin into liposomes;
[119] the method of [118], wherein the weak acid is an organic acid (e.g., acetic acid, gluconic acid, tartaric acid, glutamic acid, citric acid, formic acid, and glycînic acid);
[120] the method of [118] or [119], wherein the multivalent métal is a divalent métal (e.g., a divalent métal selected from Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+), or a trivalent métal such as Fe3+; and/or
[121] the method according to any of daims [118]-[120], wherein the weak acid is acetic acid and the multivalent métal is Ca2+ or Mg2+; and/or
[122] a pharmaceutical composition prepared according to the method according to any of[117]-[120].
[00131
Still other features Still 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 THE DRAWINGS/FIGURES
[0014] FIG. IA, FIG. IB, FIG. IC, and FIG. ID, depict the exemplary ionizable Polyene Carotenoids of the provided pharmaceutical compositions.
[0015] FIG. 2. Calcium trans-crocetînate liposome (CTC-LP) stability at 4°C over 6 months. The CTC-LP test articles contain drug/lipid (D/L) ratios of 80, 60, and 40. Each CTC-LP test article showed negligible leaching (change in D/L ratio) over the 6-month évaluation perîod.
[0016] FIG. 3. Liposomal CTC batch reproducibility. Fourbatches of liposomal CTC were reproducible and stable at 4°C, up to at least 7 months.
[0017] FIG. 4. Magnésium trans-crocetinate liposome (MTC-LP) stability at 4°C over 6 months. The MTC-LP test articles contain drug/lipid (D/L) ratios of 80, 60, and 40. Each MTC-LP test article showed negligible leaching (change in D/L ratio) over the 2 month évaluation period.
[0018] FIG. 5. Survival Study 1 (TP-936): study of CTC-LP effîcacy in mouse CLP sepsis model. Survival curve of mice treated with test articles (a) liposomal CTC (D/L80) + antibiotic, (b) liposomal CTC (D/L80) and PGPC + antibiotic, (c) saline + antibiotic, and (d) sham. Test articles (a) and (b) (in combination with imipenem) demonstrated a trend toward réduction in mortality when compared to the imipenem-treated control (c).
[0019] FIG. 6. Survival Study 2 (TP-967): mouse CLP sepsis study. Survival curve of mice treated with test articles (a) liposomal PGPC + antibiotic, (b) liposomal (PGPC and CTC) (D/L80) + antibiotic, (c) liposomal CTC (D/L80) + antibiotic, and (d) saline + antibiotic. Test article (c) demonstrated a trend toward réduction în mortality when compared to the imipenem-treated control (d).
[00201 FIG. 7. Survival Study 3 (TP-986): mouse CLP sepsis study. Survival curve of mîce treated with test articles (a) liposomal CTC (D/L80)(l mg/kg) + antibiotic, (b) liposomal CTC (D/L80)(5 mg/kg) + antibiotic, (c) liposomal CTC (D/L80)(25 mg/kg) + antibiotic, (d) liposomal CTC (D/L80)(50 mg/kg) + antibiotic, and (e) saline + antibiotic. Each of test articles (a), (c), and (d) demonstrated a trend toward réduction in mortality when compared to the imipenem-treated control (d). Test article (b) (liposomal CTC (D/L80)(5 mg/kg) + antibiotic) demonstrated a statistically significant decrease in mortality when compared to the imipenem-treated control (d)(P=0.0321).
DETAILED DESCRIPTION
[0021] The Applicants hâve surprisingly discovered that pharmaceutical compositions such as liposomes comprising multivalent ionizable carotenoid salts containing multivalent counterions substantially improves the pharmacokinetics (e.g., half-life, stabilîty, and bioavailability) and dramatically increases drug exposure via a sustained release of the ionizable carotenoid when compared to for example, carotenoid free acids and ionizable carotenoid salts containing monovalent counterions.
Définitions
[0022] Unless otherwîse defined, ail technical and scîentific terms used herein hâve the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or équivalent to those described herein can be used in the practice or testing of the provided compositions, suitable methods and materials are described below. Each publication, patent application, patent, and other reference mentioned herein is herein incorporated by reference in its entirety. In case of conflict, the présent spécification, including définitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
[0023] Other features and advantages of the disclosed compositions and methods will be apparent from the following disclosure, drawîngs, and daims.
[0024] It is understood that wherever embodîments, are described herein with the language “comprising” otherwise analogous embodîments, described in terms of “containing” “consisting of’ and/or “consisting essentially of’ are also provided. However, when used in the daims as transitional phrases, each should be interpreted separately and in the appropriate legal and factual context (e.g., in daims, the transitional phrase “comprising” is considered more of an openended phrase while “consisting of ’ is more exclusive and “consisting essentially of ’ achieves a middle ground).
[0025] As used herein, the singular form “a”, “an”, and “the”, include plural forms unless it is expressly stated or is unambiguously clear from the context that such is not intended. The singular form “a”, “an”, and “the” also includes the statistical mean composition, characteristics, or size of the particles in a population of particles (e.g., mean liposome diameter, mean liposome zêta potential, mean number of targeting moieties on liposomes in a liposomal solution, mean number of encapsulated carotenoids). The mean particle size and zêta potential of liposomes in a pharmaceutical composition can routinely be measured usîng methods known in the art, such as dynamic light scattering. The mean amount of a therapeutic agent in a nanoparticle composition may routinely be measured for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).
[0026] As used herein, the terms approximately and about, as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain embodîments, the term approximately or about refers to a range of values that fall within 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 (greater than or less than) of the stated reference value unless otherwise stated or otherwise évident from the context (except where such number would exceed 100% of a possible value). For example, when used in the context of an amount of a given compound in a lipid component of a nanoparticle composition, about may mean +/-10% of the recited value. For instance, a nanoparticle composition including a lipid component having about 40% of a given compound may include 30-50% of the compound.
[0027] The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass 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 (alone); B (alone); and C (alone).
[0028] Where embodiments, of the disclosure are described in terms of a Markush group or other grouping of alternatives, the disclosed composition or method encompasses not only the entire group listed as a whole, but also each member of the group individually and ail possible subgroups of the main group, and also the main group absent one or more of the group members. The disclosed compositions and methods also envisage the explicit exclusion of one or more of any of the group members in the disclosed compositions or methods.
[0029] The term liposome refers to a closed vesicle having an internai phase (i.e., interior space (internai solution)) enclosed by lipid bilayer. A liposome can be a small single-membrane liposome such as a small unilamellar vesicle (SUV), large single-membrane liposome such as a large unilamellar vesicle (LUV), a still larger single-membrane liposome such as a giant unilamellar vesicle (GUV), a multilayer liposome having multiple concentric membranes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), such as a multîlamellar vesicle (MLV), or a liposome having multiple membranes that are irregular and not concentric such as a multivesicular vesicle (MW). Liposomes and liposome formulations are well known in the art. Lipids which are capable of forming liposomes include ail substances having fatty or fat-like properties. Lipids which can make up the lipids in the liposomes include without limitation, glycendes, glycerophospholipids, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipîds, phospholipids, isoprenolides, steroids, stéarines, sterols, archeolîpids, synthetic cationic lipids and carbohydrate containing lipids.
[0030] A liposome composition is a prepared composition comprising a liposome and the contents within the liposome, particularly includîng the lipids which form the liposome bîlayer(s), compounds other than the lipids within the bi-layer(s) of the liposome, compounds within and associated with the aqueous interior(s) of the liposome, and compounds bound to or associated with the outer layer of the liposome. Thus, in addition to the lipids of the liposome, a liposome composition described herein suitably may include, but is not lîmited to, therapeutic agents, immunostimulating agents, vaccine antigens and adjuvants, excipients, carriers and buffering agents. In a preferred embodiment, such compounds are complementary to and/or are not significantly detrimental to the stability or AGP-incorporation efficiency of the liposome composition.
[0031] The terms liposome “internai phase, “înterior space”, and “internai core” are used interchangeably to refer to an aqueous région enclosed within (Le., encapsulated by) the lipid bilayer of the liposome. The solution of the liposomal internai phase is referred to as the “internai solution.” By contrast, the terni liposome extemal phase refers to the région not enclosed by the lipid bilayer of the liposome, such as the région apart from the internai phase and the lipid bilayer in the case where the liposome is dispersed în liquid.
[0032] The terni counterion refers to an anionic or cationic counterion.
A cationic counterion is a positively charged atom or group associated with an anionic atom or group in order to maîntaîn electronic neutrality. Exemplary cationic counterions include inorganic cations (e.g., métal cations (e.g., alkali métal cations, alkali earth métal cations, and transition métal cations)) and organic cations (e.g., ammonium cations, sulfonium cations, phosphonium cations, and pyridînium cations). An anionic counterion is a negatively charged atom or group associated with a cationic atom or group in order to maintain electronîc neutrality. Exemplary anionic counterions înclude halide anions (e.g., F, Cl', Br, and Γ), NO;, CIOF OH’, H2PO42, HSOf, sulfonate anîons (e.g., methansulfonate, trifluoromethanesuifonate, p-toluenesuifonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene1-sulfonic acid-5-sulfonate, ethan-l-sulfonic acid-2-sulfonate, and the like), and carboxylate anions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, and glycolate). A counterion may be monovalent or multivalent (e.g., divalent, trivalent, tetravalent, eic.).
[0033] The terni ionizable refers to a compound containîng at least one functional group that (a) bears a positive or négative charge (Le., is ionized) and is therefore associated with a counterion of opposite charge, or (b) is electronically neutral but ionized at a higher or lower pH. Thus, ionizable compounds înclude quatemary ammonium salts as well as uncharged amines, and carboxylate moieties as well as uncharged carboxyl groups.
[0034] The terni carotenoid, as used herein, refers to organic pigments which are structurally composed of a polyene hydrocarbon chain, and which may terminate in a ring. Carotenoids are divided into two classes, xanthophylls (which contain oxygen atoms) and carotènes (which contain no oxygen atoms). Non-limiting examples of carotenoids suitable for use in the provided compositions and methods are provided in FIGS. IA - FIG. 1D. Carotenoids with ionizable functional groups comprise naturally occurring carotenoid sulphates, carotenoid carboxylic acids / carboxylates, synthetic phosphates, blue carotenoid oxonium ions and blue carotenoproteîns.
[0035] The term Polyene Carotenoid as used herein, refers to a carotenoid containîng 3 or more conjugated double bonds, and methyl or low alkyl (C2-C3) substitutions.
[0036] The term naturally occurring refers to a compound or composition that occurs in nature, regardless of whether the compound or composition has been isolated from a natural source or chemically synthesized. Examples of naturally occurring carotenoid mono- and di-carboxylic acids include crocetin, norbixin, azafrin and neurosporaxanthin.
[0037] An apocarotenoid is a carotenoid dégradation product in whîch the normal structure (e.} C40) has been shortened by the removal of fragments from one or both ends. Examples of naturally occurring apocarotenoids include crocetin (C20), bixin (C25), Vitamin A, abscisic acid, mycorradicin and blumenin.
[0038] The term “targeting moiety” is used herein to refer to a molécule that provides an enhanced affinîty for a selected target, e.g., a cell, cell type, tissue, organ, région of the body, or a compartment, e.g., a cellular, tissue or organ compartment. The targeting moiety can comprise a wide variety of entities. Targeting moieties can include naturally occurring molécules, or recombinant or synthetic molécules. In some embodiments, the targeting moiety is an antibody, antigen-binding antibody fragment, bispecific antibody or other antibody-based molécule or compound. In some embodiments, the targeting moiety is an aptamer, avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidin binding pair, a peptide, protein, carbohydrate, lipid, vitamin, toxin, a component of a microorganism, a hormone, a receptor ligand or any dérivative thereof. Other targeting moieties are known in the art and are encompassed by the disclosure.
[0039] The terms “spécifie affinity” or “specifîcally binds” mean that a targeting moiety such as an antibody or antigen binding antibody fragment, reacts or associâtes more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molécule than with alternative substances, including proteins unrelated to the target epitope. Because of the sequenee identity between homologous proteins in different species, spécifie affmity can, in several embodiments, include a binding agent that recognizes a proteîn or target in more than one species. Likewise, because of homology within certain régions of polypeptide sequences of different proteins, the term “spécifie affmity” or “specifically binds” can include a binding agent that recognizes more than one proteîn or target. It is understood that, in certain embodiments, a targeting moiety that specifically binds a first target may or may not specifically bind a second target. As such, “spécifie affmity” does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target. Thus, a targeting moiety may, in certain embodiments, specifically bind more than one target. In certain embodiments, multiple targets may be bound by the same targeting moiety.
[0040] The term epitope refers to that portion of an antigen capable of being recognized and specifically bound by a targeting moiety (i.e., binding moiety) such as an antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a proteîn. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon proteîn denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
[0041] Expressions like “binding afïïnity for a target”, “binding to a target” and analogous expressions known in the art refer to a property of a targeting moiety which may be directiy measured through the détermination of the affmity constants, e.g., the amount of targeting moiety that associâtes and dissociâtes at a given antigen concentration. Different methods can be used to characterize the molecular interaction, such as, but not limited to, compétition analysis, equilibrium analysis and microcalorimetric analysis, and real-time interaction analysis based on surface plasmon résonance interaction (for example using a BIACORE® instrument). These methods are well-known to the skilled person 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).
[0042 ] As used herein an “effective amount” refers to a dosage of an agent sufficient to provide a medically désirable resuit. The effective amount will vary with the desired outcome, the particular condition being treated or prevented, the âge and physîcal condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent or combination therapy (if any), the spécifie route of administration and like factors within the knowledge and expertise of the health practitioner. An effective amount can be determined empirically and in a routine manner, in relation to the stated purpose. In the case of cancer, the effective amount of an agent may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relîeve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, duration of progression free survival (PFS), the response rates (RR), duration of response, and/or quality of life.
[0043] The terms “hyperproliferative disorder”, proliférative disease, and “proliférative disorder”, are used interchangeably herein to pertain to an unwanted or uncontrolled cellular prolifération of excessive or abnormal cells which îs undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo. In some embodiments, the proliférative disease is cancer or tumor disease (including benign or cancerous) and/or any métastasés, wherever the cancer, tumor and/or the metastasis is located. In some embodiments, the proliférative disease is a benign or malignant tumor. In some embodiments, the proliférative disease is a non-cancerous disease. In some embodiments, the proliférative disease is a hyperproliferative condition such as hyperplasias, fibrosis (especially pulmonary, but also other types of fibrosis, such as rénal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle prolifération in the blood vessels, such as stenosis or restenosis following angioplasty.
[0044] “Cancer,” “tumor,” or “malignancy” are used as synonymous tenus and refer to any of a number of diseases that are characterized by uncontrolled, abnormal prolifération of cells, the abilîty of affected cells to spread locally or through the bloodstream and lymphatic System to other parts of the body (metastasize) as well as any of a number of characteristic structural and/or molecular features. “Tumor,” as used herein refers to ail neoplastic cell growth and prolifération, whether malignant or benign, and ail pre-cancerous and cancerous cells and tissues. A “cancerous tumor,” or “malignant cell” is understood as a cell having spécifie structural properties, lacking différentiation and being capable of invasion and metastasis. A cancer that can be treated using a carotenoid pharmaceutical composition provided herein includes without limitation, a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous System cancer, and melanoma; and a hématologie malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias. Other types of cancer and tumors that may be treated using a trans-crocetin composition are described herein or otherwise known in the art. The tenus “cancer,” “cancerous,” “cell proliférative disorder,” “proliférative disorder,” and “tumor” are not mutually exclusive as referred to herein.
[0045]
Ternis such as treating, or treatment, or to treat refer to both (a) therapeutic measures that cure, slow down, attenuate, lessen symptoms of, and/or hait progression of a diagnosed pathologie condition or disorder and (b) prophylactic or preventative measures that prevent and/or slow the development of a targeted disease or condition. Thus, subjects in need of treatment include those already with the cancer, disorder or disease; those at risk of having the cancer or condition; and those in whom the infection or condition is to be prevented. Subjects are identifîed as “having or at risk of having” sepsis, an infectious disease, a disorder of the immune System, a metabolic disorder (e.g., diabètes), a hyperproliferative disease, or another disease or disorder referred to herein using well-known medical and diagnostic techniques. In certain embodiments, a subject is successfully treated according to the methods provided herein if the subject shows, e.g., total, partial, or transient amelioration or élimination of a symptom assocîated with the disease or condition (e.g., cancer and arthritis such as rheumatoid arthritis). In spécifie embodiments, the ternis “treating, or treatment, or to treat refer to the amelioration of at least one measurable physical parameter of a proliférative disorder, such as growth of a tumor, not necessarily discemible by the patient. In other embodiments, the terrns “treating, or treatment, or to treat refer to the inhibition of the progression of a proliférative disorder, either physically by, e.g., stabilization of a discemible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments, the terrns “treating, or treatment, or to treat refer to the réduction or stabilization of tumor size, tumor cell prolifération or survival, or cancerous cell count. Treatment can be with a provided phannaceutical composition disclosed herein (e.g., a liposomal transcrocetinate) alone, or in combination with an additional therapeutic agent.
[0046] “Subject” and “patient,” and “animal” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animais such as rabbits, rats, and mice, and other animais. Animais include ail vertebrates, e.g., mammals and non-mammals, such as chickens, amphibians, and reptiles. “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animais such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animais including rodents such as mice, rats and guinea pigs, and other members of the class Mammalia known in the art. In a particular embodiment, the patient is a human.
[0047] The term “pharmaceutically acceptable carrier” refers to an ingrédient in a pharmaceutical formulation, other than an active ingrédient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, carrier, excipient, stabilizer, diluent, or preservative. Pharmaceutically acceptable carriers can include for example, one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other subject.
[0048] “Therapeutic agent”: In some embodiments, the provided liposome compositions and liposomal formulations, comprise liposomes encapsulating or otherwise associated with one or more therapeutic agents présent anywhere in, on, or around the liposome. For example, a therapeutic agent can be embedded in the lipid bilayer of the liposome, encapsulated in the internai phase of the liposome, or tethered to the exterior of the liposome. The therapeutic agent or therapeutic agents used according to the disclosed compositions and methods can include any agent directed to treat a condition in a subject. Examples of therapeutic agents that may be suitable for use in accordance with the disclosed methods include vitamin C, thiamine, hydrocortisone or another corticosteroid (e.g., a glucocortîcoid such as, cortisone, ethamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone and méthylprednisolone; and mineralocorticoids such as fludrocortisonel), astaxanthin, abscisic acid, vitamin
A, angiotensîn II (e.g., GIAPREZA™), tissue plasminogen activator (tPA), an antimîcrobial (e.g., antibiotic) and an anti-inflammatory.
[0049] Additional examples of therapeutic agents that may be suitable for use in accordance with the disclosed methods inciude, without limitation, antirestenosis, pro- or anti-proliferative, anti-neoplastic, antimitotic, antî-platelet, anticoagulant, antifibrin, antithrombin, cytostatic, antibiotic and other antiinfective agents, anti-enzymatic, anti-metabolic, angiogenic, cytoprotective, angiotensîn converting enzyme (ACE) inhîbiting, angiotensîn II receptor antagonizing 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 Pharmacopeia (U. S.P.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Ed., McGraw Hill, 2001; Katzung, Ed., Basic and Clînical Pharmacology, McGraw-Hill/Appleton & Lange, 8* 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 animais, The Merck Veterinary Manual, 9th ed., Kahn Ed., Merck Publishing Group, 2005; ail of which are incorporated herein by reference used herein to refer to an agent or a dérivative thereof that can interact with a hyperproliferative cell such as a cancer cell or an immune cell, thereby reducing the proliférative status of the cell and/or killîng the cell. Examples of therapeutic agents inciude, but are not lîmîted to, chemotherapeutîc agents, cytotoxic agents, platinum-based agents (e.g., cisplatin, carboplatîn, oxaliplatin), taxanes (e.g., Taxol), etoposîde, alkylatîng agents (e.g., cyclophosphamide, ifosamide), metabolic antagonists (e.g, methotrexate (MTX), 5-fluorouracil, gemcitabine, pemetrexed, or dérivatives thereof), antîtumor antibiotics (e.g., mitomycin, doxorubicin), plant-derived antitumor agents (e.g., vincristine, vindesine, Taxol). Such agents may further inciude, but are not limîted to, the anticancer agents trimetrexate, TEMOZOLOMIDE™, RALTRITREXED™, S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), 6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU) and
CAMPTOTHECIN™, or a therapeutic dérivative of any thereof. “Therapeutic agents” also refer to salts, acids, and free based forms ofthe above agents.
[0050] The tenu “pharmaceutically acceptable carrier” refers to an ingrédient in a pharmaceutical formulation, other than an active ingrédient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, carrier, excipient, stabiliser, diluent, or preservative. Pharmaceutically acceptable carriers can include for example, one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other subject.
[0051] The term kit refers to a set of two or more components necessary for employîng the methods and compositions provided herein. Kit components can include, but are not limited to, liposome compositions and liposomal formulations disclosed herein, reagents, buffers, containers and/or equipment. The phrase stored separately refers to a manner of liposome storage that prevents a first population of liposomes from contactîng another population of liposomes.
[0052] The term radiosensitizing agent means a compound that makes tumor cells more sensitive to radiation therapy. Examples of radiosensîtizing agents include misonidazole, metronidazole, tirapazamine, and trans-crocetin.
Pharmaceutical Compositions
[0053] The provided pharmaceutical compositions can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readîly prepared intermediates, by employing standard synthetîc methods and procedures either known to those skilled in the art, or which will be apparent to the skilled artisan in light of the teachings herein. Standard synthetîc methods and procedures for the préparation of organic molécules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic texts such as Smith et al., March's Advanced Organic Chemistry. Reactions, Mechanisms, and Structure, 5th édition, John Wiley & Sons: New York, 2001; Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 3rd édition, 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 by reference herein, are useful and recognized reference textbooks of organic synthesis known to those in the art. The following descriptions of synthetic methods are designed to illustrate, but not to limit, general procedures for the préparation of compounds of the présent dîsclosure.
[0054] In some embodiments, the dîsclosure provides a new class of multivalent ionizable carotenoid (e.g., trans-carotenoid) salts.
[0055] In some embodiments, the dîsclosure provides a pharmaceutical composition comprising an ionizable carotenoid having the formula: Polyene Carotenoid-Q wherein, the Polyene Carotenoid comprises (a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1,2, 3, or more than 3, ionizable groups; and
Q is a multivalent counterion.
In some embodiments, the Polyene Carotenoid comprises ail trans conjugated double bonds. In some embodiments, the Polyene Carotenoid comprises 6-9 conjugated double bonds. In particular embodiments, the Polyene Carotenoid comprises 7 conjugated double bonds. The Polyene Carotenoid can be naturally occurring or synthetic. In some embodiments, the Polyene Carotenoid is naturally occurring. In other embodîments, the Polyene Carotenoid is synthetîc.
The ionizable group(s) may be anionic and/or catîonic. In some embodîments, the Polyene Carotenoid-Q comprises two or more of the same ionizable group. In some embodîments, the Polyene Carotenoid comprises two or more different ionizable groups. In some embodîments, the Polyene Carotenoid comprises one or more anionic ionizable groups. In some embodîments, the Polyene Carotenoid comprises at least one ionizable group selected from: a carboxylîc group, a sulfonate group, a sulfate group, a phosphonate, or a phosphate group, and a hydroxamate moîety. In other embodîments, the Polyene Carotenoid comprises one or more catîonic ionizable groups (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, or an imidazole group). In partîcular embodîments, the Polyene Carotenoid comprises one or more catîonic ionizable groups and the pharmaceutical composition is substantially free of nucleic acids.
In some embodîments, Q is a multivalent cation counterion. In some embodîments, Q is a multivalent métal cation. In further embodîments, Q is a multivalent transition métal cation. In some embodîments, Q is a divalent counterion. In some embodîments, Q is a divalent cation counterion. In some embodîments, Q is a divalent métal cation. In further embodîments, Q is a divalent transition métal cation. In some embodîments, Q is at least one member selected from Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+. In some embodîments, Q is Ca2+ or Mg2+. In some embodîments, Q is Ca2+. In further embodîments, the Polyene Carotenoid-Q is calcium trans-crocetinate (CTC). In some embodîments, Q is Mg:‘. In further embodîments, the Polyene Carotenoid-Q is magnésium trans-crocetinate (MTC). In other embodîments, Q is a trivalent cation counterion such as Fe3+. In other embodîments, Q is a multivalent organic counterion. In some embodîments, Q is a divalent organic cation. In some embodîments, Q is a bivalent organic cation such as protonated diamine. Liposomes comprising the Polyene Carotenoid-Q compositions and pharmaceutical compositions (e.g., liposome compositions) comprising the liposomes are also provided herem.
[0056] In some embodiments, the disclosure provides a pharmaceutical composition comprising an ionizable carotenoid having the formula: Q- Rl-Polyene Carotenoid-R2 -Q, wherein, the Rl-Polyene Carotenoid-R2 comprises (a) 3,4,5,6,7,8,9,10,3-5,6-8,9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1, 2, 3, or more than 3, ionizable groups;
Ri and R2 are ionizable groups; and
Q is a multivalent counterion.
In some embodiments, the Q- Rl-Polyene Carotenoid-R2 comprises ail trans conjugated double bonds. In particular embodiments, the Q- Rl-Polyene Carotenoid-R2 comprises 6-9 conjugated double bonds. The Q- Rl-Polyene Carotenoid-R2 can be naturally occurring or synthetic. In some embodiments, the Q- Rl-Polyene Carotenoid-R2 is naturally occurring. In other embodiments, the Q- Rl-Polyene Carotenoid-R2 is synthetic. In some embodiments, Ri and R2 are the same ionizable group. In other embodiments, Ri and R2 are different ionizable groups. In some embodiments, Ri and R2 are the same cationic ionizable group (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, and an imidazole group). In other embodiments, Ri and R2 are different cationic groups. In some embodiments, Ri and R2 are the same anionîc ionizable group (e.g., a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, a phosphate group, and a hydroxamate group). In other embodiments, Ri and R2 are different anionic groups. Tn some embodiments, Ri is a cationic ionizable group or anionic ionizable group and R2 is an anionic ionizable group or cationic group, respectively. In some embodiments, the Polyene Carotenoid comprises at least one ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, or a phosphate group, and a hydroxamate moiety. In some embodiments, Ri and/or Rz is at least one ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, or a phosphate group, and a hydroxamate moiety. In some embodiments, Rz is at least one ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, or a phosphate group, and a hydroxamate moiety. In other embodiments, Ri and/or Rz a cationic ionizable group (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, or an imidazole group). In particular embodiments, Ri is a cationic ionizable group and the pharmaceutical composition is substantially free of nucleîc acids.
In some embodiments, Q is a multivalent cation counterîon. In some embodiments, Q is a multivalent métal cation. In further embodiments, Q is a multivalent transition métal counterîon. In some embodiments, Q is a divalent counterîon. In some embodiments, Q is a divalent cation counterîon. In further embodiments, Q is a divalent métal cation. In some embodiments, Q is at least one member selected from Ca2+, Mg2\ Zn2+, Cu2+, Co2‘, and Fe2+. In further embodiments, Q îs Ca2+ or Mg2+. In further embodiments, Q is Ca2+. In some embodiments, Q is Mg2+. In other embodiments, Q is a trîvalent cation counterîon such as Fe3+. In other embodiments, Q is a multivalent organic cation. In further embodiments, Q is a divalent organic cation such as a protonated diamine. Liposomes comprising the Ri-Polyene Carotenoid-Rz compositions and pharmaceutical compositions (e.g., liposome compositions) comprising the liposomes are also provided herein.
[0057] In some embodiments, the disclosure provides a pharmaceutical composition comprising an ionizable bis-alpha, omega-carotenoid having the formula:
Q- Ri-Polyene Carotenoîd-Ri -Q, wherein, the Ri-Polyene Carotenoid-Ri -Q comprises (a) 3,4, 5, 6,7, 8, 9,10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1, 2, 3, or more than 3, ionizable groups; and
Ri is an ionizable group; and
Q is a multivalent counterion.
In some embodiments, the bis-alpha, omega-carotenoid comprises ail trans conjugated double bonds. In some embodiments, the bis-alpha, omegacarotenoid comprises 6-9 conjugated double bonds. In particular embodiments, the bis-alpha, omega-carotenoid comprises 7 conjugated double bonds. The bisalpha, omega-carotenoid can be naturally occurring or synthetic. In some embodiments, the bis-alpha, omega-carotenoid is naturally occurring. In other embodiments, the bis-alpha, omega-carotenoid is synthetic. In some embodiments, Ri is an anionic ionizable group. In some embodiments, the bisalpha, omega-carotenoid comprises an ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, a phosphate group, and a hydroxamate moiety. In other embodiments, Ri is a cationic ionizable group (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, or an imidazole group). In particular embodiments, Ri is a cationic ionizable group and the pharmaceutical composition is substantially free of nucleic acids.
In some embodiments, Q is a multivalent cation counterion. In further embodiments, Q is a multivalent métal cation. In some embodiments, Q is a multivalent transition métal counterion. In some embodiments, Q is a divalent counterion. In some embodiments, Q is a divalent cation counterion. In further embodiments, Q is a divalent métal cation. In some embodiments, Q is at least one member selected from Ca2+, Mg< Zn2+, Cu2+, Co2+, and Fe2+. In further embodiments, Q is Ca2+ or Mg2+. In some embodiments, Q is Ca2+. In some embodiments, Q is Mg2+. In other embodiments, Q is a trivalent cation counterion such as Fe . In some embodiments, Q is a multivalent organic cation. In further embodiments, Q is a divalent organic cation such as a protonated diamine or a protonated polyamine. Liposomes comprising the RiPolyene Carotenoid-Ri compositions and pharmaceutical compositions (e.g., liposome compositions) comprising the liposomes are also provided herein.
[0058] In some embodiments, the disclosure provides a pharmaceutical composition comprising an îonizable bis-alpha,omega-carotenoid having the formula: Ri-Polyene Carotenoid-Ri, wherein, the bis-alpha, omega-carotenoid comprises:
(a) 3,4, 5, 6,7, 8, 9,10, 3-5,6-8,9-10, or more than 9, conjugated double bonds, and (b) 1, 2, 3, or more than 3, îonizable groups; and the bis-alpha, omega-carotenoid is optionally substituted with 1 to n methyl or low C1-C3 alkyl substitutions, wherein η = 1 to 4; and
Ri is a polar group and/or a monocyclic functional group.
In some embodiments, the bis-alpha, omega-carotenoid comprises ail trans conjugated double bonds. In some embodiments, the bis-alpha, omegacarotenoid comprises 6-9 conjugated double bonds. In particular embodiments, the bis-alpha, omega-carotenoid comprises 7 conjugated double bonds. The bisalpha, omega-carotenoid can be naturally occurring or synthetic. In some embodiments, the bis-alpha, omega-carotenoid is naturally occurring. In other embodiments, the bis-alpha, omega-carotenoid is synthetic. In some embodiments. Ri is a polar group. In some embodiments, Ri is a monocyclic functional group. In some embodiments. Ri is a polar group and a monocyclic functional group. In some embodiments, the bis-alpha, omega-carotenoid comprises a monocyclic and/or polar functional group selected from a functional group présent in astaxanthin, lutein, xanthophyll and zeaxanthin. In some embodiments, the bis-alpha, omega-carotenoid is selected from astaxanthin, lutein, xanthophyll and zeaxanthin (e.g·, as depicted below).
Liposomes comprising the bis-alpha, omega-carotenoid compositions and pharmaceutical compositions (e.g., liposome compositions) comprising the liposomes are also provided herein.
[0059] fri some embodiments, the pharmaceutical composition comprises a trans-crocetin having the formula: Q-trans-crocetin-Q o
wherein,
Q is a multivalent cation counterion.
In some embodiments, Q is a multivalent métal cation. In further embodiments, Q is a multivalent transition métal cation. In some embodiments, Q is a divalent cation counterion. In further embodiments, Q is a divalent métal cation. In some embodiments, Q is at least one member selected from Ca2+, Mg2+, Zn2+, Cu2+, 15 Co2+, and Fe2+. In further embodiments, Q is Ca2+ or Mg2+. In some embodiments, Q is Ca2+. In some embodiments, Q is Mg2+. In other embodiments, Q is a trivalent cation counterion such as Fe3+. In some embodiments, Q is a multivalent organic cation. In further embodiments, Q is a divalent organic cation such as a protonated diamine. Liposomes comprising the trans-crocetin compositions and pharmaceutical compositions (e.g., liposome compositions) comprising the liposomes are also provided herein.
[0060] In some embodiments, the dîsclosure provides a pharmaceutical composition comprising calcium trans-crocetin (CTC). The CTC can exist in linear and/or cyclîc form (shown below)
Liposomes comprising the CTC compositions and pharmaceutical compositions (e.g., liposome compositions) comprising the liposomes are also provided herein.
[0061] In some embodiments, the dîsclosure provides a pharmaceutical composition comprising magnésium trans-crocetin (MTC). The MTC can exist in linear and/or cyclîc form (shown below).
Liposomes comprising the MTC compositions and phannaceutical compositions (e.g., liposome compositions) comprising the liposomes are also provided herein.
[0062] In some embodiments, the dîsclosure provides a pharmaceutical composition comprising trans-norbixin having the formula: Q-norbixin-Q o
wherein,
Q is a multivalent cation countenon.
In some embodiments, Q is a multivalent cation counterion. In some embodiments, Q is a multivalent métal cation. In further embodiments, Q is a multivalent transition métal cation. In some embodiments, Q is a divalent cation counterion. In further embodiments, Q is a divalent métal cation. In some embodiments, Q is at least one member selected from Ca2+, Mg2+, Zn2+, Cu2+,
Co2+, and Fe2+. In further embodiments, Q is Ca2+ or Mg2+. In some embodiments, Q is Ca2+· In some embodiments, Q is Mg2+. In other embodiments, Q is a trivalent cation counterion such as Fe3+. In some embodiments, Q is a multivalent organic cation. In further embodiments, Q is a divalent organic cation such as a protonated diamine. Liposomes comprising the trans-norbîxin compositions and pharmaceuticai compositions (e.g., liposome compositions) comprising the liposomes are also provided herein.
[0063] In some embodiments, the disclosure provides a pharmaceuticai composition comprising calcium trans-norbixin (CTN). The CTN can exist in linear and/or cyclic form (shown below), o
Liposomes comprising CTN and pharmaceuticai compositions (e.g., liposome compositions) comprising the liposomes are also provided herein.
[0064] In some embodiments, the disclosure provides a pharmaceuticai composition comprising magnésium trans-norbixin (MTN). The MTN can exist in linear and/or cyclic form (shown below),
Liposomes comprising MTN and phannaceutical compositions (e.g., liposome compositions) comprising the liposomes are also provided hereîn.
[0065] The lipids and other components of the liposomes contained in the liposome compositions can be any lipid, lipid combination and ratio, or combination of lipids and other liposome components and their respective ratios known in the art. However, it will be understood by one skilled in the art that liposomal encapsulation of any particular drug, such as, and without limitation, the carotenoid compositions discussed herein, may involve substantial routine expérimentation to achieve a useful and functional liposomal formulation. In general, the provided liposomes may hâve any liposome structure, e.g., structures havîng an inner space sequestered from the outer medium by one or more lipid bilayers, or any microcapsule that has a semi-permeable membrane with a lipophilie central part where the membrane sequesters an interior. The lipid bilayer can be any arrangement of amphiphilic molécules characterized by a hydrophilic part (hydrophilic moiety) and a hydrophobie part (hydrophobie moiety). Usually amphiphilic molécules in a bilayer are arranged into two dimensional sheets in which hydrophobie moieties are oriented inward the sheet while hydrophilic moieties are oriented outward. Amphiphilic molécules forming the provided liposomes can be any known or later discovered amphiphilic molécules, e.g., lipids of synthetic or natural origin or biocompatible lipids. The liposomes can also be formed by amphiphilic polymers and surfactants, e.g., polymerosomes and niosomes. For the purpose of this disclosure, without limitation, these liposome-forming materials also are referred to as lipids.
[0066] The liposome composition formulations provided herein can be in liquid or dry form such as a dry powder or dry cake. The dry powder or dry cake may hâve undergone primary drying under, for example, lyophilization conditions or optîonally, the dry cake or dry powder may hâve undergone both primary drying only or both primary drying and secondary drying. In the dry form, the powder or cake may, for example, hâve between 1% to 6% moisture, for example, such as between 2% to 5% moisture or between 2% to 4% moisture. One example method of drying is lyophilization (also called freeze-drying, or cryodessication). Any of the compositions and methods of the disclosure may include liposomes, lyophilized liposomes or liposomes reconstituted from lyophilized liposomes. In some embodiments, the compositions and methods include one or more lyoprotectants or cryoprotectants. These protectants are typically polyhydroxy compounds such as sugars (mono-, di-, and polysaccharides), polyalcohols, and their dérivatives, glycerol, or polyethyleneglycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycosides. In further embodiments, the lyoprotectants or cryoprotectants comprise up to 10% or up to 20% of a solution outside the liposome, inside the liposome, or both outside and inside the liposome,
[0067] The properties of liposomes are influenced by the nature of lipids used to make the liposomes. A wide variety of lipids hâve been used to make liposomes. These include cationic, anionic and neutral lipids. In some embodiments, the liposomes comprising the carotenoid compositions (e.g., CTC and MTC) are anionic or neutral. In other embodiments, the provided liposomes are cationic. The détermination of the charge (e.g., anionic, neutral or cationic) can routinely be determined by measuring the zêta potential of the liposome. The zêta potential of the liposome can be positive, zéro or négative. In some embodiments, the zêta potential of the liposome is -150 to 150 mV, or -50 to 50 mV, or any range therein between. In some embodiments, the zêta potential of the liposome is less than or equal to zéro. In some embodiments, the zêta potential of the liposome is -150 to 0, -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 therein between. In other embodiments, the zêta potential of the liposome is more than zéro. In some embodiments, the liposome has a zêta potential that 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 therein between.
[0068] In some embodiments, cationic lipids are used to make cationic liposomes which are commonly used as gene transfection agents. The positive charge on cationic liposomes enables interaction with the négative charge on cell surfaces. Following binding of the cationic liposomes to the cell, the liposome is transported inside the cell through endocytosîs.
[0069] In some preferred embodiments, a neutral to anionic liposome is used. In a preferred embodiment, 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 which are less likely to nonspecifically bind to normal cells. Spécifie binding to tumor cells can be achieved by using a tumor targeting antibody such as, for example, a folate receptor antibody, including, for example, folate receptor alpha antibody, folate receptor beta antibody and/or folate receptor delta antibody.
[0070] As an example, at least one (or some) of the lipids is/are amphipathic lipids, defined as having a hydrophilic and a hydrophobie portion (typically a hydrophilic head and a hydrophobie tail). The hydrophobie portion typically orients ïnto a hydrophobie phase (e.g., withîn the bilayer), whîle the hydrophilic portion typically orients toward the aqueous phase (e.g., outside the bilayer). The hydrophilic portion can comprise polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxy and other like groups. The hydrophobie portion can comprise apolar groups that înclude without limitation long chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted by one or more aromatic, cyclo-ahphatic or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids.
[0071] Typically, for example, the lipids are phospholipids. Phospholipids include without limitation phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and the like. It is to be understood that other lipid membrane components, such as cholestérol, sphingomyelin, and cardiolipin, can be used.
[0072] The lipids comprising the liposomes provided herein can be anionic and neutral (încludîng 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, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatîdylethanolamine, ceramide, sphingomyelin, cephalin, cholestérol, cerebrosides and diacylglycerols. Examples of zwitterionic lipids include without limitation dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids are negatively charged at physiological pH. These lipids include without limitation phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatîdic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
[0073] Collectively, anionic and neutral lipids are referred to herein as noncatîonic lipids. Such lipids may contain phosphorus but they are not so limited. Examples of non-cationic lipids include lecithin, lysolecithîn, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphos phoethanolamine (DMPE), distearoylphosphatidy 1-ethanolamine (DSPE), palmitoyloleoyl-phosphatidylethanolamine (POPE) palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholîne (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyol-phosphatidylglycerol (POPG), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans-PE, palmitoyloleoylphosphatidylethanolamîne (POPE), l-stearoyl-2-oleoylphosphatidylethanoIamine (SOPE), phosphatidylserine, phosphatidyl-inositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, and cholestérol.
[0074] The liposomes may be assembled using any liposomal assembly method using liposomal components (also referred to as liposome components) known in the art. Liposomal components include, for example, lipids such as DSPE, HSPC, cholestérol and dérivatives of these components. Other suitable lipids are commercial ly available for example, by Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). A partial listing of available negatively or neutrally charged lipids suitable for making anionic liposomes, can 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-Glutaryb(Na)2, tetramyristoyl cardiolipin *(Na)2, DSPE-mPEG-2000*Na, DSPE-mPEG-5000*Na, and DSPEmaleimide PEG-2000*Na.
[0075] In some embodiments, the provided compositions are formulated in a liposome comprising a cationic lipid. In one embodiment, the cationic lipid is selected from, but not limited to, a cationic lipid described in Intl. Appl. Publ. Nos. WO2012/040184, WO2011/153120, WO2011/149733, WO2011/090965, WO2011/043913, WO2011/022460, WO2012/061259, WO2012/054365, WO2012/044638, WO2010/080724, WO2010/21865 and WO2008/103276, U.S. Pat. Nos. 7,893,302, 7,404,969 and 8,283,333, and U.S. Appl. Publ. Nos.
US20100036115 and US20120202871; each of which is herein incorporated by reference in their entirety. In another embodiment, the cationic hpid may be seiected from, but not limited to, formula A described in Intl. Appl. Publ. Nos WO2012/040184, WO2011/153120, WO201/1149733, WO2011/090965, WO2011/ 043913, WO2011/022460, WO2012/061259, WO2012/054365 and WO2012/044638; each of which is herein incorporated by reference in their entirety. In yet another embodiment, the cationic lîpid may be seiected from, but not limited to, formula CLI-CLXXIX of International Publication No. WO2008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969 and formula I-VI of US Patent Publication No. US20100036115; each of which is herein incorporated by reference in their entirety. As a non-limiting example, the cationic lîpid may be seiected from (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z, 20Z)-N,N-dimemylhexacosa-17,20-dien-9-amine, ( 1 Z, 19Z)-N5N-dimethylpentacosa-I6, 19-dien-8-amine, (13Z, 16Z)-N,N-dimethyldocosa-13,16-dien-5amine, (12Z, 15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z, 17Z)-N,Ndimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N- dîmethyltetracosa-15,18dien-7-amine, ( 18Z,2lZ)-N,N-dimethylheptacosa-18,21 -dien-10-amine, (15Z, 18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, ( 14Z, 17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimeihyloctacosa-19,22-dien-9amine, (18Z,21 Z)-N,N-dimethylheptacosa-18,21-dien-8-amine, (17Z.20Z)Ν,Ν-dimethylhexacosa-17,20-dien-7-amine, ( 16Z, 19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10amine, (21 Z,24Z)-N,N-dimethyl- trîaconta-21,24-dien-9-amine, (18Z)-N,Ndimetylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-l7-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-l 9,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1[(11 Z, 14Z)-1 -nonylicosa-11,14-dien-1 -yl] pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-l 0-amine, (15Z)-N,N-dimethyl eptacos-15-en-l 0-amine,
(14Z)-N,N-dimethylnonacos- 14-en-l 0-amine, (17Z)-N,N-dimethylnonacos-17en-10-amîne, (24Z)-N,N-dimethyltritriacont-24-en-l 0-amîne, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacont-22-en-10amine, (16Z)-N,N-dîmethylpenta-cos-16-en-8-amine, ( 12Z,15 Z)-N,N-dimethyl
5 -2-nonylhenicosa-l 2,15-dien-l-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-l-amine, N,N-dimethyl-l-[(lS,2R)-2-octylcyclo-propyl] eptadecan-8-amine, 1 -[( 1 S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10- amine, N,N-dimethyl-1 -[( 1S ,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21 -[RI S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-
10 dimethyl-l-[(lS,2S)-2-{[(lR,2R)-2-pentylcyclopropyl]methyl}cyclopropyl] nonadecan-10-amine,N,N-dimethyl-1 -[(1 S,2R)-2-octylcyclopropyl]hexadecan -8-amine, N,N-dîmethyl-[(lR,2S)-2-undecyl-cyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(lS, 2R)-2-octylcyclopropyl]heptyl} dodecan-1-amine, 1[(lR,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, l-[(lS,2R)-2-
15 decylcyclopropyl]-N,N-dimethyl-penta-decan-6-amine, N,N-dimethyl-l-[(lS, 2R)-2-octylcyclopropyl]pentadecan-8-amine, R—N,N-dimethyl-1 -[(9Z, 12Z)octadeca-9,12-dien-1 -yloxy]-3-(octyloxy)propan-2-amine, S-N,N-dimethyl -1 [(9Z, 12Z)-octadeca-9,12-dien-1 -yloxy]-3-(octyloxy)propan-2-amine, 1 - {2-[(9 Z,12Z)-octadeca-9,12-dien-l-yloxy]-l-[(octyloxy)methyl]ethyl}pyiToHdine,
20 (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,l 2-dien-1-y loxy]-3-[(5Z-)-oct-5en-1 -yloxy]propan-2-amine, 1 - {2-[(9Z, 12Z)-octadeca-9,12-dien-1 -yloxy]-1 - [(octyloxy)methyl]ethyl}azetidine, (2S)-l-(hexyloxy)-N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9,12-dien-1 -yloxy]propan-2-amine, (2S)-1 -(heptyloxy)-N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9,12-dien-1 -yloxy]propan-2-amine, N,N-
25 dîmethyl-1 -(nonyloxy)-3-[(9Z, 12Z)-octadeca-9,12-dien-l -yloxy]propan-2amine, N,N-dimethyl-1 -[(9Z)-octadec-9-en-1 -yloxy]-3-(octyloxy) propan-2amine; (2S)-N, N-dimethyl-1 -[(6Z,9Z, 12Z)-octadeca-6,9,12-trien-1 -yloxy]-3- (octyloxy)propan-2-amine, (2S)-1 -[(11 Z, 14Z)-icosa-l 1,14-dien-l -yloxy]-N,Ndimethyl-3-(pentyloxy)propan-2-amine, (2S)-l-(hexyloxy)-3-[(l lZ,14Z)-icosa-
l,14-dien-l-yloxy]-N,N-dimethylpropan-2-amine, 1-[(1 lZ,14Z)-icosa-ll,14dien-1 -yloxy]-N,N-dimethyl 1 -3-(octyloxy)propan-2-amine, 1 -[( 13Z, 16Z)docosa-13,16-dien-l-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)1 - [( 13Z, 16Z)-docosa-13,16-dien-l -yIoxy]-3-(hexyloxy)-N,N-dimethyl-propan2-amine, (2 S)-1 -[(13Z)-docos-l 3-en-1 -yloxy]-3-(hexyloxy)-N,N-dimethyl propan-2-amine, 1-[(13Z)-docos-13-en-l -yloxy] -N,N-dimethyl-3-(octyloxy) propan-2-amine, l-[(9Z)-hexadec-9-en-l-yloxy]-N,N-dimethyl-3-(octyloxy) propan-2-amine, (2R)-N,N-dimethyl-H(l-metoylo ctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1 -yloxy]propan-2-amine, (2R)-1 -[(3,7-dimethyloctyl)oxy]-N, N-dimethyl-3-R9Z, 12Z)-octadeca-9,12-die-n-l -yloxylpropan-2-amine, N,N-dîmethyl-1 -(octyloxy)-3-({8-[(lS,2S)-2-{[(lR,2R)-2-pentylcyclopropyl]methyl} cyclopropyl]octyl}oxy) propan-2-amine, N,N-dimethyI-l-{[-(2oclylcyclo-propyl)octyl]oxy}-3-(octyloxy) propan-2-amine and(HE,20Z,23Z)N,N-dimethylnonacosa-l l,20,2-trien-10-amine or a pharmaceutically acceptable sait or acid or stereoisomer thereof.
[0076] In one embodiment, the lipid may be a cleavable lipid such as those described in in Intl. Publ. No. WO2012/170889, which is herein incorporated by reference in its entirety
[0077] The catîonic lipid can routinely be synthesized using methods known in the art (see, e.g., Intl. Publ. Nos. WO2012/040184, WO2011/153120, WO2011/149733, WO2011/090965, WO201/1043913, WO2011/022460, WO2012/061259, WO2012/ 054365, WO2012/044638, WO2010/080724 and WO2010/21865; each of which is herein incorporated by reference in its entirety.
[0078] Lipid dérivatives can include, for example, at least, the bonding (preferably covalent bonding) of one or more steric stabilizers and/or functional groups to the liposomal component after which the steric stabilizers and/or functional groups should be considered part of the liposomal components. Functional groups comprise groups that can be used to attach a liposomal component to another moiety such as a protein. Such functional groups include, at least, maleimide. These steric stabîlîzers include at least one from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxy-propyl) methacrylamide]; amphiphilic poly-Nvînylpyrrolidones; L-amino-acid-based polymer; and polyvinyl alcohol.
[0079] In some embodiments, the provided carotenoid compositions are formulated in a lipid-polycation complex. The formation of the lipid-polycation complex may be accomplîshed using methods known in the art and/or as described in U.S. Pub. No. 2012/0178702, herein incorporated by reference in its entirety. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyomithine and/or polyarginine and the cationic peptides described in International Pub. No. WO2012/013326; herein incorporated by reference in its entirety. In another embodiment, the provided carotenoid composition is formulated in a lipidpolycation complex which further includes a neutral lipid such as, but not limited to, cholestérol or dioleoyl phosphatidylethanolamine (DOPE).
[0080] Since the components of a liposome can include any molecuie(s) (i.e., chemical/reagent/protein) that is bound to it, in some embodiments, the components of the provided liposomes include, at least, a member selected from: DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholestérol; cholesterol-PEG; and cholesterol-maleimide. In some embodiments, the components of the provided liposomes include DSPE, DSPEPEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholestérol; cholesterol-PEG; and cholesterol-maleimide. In a preferred embodiment, the liposomal components that make up the liposome comprises DSPE; DSPEFITC; DSPE-maleimide; cholestérol; and HSPC.
[0081] In additional embodiments, the liposomes ofthe liposome compositions provided herein comprise oxidized phospholipids. In some embodiments, the liposomes comprise an oxidize phospholipid of a member selected from phosphatidylserines, phosphatidylinositols, phosphatidylethanolammes, phosphatidylcholines and 1 -palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments, the phospholipids hâve unsaturated bonds. In some embodiments, the phospholipids are arachidonic acid containing phospholipids. In additional embodiments, the phospholipids are sn-2-oxygenated. In additional embodiments, the phospholipids are not fragmented.
[0082] In some embodiments, the liposomes of the disclosed liposome compositions comprise oxidîzed 1 -palmitoyl-2-arachidonoyl-sn-glycero-3phosphorylcholine (OxPAPC). The term oxPAPC, as used herein, refers to lipids generated by the oxîdation of l-palmîtoyl-2-arachîdonyl-sn-glycero-3phosphorylcholine (PAPC), which résulte in a mixture of oxidized phospholipids containing either fragmented or full length oxygenated sn-2 residues. Wellcharacterized oxidatively fragmented species contain a five- carbon sn-2 residue bearing omega-aldehyde or omega-carboxyl groups. Oxîdation of arachidonic acid residue also produces phospholipids containing esterified isoprostanes. oxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC species, among other oxidized products présent in oxPAPC. In further embodiments, the oxPAPCs are epoxyisoprostane-containing phospholipids. In further embodiments, the oxPAPC is l-palmitoyl-2-(5,6-epoxyisoprostane E2)-snglycero-3-phosphocholine (5,6-PEIPC), I-palmitoyl-2-(epoxy-cyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC) and/or 1 -palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, the phospholipids hâve unsaturated bonds. In some embodiments, the phospholipids are arachidonic acid containing phospholipids. In additional embodiments, the phospholipids are sn-2-oxygenated. In additional embodiments, the phospholipids are not fragmented.
[0083] In some embodiments, the liposomes of the disclosed liposome compositions comprise a lipid selected from: l-palmitoyl-2-glutaroyl-sn20783 glycero-3-phosphocholîne (PGPC); 1 -palmitoyl-2-(9'oxo-nonanoyl)-sn-glycero
-3-phosphocholine; 1 -palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholme; 1 - palmitoyl-2-myristoyl-sn-glycero-3-phosphochoIine; l-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and l-palmitoyl-2-acetoyl-sn-glycero-3-phospho-choline. In further embodiments, the liposome comprises PGPC.
[0084] In some embodiments, at least one component of the liposome lipid bilayer is functionalized (or reactive). As used herein, a functionalized component is a component that comprises a reactive group that can be used to crosslink reagents and moîeties to the lipid. If the lipid is functionalized, any liposome that it forms is also functionalized. In some embodiments, the reactive group is one that will react with a crosslinker (or other moîety) to form crosslinks. The reactive group in the liposome lipid bilayer is located anywhere on the lipid that allows it to contact a crosslinker and be crosslinked to another moiety (e.g., a steric stabilizer or targeting moiety). In some embodiments, the reactive group is in the head group of the lipid, including for example a phospholipid. In some embodiments, the reactive group is a maleimide group. Maleimide groups can be crosslinked to each other in the presence of dithîol crosslinkers including but not limîted to dithîothreitol (DTT).
[0085] It is to be understood that the use of other functionalized lipids, other reactive groups, and other crosslinkers beyond those described above is further contemplated. In addition to the 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, aldéhyde groups, alkyne groups, azide groups, carbonyls, halo acetyl (e.g., iodoacetyl) groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydryl groups, and pyridyl disulfide groups.
[0086] Functionalized and non-functîonalized lipids are avaîlable from a number of commercial sources including Avanti Polar Lipids (Alabaster, AL) and Lipoid LLC (Newark, NJ).
[0087] In some embodiments, the liposomes include a steric stabilizer that increases their longevity in circulation. One or more steric stabilizers such as a hydrophilic polymer (polyethylene glycol (PEG)), a glycolipid (monosialoganglioside (GM1)) or others occupies the space immediately adjacent to the liposome surface and excludes other macromolecules from this space. Consequently, access and bînding of blood plasma opsonins to the liposome surface are hindered, and thus interactions of macrophages with such liposomes, or any other clearing mechanism, are inhibited and longevity of the liposome in circulation is enhanced. In some embodiments, the steric stabilizer or the population of steric stabilizers is a PEG or a combination comprising PEG. In further embodiments, the steric stabilizer is a PEG or a combination comprising PEG with a number average molecular weight (Mn) of 200 to 5000 Daltons. These PEG(s) can be of any structure such as linear, branched, star or comb structure and are commercially avaîlable.
[0088] In some embodiments, liposomes of the provided liposome compositions are pegylated (e.g., pegylated liposomal CTC and pegylated liposomal MTC). In some embodiments, the pegylated liposomes are water soluble. That is, the pegylated liposomes are in the form of an aqueous solution.
[0089] The dîameter of the provided liposomes is not particularly limited. In some embodiments, the liposomes hâve a mean dîameter of for example, 20 nm to 500 nm (nanometer), or 20 nm to 200 nm, or any range thereîn between. In some embodiments, the liposomes hâve a mean dîameter of 80 nm to 120 nm, or any range thereîn between.
[0090] In some embodiments, the pH of solutions comprising the liposome composition is from pH 2 to 8, or any range thereîn between. In some embodiments, the pH of solutions comprising the liposome composition is from pH 5 to 8, or 6 to 7, or any range thereîn between. In some embodiments, the pH of solutions comprising the liposome composition is from pH 6 to 7, or any range thereîn between. In some embodiments, the pH of solutions comprising the liposome composition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, or any range thereîn between.
[0091] In additional embodiments, the provided liposome composition comprises a buffer. In further embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., monobasic and/or dibasic sodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monobasic and/or dibasic sodium phosphate). In some embodiments, the buffer is at a concentration of 15 to 200 mM, or any range thereîn between. In yet further embodiments, the buffer is at a concentration of 5 to 200 mM, 15 to 200, 5 to 100 mM, 15 to 100 mM, 5 to 50 mM, 15 to 50 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, or any range thereîn between. In some embodiments, the buffer is HEPES at a concentration of 5 to 200 mM, or any range therein between. In some embodiments, the buffer is citrate at a concentration of 5 to 200 mM, or any range therein between. In some embodiments, the buffer is sodium phosphate at a concentration of 5 to 200 mM, or any range therein between.
[0092] In additional embodiments, the liposome composition contains one or more lyoprotectants or cryoprotectants. In some embodiments, the cryoprotectant is mannitol, trehaiose, sorbitol, or sucrose. In some embodiments, the lyoprotectant and/or cryoprotectant is présent in the composition at 1 to 20%, or 5 to 20% weight percent, or any range therein between.
[0093] In additional embodiments, the provided liposome composition comprises a tonicity agent. In some embodiments, the concentration (weight percent) ofthe tonicity agent is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range thereîn between. In some embodiments, the liposome composition includes a sugar (e.g., trehaiose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.). In further embodiments, the concentration (weight percent) of the sugar is 0.1-20%, 1-20%, 0.5-15%, 1%-15%, or 1-50%, or any range therein between.
[0094] In some embodiments, the provîded liposome composition comprises trehalose. In further embodiments, the concentration weight percent of trehalose is 0.1-20%, 1-20%, 0.5-15%, 1%-15%, 5-20%, or 1-50%, or any range therein between. In yet further embodiments, the concentration (weight percent) of trehalose is 1-15%, or any range therein between. In an additional embodiment, the trehalose is présent at about 5% to 20% weight percent of trehalose or any combination of one or more lyoprotectants or cryoprotectants at a total concentration of 5% to 20%. In some embodiments, the pH of the liposome composition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, or any range therein between.
[0095] In some embodiments, the liposome composition comprises dextrose. In some embodiments, the concentration weight percent of dextrose is 0.1 -20%, 1-20%, 0.5-15%, 1-15%, 5-20%, or 1-50%, or any range therein between. In particular embodiments, the concentration (weight percent) of dextrose is 1-20%, or any range therein between. In an additional embodiment, the dextrose is présent at 1 to 20% weight percent of dextrose or any combination of one or more lyoprotectants or cryoprotectants at a total concentration of 1% to 20%, or 5% to 20%, or any range therein between.
[0096] In some embodiments, the disclosure provides a liposome composition that comprises a liposome encapsulating an ionizable carotenoid sait. In some embodiments, the composition comprises a liposome encapsulating an ionizable carotenoid sait of any of [l]-[28]. In some embodiments, the liposome is pegylated. In some embodiments, the liposome is targeted. In some embodiments, the liposome is unpegylated and targeted. In some embodiments, the liposome is unpegylated and nontargeted. In some embodiments, the 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, molécules of the îonizable carotenoid. In some embodiments, the liposome contams 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules ofthe îonizable carotenoid, or any range therein between. In additional embodiments, the encapsulated îonizable carotenoid is trans-crocetin, trans-norbixin, or an îonizable carotenoid provided in [l]-[28] and/or FIGS. 1A-1D, herein. In some embodiments, the liposome encapsulâtes a pluralîty of carotenoids In further embodiments, the liposome encapsulâtes a pluralîty of îonizable carotenoids (e.g., a combination of trans-crocetin, trans-norbixin, and/or one or more îonizable carotenoid provided in [l]-[28] and/or FIGS. 1A-1D, herein).
[0097] In some embodiments, the disclosure provides a liposome composition that comprises an unpegylated nontargeted liposome encapsulating an îonizable carotenoid sait. In some embodiments, the liposome encapsulâtes the îonizable carotenoid sait of any of [l]-[28]. In some embodiments, the encapsulated îonizable carotenoid is trans-crocetin. In some embodiments, the encapsulated îonizable carotenoid is trans-norbixin. In some embodiments, the encapsulated îonizable carotenoid is a carotenoid provided in [l]-[28] and/or FIGS. 1A-1D, herein. In some embodiments, the unpegylated nontargeted liposome contaîns 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, molécules of the carotenoid. In some embodiments, the liposome contaîns lOto 100,000, lOOto 10,000, or 1,000 to 5,000 molécules ofthe îonizable carotenoid, or any range therein between. In further embodiments, the liposome encapsulâtes a pluralîty of carotenoids. In some embodiments, the liposome encapsulâtes a pluralîty of îonizable carotenoids (e.g., a combination of trans-crocetin, trans-norbixin, and/or one or more îonizable carotenoid provided in [l]-[28] and/or FIGS. 1A-1D, herein).
[0098] In some embodiments, the disclosure provides a liposome composition that comprises a pegylated liposome encapsulating an îonizable carotenoid sait. In some embodiments, the liposome encapsulâtes the îonizable carotenoid sait of any of [l]-[28], In some embodiments, the pegylated liposome contaîns 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, molécules of the ionizable carotenoid. In some embodiments, the pegylated liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000, molécules of the carotenoid, or any range therein between. In some embodiments, the encapsulated ionizable carotenoid is trans-crocetin. In some embodiments, the encapsulated ionizable carotenoid is trans-norbixin. In some embodiments, the encapsulated ionizable carotenoid is a carotenoid provided in [l]-[28] and/or FIGS. 1A-1D, herein. In some embodiments, the pegylated liposome encapsulâtes a plurality of carotenoids In further embodiments, the liposome encapsulâtes a plurality of ionizable carotenoids (e.g., a combination of trans-crocetin, trans-norbixin, and/or one or more ionizable carotenoid provided in [ 1 ]-[28] and/or FIGS. 1A1D, herein). In some embodiments, the pegylated liposome is targeted. In some embodiments, the pegylated liposome is nontargeted.
[0099] In some embodiments, the disclosure provides a liposome composition that comprises a targeted liposome encapsulating an ionizable carotenoid sait. In some embodiments, the liposome encapsulâtes the ionizable carotenoid sait of any of [l]-[28]. In some embodiments, the targeted liposome comprises a targeting moiety having a spécifie affinity for a surface antîgen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or an antigen binding fragment of an antibody. In some embodiments, the targeting moiety binds the surface antigen with an equilibrium dissociation constant (Kd) in a range of 50 x 10'12 to 10 x 10‘6 as determined using BIACORE® analysis. In further embodiments, the Kd is determined using a surface plasmon résonance technique in which an antigen containing the epitope is immobilized, the targeting moiety serves as analyte, and the following conditions are used: lOmM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150mM NaCI at 37°C. In some embodiments, the targeting moiety comprises a protein or folate conjugale with spécifie affinity for one or more folate receptors selected from: folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR δ). In some embodiments, the targeted liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. The targeted liposome can be pegylated or unpegylated.
[0100] In some embodiments, the targeted liposome is pegylated and encapsulâtes an ionizable carotenoid sait. In some embodiments, the liposome encapsulâtes the ionizable carotenoid sait of any of [l]-[28], In some embodiments, the targeted pegylated liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the targeted pegylated liposome contains lOto 100,000,100 to 10,000, or 1,000 to 5,000, molécules of the ionizable carotenoid, or any range therein between. In some embodiments, the encapsulated ionizable carotenoid is trans-crocetin. In some embodiments, the encapsulated ionizable carotenoid is trans-norbixin. In some embodiments, the encapsulated ionizable carotenoid is a carotenoid provided in [1 ]-[28] and/or FIGS. 1A-1D, herein.
[0101] In other embodiments, the targeted liposome is unpegylated and encapsulâtes an ionizable carotenoid sait. In some embodiments, the liposome encapsulâtes the ionizable carotenoid sait of any of [l]-[28]. In some embodiments, the targeted unpegylated liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In further embodiments, the liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000, molécules of the ionizable carotenoid, or any range therein between. In some embodiments, the encapsulated ionizable carotenoid is transcrocetin. In some embodiments, the encapsulated ionizable carotenoid is transnorbixin. In some embodiments, the encapsulated ionizable carotenoid is a carotenoid provided in [l]-[28] and/or FIGS. 1A-1D, herein. In additional embodiments, the targeted unpegylated liposome encapsulâtes a plurality of carotenoids. In further embodiments, the liposome encapsulâtes a plurality of ionizable carotenoids (e.g., a combination of trans-crocetin, trans-norbixin, and/or one or more ionizable carotenoid provided in [l]-[28] and/or FIGS. 1A1D, herein).
[01021 In some embodiments, the disclosure provides a pegylated liposome composition that comprises a liposome encapsulatîng an ionizable carotenoid sait provided in any of and encapsulâtes an ionizable carotenoid sait. In some embodiments, the liposome encapsulâtes the ionizable carotenoid sait of any of [l]-[28]. In some embodiments, the 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, molécules of the ionizable carotenoid. In some embodiments, the pegylated liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of the ionizable carotenoid, or any range therein between. In additional embodiments, the liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as two or more of the ionizable carotenoids provided in [l]-[28] and/or FIGS. 1A-1D, herein.
[0103] In additional embodiments, the disclosure provides a liposome composition that comprises a pegylated and targeted liposome encapsulatîng an ionizable carotenoid sait. In some embodiments, the liposome encapsulâtes the ionizable carotenoid sait of any of [l]-[28]. In some embodiments, the pegylated and targeted liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the pegylated and targeted liposome contains lOto 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of the ionizable carotenoid, or any range therein between. In additional embodiments, the liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as two or more of the ionizable carotenoids provided in [1]-
[28] and/or FIGS. 1A-1D, herein.
[0104] In additional embodiments, the disclosure provides a liposome composition that comprises a pegylated and untargeted liposome encapsulating an ionizable carotenoid sait. In some embodiments, the liposome encapsulâtes the ionizable carotenoid sait of any of [l]-[28]. In some embodiments, the pegylated and untargeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of the ionizable carotenoid, or any range therein between. In additional embodiments, the pegylated and untargeted liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more ionizable carotenoids such as two or more of the ionizable carotenoids provided in [1 ]-[28] and/or FIGS. 1A-1D, herein.
[0105] In additional embodiments, the disclosure provides a liposome composition that comprises an unpegylated liposome encapsulating an ionizable carotenoid sait. In some embodiments, the liposome encapsulâtes the ionizable carotenoid sait of any of [l]-[28]. In some embodiments, the 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, molécules of the ionizable carotenoid. In some embodiments, the unpegylated liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of the ionizable carotenoid, or any range therein between. In additional embodiments, the unpegylated liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as two or more of the ionizable carotenoids provided in [ l |-[28] and/or FIGS. 1A-1D, herein.
[0106] In additional embodiments, the disclosure provides a liposome composition that comprises an unpegylated and targeted liposome encapsulating an ionizable carotenoid sait, and encapsulâtes an ionizable carotenoid sait. In some embodiments, the liposome encapsulâtes the ionizable carotenoid sait of any of
[11-(28]. In some embodiments, the unpegylated and targeted liposome contains to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range thereîn between. In some embodiments, the unpegylated and targeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of the ionizable carotenoid, or any range thereîn between. In additional embodiments, the unpegylated and targeted liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as two or more of the ionizable carotenoids provided in [1][28] and/or FIGS. 1A-1D, herein.
[0107] In additional embodiments, the disclosure provides a liposome composition that comprises an unpegylated and nontargeted liposome encapsulatîng an ionizable carotenoid sait and encapsulâtes an ionizable carotenoid sait. In some embodiments, the liposome encapsulâtes the ionizable carotenoid sait of any of [l]-[28]. In some embodiments, the unpegylated and nontargeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of the ionizable carotenoid, or any range thereîn between. In additional embodiments, the unpegylated and nontargeted liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as two or more of the ionizable carotenoids provided in [ 1 ]-[28] and/or FIGS. 1A-1D, herein.
[0108] In some embodiments, the disclosure provides a pegylated liposome composition that comprises a liposome encapsulatîng a trans-crocetin sait. In some embodiments, the 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, molécules of trans-crocetin. In some embodiments, the pegylated liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of trans-crocetin, or any range thereîn between. In additional embodiments, the liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more ionizable carotenoids provided in [ 1 ]-[28] and/or FIGS. 1A-1D, herem.
[0109] In additional embodiments, the disclosure provides a liposome composition that comprises a pegylated and targeted liposome encapsulating a trans-crocetîn sait. In some embodiments, the pegylated and targeted liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the pegylated and targeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of trans-crocetin, or any range therein between. In additional embodiments, the pegylated and targeted liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more ionizable carotenoids provided in [ 1 ]-[28] and/or FIGS. 1A-1D, herein.
[0110] In additional embodiments, the disclosure provides a liposome composition that comprises a pegylated and untargeted liposome encapsulating a trans-crocetin sait. In some embodiments, the pegylated and untargeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of trans-crocetin, or any range therein between. In additional embodiments, the pegylated and untargeted liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more ionizable carotenoids provided in [l]-[28] and/or FIGS. 1A-1D, herein.
[0111] In additional embodiments, the disclosure provides a liposome composition that comprises an unpegylated liposome encapsulating trans-crocetin sait. In some embodiments, the liposome contains less than 6 million, le s s than 500,000, less than 200,000, less than 100,000, less than 50,000, less than 10,000, or less than 5,000, molécules of trans-crocetin. In some embodiments, the unpegylated liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of trans-crocetin, or any range therein between. In additional embodiments, the unpegylated liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more ionizable carotenoids provided în [l]-[28] and/or FIGS. 1A-ID, herein.
[0112] In additional embodiments, the disclosure provides a liposome composition that comprises an unpegylated and targeted liposome encapsulating a trans-crocetin sait. In some embodiments, the unpegylated and targeted liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targetîng moieties, or any range therein between. In some embodiments, the unpegylated and targeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of trans-crocetin, or any range therein between. In additional embodiments, the unpegylated and targeted liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more carotenoids provided in [l]-[28] and/or FIGS. 1A-1D, herein.
[0113] In additional embodiments, the disclosure provides a liposome composition that comprises an unpegylated and nontargeted liposome encapsulating a transcrocetin sait. In some embodiments, the unpegylated and nontargeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of transcrocetin, or any range therein between. In additional embodiments, the unpegylated and nontargeted liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more carotenoids provided in [ 1 ]-[28] and/or FIGS. 1A-1D, herein.
[0114] In some embodiments, the disclosure provides a pegylated liposome composition that comprises a liposome encapsulating a trans-norbixin sait. In some embodiments, the 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, molécules of trans-norbixin. In some embodiments, the pegylated liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of trans-norbixin, or any range therein between. In additional embodiments, the liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more ionizable carotenoids provided in [ 1 ]-[28] and/or FIGS. 1A-1D, herein.
[0115] In additional embodiments, the disclosure provides a liposome composition that comprises a pegylated and targeted liposome encapsulating a trans-norbixin sait. In some embodiments, the pegylated and targeted liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting molettes, or any range therein between. In some embodiments, the pegylated and targeted liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of trans-norbixin, or any range therein between. In additional embodiments, the pegylated and targeted liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more ionizable carotenoids provided in [ 1 ]-[28] and/or FIGS. 1A-ID, herein.
[0116] In additional embodiments, the disclosure provides a liposome composition that comprises a pegylated and untargeted liposome encapsulating a trans-norbixin sait. In some embodiments, the pegylated and untargeted liposome contains 10 to 100,000,100 to 10,000, or 1,000 to 5,000 molécules of trans-norbixin, or any range therein between. In additional embodiments, the pegylated and untargeted liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more ionizable carotenoids provided in [l]-[28] and/or FIGS. 1A-1D, herein.
[0117] In additional embodiments, the disclosure provides a liposome composition that comprises an unpegylated liposome encapsulating trans-norbixin sait. In some embodiments, the 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, molécules of trans-norbixin. In some embodiments, the unpegylated liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of trans-norbixin, or any range therein between. In additional embodiments, the unpegylated liposome comprises a plurality of carotenoids. In further embodiments, the liposome comprises a plurality of ionizable carotenoids such as one or more îonizable carotenoids provided in [l]-[28] and/or FIGS. 1A-1D, herein.
[0118] In additional embodiments, the disclosure provides a liposome composition that comprises an unpegylated and targeted liposome encapsulating a trans-norbixin sait. In some embodiments, the unpegylated and targeted liposome contaîns 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the unpegylated and targeted liposome contaîns 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of trans-norbixin, or any range therein between. In additional embodiments, the unpegylated and targeted liposome comprises a pluralîty of carotenoids. In further embodiments, the liposome comprises a pluralîty of îonizable carotenoids such as one or more carotenoids provided in [ 1 ]-[28] and/or FIGS. 1A-1D, herein.
[0119] In additional embodiments, the disclosure provides a liposome composition that comprises an unpegylated and nontargeted liposome encapsulating a transnorbixin sait. In some embodiments, the unpegylated and nontargeted liposome contaîns 10 to 100,000, 100 to 10,000, or 1,000 to 5,000 molécules of transnorbixin, or any range therein between. In additional embodiments, the unpegylated and nontargeted liposome comprises a pluralîty of carotenoids. In further embodiments, the liposome comprises a pluralîty of îonizable carotenoids such as one or more carotenoids provided in [l]-[28] and/or FIGS. 1A-1D, herein.
[0120] In some embodiments, the provided liposome compositions comprise a liposome encapsulating one or more îonizable carotenoid salts (e.g., divalent, trivalent or tetravalent sait of an îonizable carotenoid of any of [l]-[28], and/or FIGs. 1A-1D)) and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, the liposome composition contaîns trehalose. In some embodiments, the liposome composition contaîns 1% to 50% weight of trehalose. In some embodiments, the liposome composition contaîns HBS at a concentration of between 1 to 200 mM and a pH of between 2 to 8. In some embodiments, liposome composition has a pH 5-8. or any range therein between.
In some embodiments, liposome composition has a pH 6-7, or any range therem between.
[0121] In further embodiments, the provided liposome compositions comprise a liposome encapsulating a trans-crocetin sait, and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, the liposome solution contains trehalose. In some embodiments, the liposome solution contaîns 1 % to 50% weight of trehalose. In some embodiments, the liposome solution contains HBS at a concentration of 1 to 200 mM and a pH of 2 -8, or any range therein between. In some embodiments, liposome solution has a pH 5-8, or any range therein between. In some embodiments, liposome solution has a pH 6-7, or any range therein between. In some embodiments, the provided trans-crocetin sait is a multivalent sait (e.g., divalent, trivalent, or tetravalent). In some embodiments, the trans-crocetin sait is CTC. In some embodiments, the trans-crocetin sait is MTC.
[0122] In further embodiments, the provided liposome compositions comprise a liposome encapsulating a trans-norbixin sait, and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, the liposome solution contains trehalose. In some embodiments, the liposome solution contains 1% to 50% weight of trehalose. In some embodiments, the liposome solution contains HBS at a concentration of 1 to 200 mM and a pH of 2 -8, or any range therein between. In some embodiments, liposome solution has a pH 5-8, or any range therein between. In some embodiments, liposome solution has a pH 6-7, or any range therein between. In some embodiments, the provided trans-norbixin sait is a multivalent sait (e.g., divalent, trivalent, or tetravalent). In some embodiments, the trans-norbixin sait is CTN. In some embodiments, the trans-norbixin sait is MTN.
[0123] The provided liposomes comprise an aqueous compartment enclosed by at least one lipid bilayer. When lipids that include a hydrophilîc headgroup are dispersed in water they can spontaneously form bilayer membranes referred to as lamellae. The lamellae are composed of two monolayer sheets of lipid molécules with their non-polar (hydrophobie) surfaces facing each other and their polar (hydrophilic) surfaces facing the aqueous medium. The term liposome includes unilamellar vesicles which are comprised of a single lipid bilayer and generally hâve a dîameter in the range of about 20 to about 500 nm, about 50 to about 300 nm, about 50 to about 150 nm, 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 generally hâve a dîameter in the range of 0.5 to 10 um with anywhere from two to hundreds of concentric lipid bilayers altemating with layers of an aqueous phase. In some embodiments, liposomes can include multilamellar vesicles (MLV), large unilamellar vesicles (LUV), and small unilamellar vesicles (SUV). The lipids of the liposome can be cationic, zwitterionic, neutral or anionic, or any mixture thereof.
[0124] Any suitable combination of lipids can be used to provide the liposomes and lipid nanoparticles provided herein. The lipid compositions can be tailored to affect characteristics such as leakage rates, stabîlîty, particle size (e.g., liposome dîameter), zêta potential, proteîn binding, in vivo circulation, and/or accumulation in tissues or organs. For example, DSPC and/or cholestérol can be used to decrease leakage from liposomes. Negatively or positively lipids, such as DSPG and/or DOTAP, can be included to affect the surface charge of a liposome or lipid nanoparticle. In some embodiments, the lipid compositions can include about ten or fewer types of lipids, or about five or fewer types of lipids, or about three or fewer types of lipids. In some embodiments, the molar percentage (mol %) of a spécifie type of lipid présent typically comprises from about 0% to about 10%, from about 10% to about 30%, from about 30% to about 50%, from about 50% to about 70%, from about 70% to about 90%, from about 90% to 100% of the total lipid présent in a liposome or lipid nanoparticle. In some embodiments, the therapeutic liposome comprises 40-80 mol % DSPC, 520783 mol % cholestérol, 0-30 mol % DSPG, and 0-10 mol % DSPE-PEG(2000).
In some embodiments, the attacking liposome comprises 40-70 mol % DPPC, 520 mol % cholestérol, 0-20 mol % DOTAP, and 20-40 mol % TPGS.
[0125] Depending on the desired application, the particle size (diameter) of the liposome can be regulated. For example, when it is intended to deliver the liposome to cancerous tissue or inflamed tissue by the Enhanced Permeability and Rétention (EPR) effect as an injection product or the like, it is préférable that liposome diameter is 20-500 nm, 30-175 nm, or 50-150 nm, or any range therein between. In the case where the intention is to transmit liposome to macrophage, it is préférable that liposome diameter is 30 to 1000 nm, or 80 to 400 nm, or any range therein between. In the case where liposome composition is to be used as an oral préparation or transdermal préparation, the particle size of liposome can be set at several microns. It shouldbe noted that in normal tissue, vascular walls serve as barriers (because the vascular walls are densely constituted by vascular endothélial cells), and microparticles such as supermolecules and liposome of specifîed size cannot be distributed within the tissue. However, in diseased tissue, vascular walls are loose (because interstices exist between vascular endothélial cells), increasing vascular permeability, and supermolecules and microparticles can be distributed to extravascular tissue (enhanced permeability). Moreover, 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 recycled through the general System, and are retained in the diseased tissue (enhanced rétention), which fonns the basîs of the EPR effect (Wang et al., Ann. Rev. Med. 63:185-198 (2012); Peer et al., Nat. Nanotech. 2:751-760 (2007); Gubemator, Exp. Opin. Drug Deliv. 8:565-580 (2011); Huwyler et al., Int. J. Nanomed. 3:21-29 (2008); Maruyama et al. Adv. Drug Deliv. Rev. 63:161-169 (2011); Musacchio and Torchilin Front. Bioscî. 16:1388-1412 (2011); Baryshnikov Vest. Ross. Akad. Med. Nauk. 23-31 (2012); and Torchilin
Nat. Rev. Drug Disc. 4:145-160 (2005)). Thus, it is possible to control liposome pharmacokinetics by adjusting liposome particle size (diameter).
[0126] The size of the liposomes in the provided liposome compositions may vary from for example, 0.5 nm to 10 um, or 20 nm to 5 um, depending on the phospholipid composition, the method used for their préparation, and the intended therapeutic use of the liposomes. In some embodiments, the médian diameter of the liposomes in the provided liposome composition is 20 nm to 500 nm, 50 nm to 200 nm, or 20 nm to 200 nm, or any range therein between. In some embodiments, the liposome the liposome médian diameter is 80 nm to 120 nm, or any range therein between (e.g., 85-115 nm, 90-110 nm, 95-110 nm, or 95-105 nm). In some embodiments, the médian diameter of the liposomes in the provided liposome composition is 10-250 nm, or any range therein 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, ΙΟΙ 00 nm, 10-75 nm, 10-50 nm, 50-100 nm, 50-90 nm, 50-80 nm, 50-70 nm, 5060 nm, 60-100 nm, 60-90 nm, 60-80 nm, 60-70 nm, 70-100 nm, 70-90 nm, 7080 nm, 80-100 nm, 80-90 nm, or 90-100 nm). In some embodiments, the médian diameter of the liposomes in the provided liposome composition is 100-250 nm, or any range therein between (e.g., 100-225 nm, 100-200 nm, 100-175 nm, or 100-150 nm). In other embodiments, the médian diameter of the liposomes in the provided liposome composition is 10-100 nm, or any range therein between (e.g., from about 10-90 nm, 10-80 nm, 10-70 nm, 10-60 nm, or 10-50 nm). In some embodiments, the médian diameter of the liposomes in the provided liposome composition is less than, about 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 150 nm, 145 nm, 150 nm, 135 nm, 130 nm, 125 nm, 120 nm, 115 nm, 110 nm, 105 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, or 50 nm, 45 nm, or 40 nm. Dynamic laser light scattering is a method used to measure the diameter of liposomes that is well known to those skilled in the art. The diameter of the liposomes (DLP) can routinely be determined using any techniques and equipment known in the art including for example, dynamic laser light scattering (Coulter N4 particle size analyzer), the Zetasizer Nano ZSP (Malvem, UK), and an ELS-8000 (Otsuka Electronics Co., Ltd.)).
[0127] In some embodiments, the provided liposome compositions hâve a monodisperse size (diameter) distribution. Monodisperse and homogeneous size distribution, are used interchangeably herein and describe a plurality of liposomal nanoparticles or microparticles where the particles hâve the same or nearly the same diameter. As used herein, a monodisperse distribution refers to particle distributions in which 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or greater 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 médian diameter.
[0128] In some embodiments, the liposome population in a provided liposome composition is relatively homogenous. In some embodiments, the liposome population in a provided liposome composition is heterogeneous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size (diameter) distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. In some embodiments, the liposome population in a provided liposome composition has a polydispersity index from 0 to 0.25, or 0.01 to 0.1, or any range therein between (e.g., 0.001 to 0.2, 0.005 to 0.1, 0.005 to 0, 0.005 to 0.09, 0.009 to 0.09, 0.01 to 0.08, 0.02 to 0.09, or 0.02 to 0.07, or any range therein between.
[0129] In some embodiments, liposomes in the liposome population in a provided liposome composition differ in their lipid composition, molar ratio of lipid components, size, charge (zêta potential), targeting ligands and/or combinations thereof.
[0130] The zêta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the zêta potential may describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or négative, are generally désirable, as more highly charged species may interact undesirably with cells, tissues, and other éléments in the body. In some embodiments, the zêta potential of a nanoparticle composition can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV. Liposome zêta potential can routinely be determined using techniques and equipment known in the art including for example, dynamic light scattering (Zetasizer Nano ZSP, Malvem, UK) and laser Doppler electrophoresis.
[0131] The encapsulation efficiency of a therapeutic and/or prophylactic such as an ionizable carotenoid (e.g., trans-crocetin), describes the amount of therapeutic and/or prophylactic that is encapsulated or otherwise associated with a nanoparticle composition after préparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and/or prophylactic in a solution containing the nanoparticle composition before and after removing the unencapsulated therapeutic and/or prophylactic drug. For the liposome compositions described herein, the encapsulation efficiency of a ionizable carotenoid (e.g., transcrocetin), can be at least 50%, for example 60%, 70%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the encapsulation efficiency is at least encapsulation efficiency is at least encapsulation efficiency is at least
80%. In certain embodiments, the
90%. In certain embodiments, the
95%. In certain embodiments, the encapsulation efficiency is at least 98%.
[0132] In additional embodiments, the provided liposome compositions contain liposomes encapsulating a sait of an ionizable carotenoid. In some embodiments, the ionizable carotenoid (e.g., trans-crocetin)/lipid ratio of the provided liposome composition is 1 to 1000 g/mol, or any range therein between. In some embodiments, the ionizable carotenoid/lipid ratio of the liposome composition îs 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, 20 to 100 g/mol, 30 to 200 g/mol, 30 to 150 g/mol, 30 to 100 g/mol, 40 to 200 g/mol, 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 therein between. In some embodiments, the ionizable carotenoid/lipid ratio is 30 to 90 g/mol, or any range therein between. In some embodiments, the 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 therein between. In some embodiments, the liposome encapsulâtes an ionizable carotenoid sait of any of [1] to [27]. In some embodiments, the liposome encapsulâtes an ionizable carotenoid sait presented in any of FIGs. 1A-1D.
[0133] In some embodiments, the liposome composition contains liposomes encapsulating a trans-crocetin sait. In some embodiments, the transcrocetin/lipid ratio of the provided liposome composition is 1 to 1000 g/mol, or any range therein between. In some embodiments, the trans-crocetin /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, 20 to 100 g/mol, 30 to 200 g/mol, 30 to 150 g/mol, 30 to 100 g/mol, 40 to 200 g/mol, 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 therein between. In some embodiments, the trans-crocetin/lipîd ratio is 30 to 90 g/mol, or any range therein between. In some embodiments, the trans-crocetin /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 therein between.
[0134] In some embodiments, the liposome composition contains liposomes encapsulating a trans-norbixin sait. In some embodiments, the transnorbîxin/lipid ratio of the provided liposome composition is 1 to 1000 g/mol, or any range therein between. In some embodiments, the trans-norbixin /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, 20 to 100 g/mol, 30 to 200 g/mol, 30 to 150 g/mol, 30 to 100 g/mol, 40 to 200 g/mol, 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 therein between. In some embodiments, the trans-norbixin/lipid ratio is 30 to 90 g/mol, or any range therein between. In some embodiments, the trans-norbixin /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 therein between.
[0135] In some embodiments, the liposome composition is buffered using a zwitterionic buffer. Suitably, the zwitterionic buffer is an aminoalkanesulfonic acid or suitable sait. Examples of aminoalkanesulfonic buffets înclude but are not limited to HEPES, HEPPS/EPPS, MOPS, MOBS and PIPES. Preferably, the buffer is a pharmaceutically acceptable buffer, suitable for use in humans, such as in for use in a commercial injection product. Most preferably the buffer is HEPES. The liposome composition may suitable include an AGP.
[0136] In some embodiments, the liposome composition is buffered using HEPES. In some embodiments, he liposome composition is buffered using HEPES having a pH of about 7.
[0137] In some embodiments, the pharmaceutical composition is a liposome composition comprising a cationic liposome. In some embodiments, the liposome composition comprises a liposome that has a zêta potential that is more than zéro. In some embodiments, the liposome has a zêta potential of 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 therein between. In some embodiments, the liposome has a diameter of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 200 nm, or 50 nm to 150 nm, or any range therein between. In some embodiments, the cationic liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of an ionizable carotenoid (e.g., transcrocetin). In some embodiments, during the process of preparing 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 an ionizable carotenoid starting material is encapsulated (entrapped) in the liposomes of the liposome composition. In additional embodiments, the ionizable carotenoid (e.g., trans-crocetin) encapsulated by the liposome is in a HEPES buffered solution within the liposome. In further embodiments, the liposome comprises at least one OxPAPC.
[0138] In some embodiments, the provided pharmaceuticai composition is a liposome composition comprising an anionic or neutral liposome. In some embodiments, the liposome composition comprises a liposome that has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, -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 therein between. In some embodiments, the anionic or neutral liposome has a diameter of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therein between. In other embodiments, the anionic or neutral liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the anionic liposome has a diameter of nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therein between. In further embodiments, the anionic liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the neutral liposome has a diameter of 20 nm to 500 nm, 20 nm to 200 nm, 30nmto 175 nm, or 50nmto 150 nm, or any range therein between. In some embodiments, the neutral liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the pharmaceutical composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w ionizable carotenoid (e.g., trans-crocetin). In some embodiments, during the process of preparing the liposome composition, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, of the starting material of ionizable carotenoid (e.g., trans-crocetin) is encapsulated (entrapped) in the liposomes. In some embodiments, the liposome composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the ionizable carotenoid (e.g., trans-crocetin). In some embodiments, the 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 more than 75%, w/w of the ionizable carotenoid (e.g., trans-crocetin). In some embodiments, liposome composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the ionizable carotenoid (e.g., trans-crocetin). In additional embodiments, the ionizable carotenoid (e.g., trans-crocetin) is encapsulated by the anionic or neutral liposome is in a HEPES buffered solution within the liposome. In further embodiments, the liposome comprises at least one OxPAPC.
[0139] In some embodiments, the provided pharmaceutical composition is a liposome composition comprising a liposome that comprises at least one OxPAPC. In some embodiments, the OxPAPC is an oxidized and/or phospholipid containing fragmented oxygenated sn-2 residues. In some embodiments, the OxPAPC is an oxidized phospholipid containing a five-carbon sn-2 residue bearing an omega-aldehyde or omega-carboxyl group. In some embodiments, the OxPAPC is an oxidized phospholipid selected from HOdiAPC, KOdiA-PC, HOOA-PC and KOOA-PC. In some embodiments, the OxPAPC is a epoxyisoprostane-containing phospholipid. In some embodiments, the OxPAPC is PGPC. In some embodiments, the liposome comprises at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, or at least 30%, OxPAPC. In some embodiments, the liposome composition has a cationic liposome that comprises 0.01%-35%, 0.1%-30%, l%-25%, 3-20%, or 5-15%, OxPAPC, or any range therein between. 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, the liposome composition comprises at least one liposome containing an OxPAPC that has a diameter of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therein between. In further embodiments, the liposome composition comprises a at least one liposome containing an OxPAPC that has a diameter of 80 nm to 120 nm, or any range therein between.
[0140] In some embodiments, the provided pharmaceutical composition is a liposome composition comprising a cationic liposome that comprises at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, or at least 30%, OxPAPC. In some embodiments, the liposome composition has a cationic liposome that comprises 0.01%-35%, 0.1%-30%, l%-25%, 3-20%, or 5-15%, OxPAPC, or any range therein between. In some embodiments, the liposome comprises at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, or at least 30%, OxPAPC. In some embodiments, the liposome composition has a cationic liposome that contains about 10% OxPAPC. In some embodiments, the liposome composition has a cationic liposome that comprises at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%,
25%, or at least 30%, PGPC. In some embodiments, the liposome comprises 0.01 %-35%, 0.1 %-30%, 1 %-25%, 3-20%, or 5-15%, PGPC, or any range therein between. In some embodiments, the liposome composition has a cationic liposome that comprises about 10% PGPC.
[0141] In some embodiments, the pharmaceutical composition is a liposome composition comprising an anionic or neutral liposome that comprises at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, or at least 30%, OxPAPC. In some embodiments, the liposomal composition has a anionic or neutral liposome that comprises 0.01%-35%, 0.1%-30%, l%-25%, 3-20%, or 5-15%, OxPAPC, or any range therein between. In some embodiments, the liposome comprises at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, or at least 30%, OxPAPC. In some embodiments, the liposomal composition has a anionic or neutral liposome that contains about 10% OxPAPC. In some embodiments, the liposomal composition comprises has a anionic or neutral liposome that comprises at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, or at least 30%, PGPC. In some embodiments, the liposome comprises 0.01%-35%, 0.1%-30%, l%-25%, 3-20%, or 5-15%, PGPC, or any range therein between. In some embodiments, the liposomal composition has a anionic or neutral liposome that contains about 10% PGPC.
[0142] In some embodiments, the pharmaceutical composition is a liposomal composition comprising a neutral liposome that comprises at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, or at least 30%, OxPAPC. In some embodiments, the neutral OxPAPC containing liposomal composition comprises 0.01%-35%, 0.1%-30%, l%-25%, 3-20%, or 5-15%, OxPAPC, or any range therein between. In some embodiments, the neutral OxPAPC containing liposomal composition comprises about 10% OxPAPC. In some embodiments, the neutral OxPAPC containing liposomal composition comprises at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, or at least 30%, PGPC. In some embodiments, the neutral PGPC containing liposomal composition comprises
0.01%-35%, 0. l%-30%, l%-25%, 3-20%, or 5-15%, PGPC, or any range therein between. In some embodiments, the neutral OxPAPC containing liposomal composition comprises about 10% PGPC.
[0143] In additional embodiments, a liposome in the liposomal composition is pegylated.
[0144] In some embodiments, the provided pharmaceutical composition is a non-targeted liposomal composition. That is, the liposomes in the liposomal composition do not hâve spécifie affmity towards an epitope (e.g., an epitope on a surface antigen) expressed on the surface of a target cell of interest. In further embodiments, the non-targeted liposomal composition is pegylated.
[0145] In some cases, liposome accumulation at a target site may be due to the enhanced permeability and rétention characteristics of certain tissues such as cancer tissues. Accumulation in such a manner often résulte in part because of liposome size and may not require spécial targeting fimctionality. In other embodiments, the provided liposomes include a targeting agent. Generally, the targeting agents can associate with any target of interest, such as a target associated with an organ, tissues, cell, extracellular matrix, or intracellular région. In certain embodiments, a target can be associated with a particular disease State, such as a cancerous condition. In some embodiments, the targeting component can be spécifie to only one target, such as a receptor. Suitable targets can include but are not limited to a nucleic acid, such as a DNA, RNA, or modified dérivatives thereof. Suitable targets can also include but are not limited to a protein, such as an extracellular protein, a receptor, a cell surface receptor, a tumor-marker, a transmembrane protein, an enzyme, or an antibody. Suitable targets can include a carbohydrate, such as a monosaccharide, disaccharide, or polysaccharide that can be, for example, présent on the surface of a cell.
[0146] In certain embodiments, a targeting agent can include a target ligand (e.g., an RGD-contaîning peptide), a small molécule mimic of a target ligand (e.g., a peptide mimetic ligand), or an antibody or antibody fragment spécifie for a particular target. In some embodiments, a targeting agent can further înclude folie acid dérivatives, B-12 dérivatives, integrin RGD peptides, NGR dérivatives, somatostatin dérivatives or peptides that bind to the somatostatin receptor, e.g., octreotide and octreotate, and the like. In some embodiments, the targeting agents înclude an aptamer. Aptamers can be desîgned to associate with or bind to a target of interest. Aptamers can be comprised of, for example, DNA, RNA, and/or peptides, and certain aspects of aptamers are known in the art. (See, e.g., Klussman, Ed., The Aptamer Handbook, Wiley-VCH (2006); Nîssenbaum, Trends in Biotech. 26(8); 442-449 (2008)).
[0147] In other embodiments, the liposomal composition comprises a targeted liposome. That is, the liposome contains a targeting moiety that has spécifie affmity for an epitope (e.g., a surface antigen or other molécule) on a target cell of interest. In some embodiments, the targeting moiety of the liposome is not attached to the liposome through a covalent bond. In other embodiments, the targeting moiety of the liposome is attached to one or both of a PEG and the exterior of the liposome. In further embodiments, the targeted liposome is pegylated. The fonctions of the targeting moiety of the targeted liposome may înclude but is not lîmited to, targeting the liposome to the target cell of interest in vivo or in vitro; interacting with the surface antigen for which the targeting moiety has spécifie affmity, and delivering the liposome payload (e.g., transcrocetin) to the location of or into the cell.
[0148] Suitable targeting moieties are known in the art and înclude, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is a polypeptide that comprises at least 3, 5, 10, 15, 20, 30, 40, 50, or 100, amino acid residues. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In further embodiments, the targeting moiety comprises one or more of an antibody, a humanized antibody, an antigen binding fragment of an antibody, a single chain antibody, a single-domain antibody, a bi-specinc antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody. In some embodiments, the targeting moiety has spécifie affinity for an epitope that is preferentially expressed on a target cell such as a tumor cell, compared to normal or non-tumor cells. In some embodiments, the targeting moiety has spécifie affinity for an epitope on a tumor cell surface antigen that is présent on a tumor cell but absent or inaccessible on a non-tumor cell. In some embodiments, the targeting moiety binds an epitope of interest with an equilibrium dissociation constant (Kd) in a range of 50 x 10'12 to 10 x 10-6 as determined using BIACORE® analysis. In further embodiments, the Kd is determined using a surface plasmon résonance technique in which an antigen containing the epitope is immobilized, the targeting moiety serves as analyte, and the following conditions are used: lOmM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150mM NaCl at 37°C.
[0149] In particular embodiments, the targeting moiety comprises a polypeptide that specifically binds a folate receptor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the targeting moiety is a liposome surface-conjugated folate (e.g., a folate-PEG conjugate) or folate dérivative. In some embodiments, the folate receptor bound by the targeting moiety is one or more folate receptors selected from: folate receptor alpha (FR-α, FOLR1), folate receptor beta (FR-β, FOLR2), and folate receptor delta (FR-δ, FOLR4). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor alpha (FR-α). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor beta (FR-β). In some embodiments, the targeting moiety specifically binds FRα and FR-β.
[0150] In additional embodiments, the liposome composition comprises one or more of an immunostîmulatory agent, a détectable marker, and a maleimide, disposed on at least one of the PEG and the exterior of the liposome. In some embodiments, a liposome of the liposome composition is cationic. In other embodiments, a liposome of the liposome composition is anionic or neutral. In additional embodiments, a liposome of the liposomal composition has a dîameter of 20 nm to 500 nm, or any range thereîn between. In further embodiments, a liposome of the liposomal composition has a dîameter of 80 nm to 120 nm, or any range thereîn between. In some embodiments, a liposome of the liposomal composition is pegylated. In some embodiments, a liposome of the liposomal composition is targeted. In further embodiments, a liposome of the liposomal composition is pegylated and targeted.
[0151] In some embodiments, the disclosure provides a pharmaceutical composition comprising an ionizable carotenoid having the formula: Polyene Carotenoid-Q encapsulated by a liposome, wherein, the Polyene Carotenoid comprises (a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1, 2, 3, or more than 3, ionizable groups; and
Q is a (a) a multivalent counterion or (b) a monovalent cation.
In some embodiments, the Polyene Carotenoid comprises ail trans conjugated double bonds. In some embodiments, the Polyene Carotenoid comprises 6-9 conjugated double bonds. In particular embodiments, the Polyene Carotenoid comprises 7 conjugated double bonds. The Polyene Carotenoid can be naturally occurring or synthetic. In some embodiments, the Polyene Carotenoid is naturally occurring. In other embodiments, the Polyene Carotenoid is synthetic. The ionizable group(s) may be anionic and/or cationic. In some embodiments, the Polyene Carotenoid-Q comprises two or more of the same ionizable group. In some embodiments, the Polyene Carotenoid comprises ail trans conjugated double bonds. In some embodiments, the Polyene Carotenoid comprises 6-9 conjugated double bonds. In some embodiments, the Polyene Carotenoid comprises two or more different ionizable groups. In some embodiments, the Polyene Carotenoid-Q comprises one or more anionic ionizable groups. In some embodiments, the Polyene Carotenoid comprises at least one ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, or a phosphate group, and a hydroxamate moiety. In other embodiments, the Polyene Carotenoid-Q comprises one or more cationic ionizable groups (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, or an imidazole group). In particular embodiments, the Polyene Carotenoid comprises at least one 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 cation counterion. In some embodiments, Q is a multivalent métal cation. In some embodiments, Q is a multivalent transition métal cation. In some embodiments, Q is a divalent counterion. In some embodiments, Q is a divalent cation counterion. In some embodiments, Q is a divalent métal cation. In some embodiments, Q is a divalent transition métal cation. In some embodiments, Q is at least one member selected from Ca2+, Mg';', Zn2+, Cu2+, Co2+, and Fe2+. In some embodiments, Q is Ca2+ or Mg2+. In some embodiments, Q is Ca2+. In further embodiments, the Polyene Carotenoid-Q is calcium transcrocetinate (CTC). In some embodiments, Q is Mg2+. In further embodiments, the Polyene Carotenoid-Q is magnésium trans-crocetinate (MTC). In other embodiments, Q is a trivalent cation counterion such as Fe3+. In some embodiments, Q is a multivalent organic counterion. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a bivalent organic cation such as protonated diamine.
In further embodiments, Q is a monovalent counterion. In some embodiments, Q is a monovalent cation counterion. In some embodiments, Q is a monovalent métal 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 divalent organic cation. In some embodiments, Q is a monovalent organic cation such as a protonated amine (e.g., a protonated dîamine or a protonated polyamine). In some embodiments, Q is an organic cation such as NHZ, a protonated diamine or a protonated polyamine.
In some embodiments, the liposome contaîns 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, molécules of îonizable carotenoid. In some embodiments, the liposome contaîns 10 to 100,000 molécules of îonizable carotenoid, or any range therein between. In some embodiments, the îonizable carotenoid /lîpid ratio of the liposomal composition is lg/mol and about 1000 g/mol, or any range therein between. In some embodiments, the îonizable carotenoid /lîpid 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 therein between. In some embodiments, the liposome comprises at least 0.1 % to 97% trans-crocetin. In some embodiments, the liposome has a diameter of 20 nm to 500 nm, 20 nm to 200 nm, or 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lîpid and a neutral lipid. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimide. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and HSPC. In additional embodiments, the liposome further comprises an oxidized phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that 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 omega-aldehyde or omega-carboxyl groups. In some embodiments, the liposome comprises an
OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxy-isoprostane-containing phospholipid. In some embodiments, the liposome comprises an OxPAPC selected from 1 -palmitoyl2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholme (5,6 PEIPC), 1palmitoyl-2-(epoxycyclopentenOne)-sn-glycero-3-phosphorylcholine (PECPC), 1 -palmitoyl-2-(epoxyisoprost-ane E2)-sn-glycero-4-phospho-choline (PEIPC), l-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC); 1palmitoyl-2-(9'oxo-nonanoyl)-sn-glycer-o-3-phosphocholine; l-palmitoyl-2arachinodoyl-sn-glycero-3-phosphocholine; 1 -palmitoyl-2-myristoyl-snglycero-3-phosphocholine; l-palmitoyl-2-hexadec-yl-sn-glycero-3phosphocholine; l-palmitoyl-2-azelaoyl-sn-glycero-3-phos-phocholine; and 1palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine. In some embodiments, the liposome comprises PGPC. In some embodiments, the OxPAPC within the liposome lipid bilayer is 0%-100% of total lipids, or any range therein between. In some embodiments, the liposome comprises a targeting moiety having a spécifie affinity for a surface antigen or other molécules on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or an antigen binding fragment of an antibody. In some embodiments, the liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the liposome further comprises an immunostîmulating agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabilizer. In some embodiments, the steric stabîlîzer is polyethylene glycol (i.e., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weight (Mn) of 200 to
5000 Daltons. In addîtional embodiments, the liposome is anionic or neutral. In some embodiments, the liposome has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, -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 therein between. In other embodiments, the liposome is cationic. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that is more than zéro. In some embodiments, the liposome has a zêta potential that 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 therein between.
[0152] In some embodiments, the disclosure provides a pharmaceutical composition comprising an ionizable carotenoid having the formula: Q- Rl-Polyene Carotenoid-R2 -Q, encapsulated by a liposome, wherein, the Polyene Carotenoid comprises (a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1, 2, 3, or more than 3, ionizable groups;
Ri and R; are ionizable groups; and
Q is a (a) multivalent counterion or (b) monovalent cation.
In some embodiments, the Polyene Carotenoid comprises ail trans conjugated double bonds. In particular embodiments, the Polyene Carotenoid comprises 69 conjugated double bonds. The Polyene Carotenoid can be naturally occurring or synthetic. In some embodiments, the Polyene Carotenoid is naturally occurring. In other embodiments, the Polyene Carotenoid is synthetic. In some embodiments, Ri and R^are the same ionizable group. In other embodiments, Ri and R2 are different ionizable groups. In some embodiments, Ri and R2 are
the same cationic ionizable group. In other embodiments, Ri and R: are different cationic groups. In some embodiments, Ri and R; are the same anionic ionizable group. In other embodiments, Ri and R2 are different anionic groups. In some embodiments, Ri is a cationic ionizable group or anionic ionizable group and R2
5 is an anionic ionizable group or cationic group, respectively. In some embodiments, the Polyene Carotenoid comprises at least one anionic ionizable group. In some embodiments, the Polyene Carotenoid comprises at least one ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, or a phosphate group, and a hydroxamate moiety. In some
10 embodiments, Ri is at least one ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, or a phosphate group, and a hydroxamate moiety. In some embodiments, R2 is at least one ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, or a phosphate group, and a hydroxamate moiety. In other
15 embodiments, the Polyene Carotenoid-Q comprises one or more cationic ionizable groups (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, or an imidazole group). In particular embodiments, the Polyene Carotenoid comprises at least one cationic ionizable group and the pharmaceutical composition is
20 substantially free of nucleic acids. In some embodiments, Q is a multivalent counterîon. In some embodiments, Q is a multivalent cation counterîon. In further embodiments, Q is a multivalent métal cation. In some embodiments, Q is a multivalent transition métal counterîon. In some embodiments, Q is a divalent counterîon. In some
25 embodiments, Q is a divalent cation counterion. In further embodiments, Q is a divalent métal cation. In some embodiments, Q is at least one member selected from Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+. In further embodiments, Q is Ca2+ or Mg2+. In further embodiments, Q is Ca2+. In some embodiments, Q is Mg2+. In other embodiments, Q is a trivalent cation counterion such as Fe3+. In other
embodiments, Q is a multivalent organic counterion. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a bivalent organic cation such as protonated dîamine.
In some embodiments, Q is a monovalent cation counterion. In further embodiments, Q is at least one member selected from Na+, or Li+, or K+. In some embodiments, Q is an organic counterion. In some embodiments, Q is a multivalent organic cation. In further embodiments, Q is a divalent organic cation such as a protonated diamine or a protonated polyamîne. In other embodiments, Q is a monovalent organic cation such as NH4+, a protonated diamine or a protonated polyamîne.
In some embodiments, the 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, molécules of ionizable carotenoid. In some embodiments, the liposome contains between 10 to 100,000 molécules of ionizable carotenoid, or any range therein between. In some embodiments, the ionizable carotenoid/lipid ratio of the liposomal composition is 1 g/mol and about 1000 g/mol, or any range therein between. In some embodiments, the 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 therein between. In some embodiments, the liposome comprises at least 0.1 % to 97% ionizable carotenoid. In some embodiments, the liposome has a diameter of 20 nm to 500 nm, 20 nm to 200 nm, or 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimide. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and HSPC. In additional embodiments, the liposome further comprises an oxidized phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that 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 omegaaldehyde or omega-carboxyl groups. In some embodiments, the liposome comprises an OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, the liposome comprises an OxPAPC selected from 1palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6 PEIPC), l-palmitoyl-2-(epoxycyclopenten-one)-sn-glycero-3phosphorylcholine (PECPC), l-palmitoyl-2-(epoxyîsoprost-ane E2)-sn-glycero4-phosphocholine (PEIPC), i -palmitoyl-2-glutaroyl-sn-glycero-3phosphocholine (PGPC); l-palmitoyl-2-(9'oxo-nonanoyl)-sn-glyc-ero-3phosphocholine; 1 -palmitoyl-2-arachinodoyl-sn-gIycero-3-phosphochol-ine; 1 palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1 -palmitoyl-2-hexadecy 1sn-glycero-3-phosphocholine; l-palmitoyl-2-azelaoyl-sn-glycero-3phosphocholine; and l-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine. In some embodiments, the liposome comprises PGPC. In some embodiments, the OxPAPC within the liposome lipid bilayer is 0%-100% of total lipids, or any range therein between. In some embodiments, the liposome comprises a targeting moiety having a spécifie affmity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or an antigen binding fragment of an antibody. In some embodiments, the liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range thereîn between. In some embodiments, the liposome further comprises an immunostimulating agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabilizer. In some embodiments, the steric stabilizer is polyethylene glycol (Le., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In some embodiments, the zêta potential of the liposome is in a range of -150 to 150 mV, or -50 to 50 mV, or any range thereîn between. In some embodiments, the liposome is cationic. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that is more than zéro. In some embodiments, the liposome has a zêta potential that 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 thereîn between. In some embodiments, the liposome is anionic or neutral. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, -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 thereîn between.
[0153] In some embodiments, the disclosure provides a pharmaceutical composition comprising an ionizable bis-alpha, omega-carotenoid having the formula:
Q- Ri-Polyene Carotenoid-Ri -Q, encapsulated by a liposome, wherein, the Polyene Carotenoid comprises (a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 1, 2, 3, or more than 3, ionizable groups; and
Ri is an ionizable group; and
Q is a (a) multivalent counterion or (b) monovalent cation.
In some embodiments, Ri-Polyene Carotenoîd-Ri comprises ail trans conjugated double bonds. In some embodiments, the Ri-Polyene Carotenoid-Ri comprises 6-9 conjugated double bonds. In particular embodiments, the Ri-Polyene Carotenoid-Ri comprises 7 conjugated double bonds. The Ri-Polyene Carotenoid-Ri can be naturally occurring or synthetic. In some embodiments, the Ri -Polyene Carotenoid-Ri is naturally occurring. In other embodiments, the b Ri-Polyene Carotenoîd-Ri is synthetic. In some embodiments, Ri is an anionic ionizable group. In some embodiments, the Ri-Polyene Carotenoid-Ri comprises an ionizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, a phosphate group, and a hydroxamate moîety. In other embodiments, Ri is a cationic ionizable group (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, or an imidazole group). In particular embodiments, Ri 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 cation counterion. In further embodiments, Q is a multivalent métal cation. In some embodiments, Q is a multivalent transition métal counterion. In some embodiments, Q is a divalent counterion. In some embodiments, Q is a divalent cation counterion. In further embodiments, Q is a divalent métal cation. In some embodiments, Q is at least one member selected from Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+. In further embodiments, Q is Ca2+ or Mg?T In further embodiments, Q is Ca2+. In some embodiments, Q is Mg2+. In other embodiments, Q is a trivalent cation counterion such as Fe3+. In other embodiments, Q is a multivalent organic counterion. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a bivalent organic cation such as protonated diamine.
Τη some embodiments, Q is a monovalent cation counterion. In further embodiments, Q is at least one member selected from Na , or Li , or K .In some embodiments, Q is an organic counterion. In some embodiments, Q is a multivalent 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 NHZ, a protonated diamine or a protonated polyamine.
In some embodiments, the 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, molécules of ionizable carotenoid. In some embodiments, the liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000, molécules of ionizable carotenoid, or any range therein between. In some embodiments, the ionizable carotenoid /lipid ratio of the liposomal composition is 1 g/mol and about 1000 g/mol, or any range therein between. In some embodiments, the 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 therein between. In some embodiments, the liposome comprises at least 0.1 % to 97% ionizable carotenoid. In some embodiments, the liposomes hâve a mean diameter of for example, 20 nm to 500 nm (nanometer), or 20 nm to 200 nm, or any range therein between. In some embodiments, the liposomes hâve a mean diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimide. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and HSPC. In additional embodiments, the liposome further comprises an oxidized phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that 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 fivecarbon sn-2 residue bearing omega-aldehyde or omega-carboxyl groups. In some embodiments, the liposome comprises an OxPAPC selected from HOdiAPC, KOdiA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, the liposome comprises an OxPAPC selected from l-palmitoyl-2-(5,6-epoxyisoprostane E2)sn-glycero-3-phosphocholine (5,6 PEIPC), l-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphoryl-choline (PECPC),l-palmitoyl-2-(epoxy-îsoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), l-palmitoyl-2-glutaroylsn-glycero-3-phospho-choline (PGPC); l-palmitoyl-2-(9'oxo-nonanoyl)-snglycero-3-phospho-choline; l-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine; 1 -palmitoyl-2-myristoyI-sn-glycero-3-phosphochoIine; 1 -palmitoyl-2hexa-dec-yl-sn-glycero-3-phosphocholine; l-palmitoyl-2-azelaoyl-sn-glycero3-phos-phocholine; and l-palmitoyl-2-acetoyl-sn-glycero-3-phospho-choline. In some embodiments, the liposome comprises PGPC. In some embodiments, the OxPAPC within the liposome lipid bilayer is 0%-100% of total lipids, or any range therein between. In some embodiments, the liposome comprises a targeting moiety having a spécifie affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety îs an antibody or an antigen binding fragment of an antibody. In some embodiments, the liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the liposome further comprises an immunostimulatîng agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabilizer. In some embodiments, the steric stabilizer is polyethylene glycol (Le., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In addîtional embodiments, the liposome is anionic or neutral. In some embodiments, the liposome has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, -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 therein between. In other embodiments, the liposome is cationic. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that is more than zéro. In some embodiments, the liposome has a zêta potential that 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 therein between.
[0154] In some embodiments, the disclosure provides a pharmaceutical composition comprising an bis-alpha, omega-carotenoid having the formula: Ri-Polyene Carotenoid-Ri.encapsulatedby a liposome, wherein, the bis-alpha, omega-carotenoid comprises:
(a) 3, 4, 5, 6, 7, 8, 9, 10, 3-5, 6-8, 9-10, or more than 9, conjugated double bonds, and (b) 1, 2, 3, or more than 3, ionizable groups; and the Polyene Carotenoid is optionally substituted with 1 to n methyl or low ClC3 alkyl substitutions, wherein η = 1 to 4; and
Ri is a polar group and/or a monocyclic functional group.
In some embodiments, the bis-alpha, omega-carotenoid comprises ail trans conjugated double bonds. In some embodiments, the bis-alpha, omegacarotenoid comprises 6-9 conjugated double bonds. In particular embodiments, the bis-alpha, omega-carotenoid comprises 7 conjugated double bonds. The bisalpha, omega-carotenoid can be naturally occurring or synthetîc. In some embodiments, the bis-alpha, omega-carotenoid is naturally occurring. In other embodiments, the bis-alpha, omega-carotenoid is synthetîc. In some embodiments. Ri is a polar group. In some embodiments, Ri is a monocyclic functional group. In some embodiments, Ri is a polar group and a monocyclic functional group. In some embodiments, the bis-alpha, omega-carotenoid comprises a monocyclic and/or polar functional group selected from a functional group présent in astaxanthin, lutein, xanthophyll and zeaxanthin. In some embodiments, the bis-alpha, omega-carotenoid is selected from astaxanthin, lutein, xanthophyll and zeaxanthin (e.g., as depicted below).
In some embodiments, the liposome contaîns 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, molécules of bis-alpha, omega-carotenoid. In some embodiments, the liposome contaîns 10 to 100,000, 100 to 10,000, or 1,000 to 5,000, molécules of bis-alpha, omega-carotenoid, or any range therein between. In some embodiments, the bis-alpha, omega-carotenoid/lipid ratio of the liposomal composition is 1 g/mol and about 1000 g/mol, or any range therein between. In some embodiments, the 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 therein between. In some embodiments, the liposome comprises at least 0.1 % to 97% bis-alpha, omega-carotenoid. In some embodiments, the liposome has a diameter of 20 nm to 500 nm, or 20 nm to 200 nm, or any range therein between. In some embodiments, the liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimide. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and HSPC. In additional embodiments, the liposome further comprises an oxidized phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that 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 omegaaldehyde or omega-carboxyl groups. In some embodiments, the liposome comprises an OxPAPC selected from HOdiA-PC, KOdîA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, the liposome comprises an OxPAPC selected from 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6 PEIPC), l-palmitoyl-2-(epoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), 1 -palmitoyl-2-(epoxy-isoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), l-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholîne (PGPC); 1paimitoyl-2-(9'oxo-nonanoyl)-sn-glycero-3-phos-phocholine; l-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-myristoyl-sn-glyc-ero3-phosphocholine; l-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; 120783 palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholîne; and 1 -palmitoyl-2-acetoylsn-glycero-3-phosphocholine. In some embodiments, the liposome compnses PGPC. In some embodiments, the OxPAPC within the liposome lîpid bilayer is 0%-100% of total lipids, or any range therein between. In some embodiments, the liposome comprises a targeting moiety having a spécifie affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or an antigen binding fragment of an antibody. In some embodiments, the liposome contaîns 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moietîes, or any range therein between. In some embodiments, the liposome further comprises an immunostimulating agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabilizer. In some embodiments, the steric stabilizer is polyethylene glycol (z.e., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In additional embodiments, the liposome îs anionic or neutral. In some embodiments, the liposome has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, -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 therein between. In other embodiments, the liposome is cationic. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that îs more than zéro. In some embodiments, the liposome has a zêta potential that 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 therein between.
100
[0155] In some embodiments, the pharmaceutical composition comprises a trans-crocetin sait having the formula: Q-trans-crocetm-Q o
encapsulated by a liposome, wherein,
Q is a (a) multivalent counterion or (b) monovalent cation.
In some embodiments, Q is a multivalent cation counterion. In some embodiments, Q is a multivalent métal cation. In further embodiments, Q is a multivalent transition métal cation. In some embodiments, Q is a divalent cation counterion. In further embodiments, Q is a divalent métal cation. In some embodiments, Q is at least one member selected from Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+. In further embodiments, Q is Ca2+ or Mg2+. In some embodiments, Q is Ca2+. In some embodiments, Q is Mg2+. In some embodiments, Q is a divalent organic counterion. In other embodiments, Q is a trivalent cation counterion such as Fe3+. In other embodiments, Q is a multivalent organic counterion. In some embodiments, Q is a divalent organic cation. In some embodiments, Q is a bivalent organic cation such as protonated diamine.
In further embodiments, Q is a monovalent cation counterion. In some embodiments, Q is a monovalent métal 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 (e.g., a protonated diamine or a protonated polyamine). In some embodiments, Q is an organic cation such as NHZ, a protonated diamine or a protonated polyamine.
In some embodiments, the 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, molécules of trans-crocetin. In some embodiments, the liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000, molécules of trans20783
101 crocetin., or any range therein between. In some embodiments, the transcrocetin/lipid ratio of the liposomal composition is 1 g/mol and about 1000 g/mol, or any range therein between. In some embodiments, the transcrocetin/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 therein between. In some embodiments, the liposome comprises at least 0.1 % to 97% trans-crocetin. In some embodiments, the liposome has a diameter of 20 nm to 500 nm, or 20 nm to 200 nm, or any range therein between. In some embodiments, the liposome has a diameter of 80 nmto 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG, DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesteroI-PEG; and cholestérol-maleîmide. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and HSPC. In addîtional embodiments, the liposome further comprises an oxidized phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that 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 fivecarbon sn-2 residue bearing omega-aldehyde or omega-carboxyl groups. In some embodiments, the liposome comprises an OxPAPC selected from HOdiAPC, KOdiA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxyisoprostane-contaîning phospholipid. In some embodiments, the liposome comprises an OxPAPC selected from l-paImitoyl-2-(5,6-epoxyisoprostane E2)sn-glycero-3-phosphocholine (5,6 PEIPC), l-paImitoyl-2-(epoxy-cyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), 1 -palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), 1 -palmitoyl-2-glutaroyl-sn20783
102 glycero-3-phosphocholine (PGPC); l-palmitoyl-2-(9'oxo-nonanoyl)-sn-glycero-3-phosphocholine; l-palmitoyl-2-ar-achinodoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-myrisÎoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2hexadecyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-azelaoyl-sn-glycero-3phosphocholine; and 1 -palmitoyl-2-acetoyl-sn-glycero-3 -phosphocholine. In some embodiments, the liposome comprises PGPC. In some embodiments, the OxPAPC within the liposome lipid bilayer is 0%-100% of total lipids, or any range thereîn between. In some embodiments, the liposome comprises a targeting moiety having a spécifie affinîty for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or an antigen binding fragment of an antibody. In some embodiments, the liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range thereîn between. In some embodiments, the liposome further comprises an immunostimulating agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabilizer. In some embodiments, the steric stabilizer is polyethylene glycol (i.e., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In additional embodiments, the liposome is anionic or neutral. In some embodiments, the liposome has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, 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 thereîn between. In other embodiments, the liposome is cationic. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that is
103 more than zéro. In some embodiments, the liposome has a zêta potential that 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 therein between.
[0156] In some embodiments, the disclosure provides a pharmaceutical composition comprising calcium trans-crocetinate (CTC) encapsulated by a liposome. The CTC can exist in linear and/or cyclic form (shown below).
In some embodiments, the 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, molécules of trans-crocetin. In some embodiments, the liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000, molécules of transcrocetin, or any range therein between. In some embodiments, the transcrocetin/lipid ratio of the liposomal composition is 1 g/mol and about 1000 g/mol, or any range therein between. In some embodiments, the transcrocetin/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 therein between. In some embodiments, the liposome comprises at least 0.1% to 97% trans-crocetin. In some embodiments, the liposome has a diameter of 20 nm to 500 nm, or 20 nm to 200 nm, or any range therein between. In some embodiments, the liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, the liposomal components comprise at least one
104 selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG;
cholestérol; cholesteroI-PEG; and cholesterol-maleimide. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and HSPC. In additional embodiments, the liposome further comprises an oxidized phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that 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 fivecarbon sn-2 residue bearing omega-aldehyde or omega-carboxyl groups. In some embodiments, the liposome comprises an OxPAPC selected from HOdiAPC, KOdiA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, the liposome comprises an OxPAPC selected from l-palmitoyl-2-(5,6-epoxyisoprostane E2)sn-glycero-3-phosphocholine (5,6 PEIPC), l-palmitoyl-2-(epoxy-cyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), l-palmitoyl-2-(epoxy-isoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), l-palmitoyl-2-glutaroyl-sngIycero-3-phosphocholine (PGPC); l-paimitoyl-2-(9'oxo-nonanoyI)-sn-glycero-3-phosphocholine; I-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-myristoyl-sn-glycer-o-3-phosphocholine; 1 -palmitoyl-2hexadecyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-azelaoyl-sn-glycero-3phosphocholine; and l-palmitoyl-2-acetoyl-sn-glycero-3-phos-phocholine. In some embodiments, the liposome comprises PGPC. In some embodiments, the OxPAPC within the liposome lipid bilayer is 0%-100% of total lipids, or any range therein between. In some embodiments, the liposome comprises a targeting moiety having a spécifie afïïnity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by
105 a covalent bond. In some embodiments, the targeting moiety is a polypeptide.
In further embodiments, the targeting moiety is an antibody or an antigen binding fragment of an antibody. In some embodiments, the liposome contaîns 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the liposome contaîns less than 500,000 or less than 200,000 molécules of trans-crocetin. In some embodiments, the liposome contaîns between 10 to 100,000 molécules of trans-crocetin, or any range therein between. In some embodiments, the liposome further comprises an immunostimulating agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabilizer. In some embodiments, the steric stabilizer is polyethylene glycol (Le., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In additional embodiments, the liposome is anionic or neutral. In some embodiments, the liposome has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, 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 therein between. In other embodiments, the liposome is cationic. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that is more than zéro. In some embodiments, the liposome has a zêta potential that 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 therein between.
[0157] In some embodiments, the disclosure provides a pharmaceutical composition comprising magnésium trans-crocetinate (MTC) encapsulated by a liposome. The MTC can exîst in linear and/or cyclic form (shown below).
106
In some embodiments, the 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, molécules of trans-crocetin. In some embodiments, the liposome 5 contains 10 to 100,000, 100 to 10,000, or 500 to 5,000, molécules of transcrocetin, or any range therein between. In some embodiments, the transcrocetin/Iipid 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 therein between. In some embodiments, the liposome comprises at least 0.1 % to 97% trans-crocetin. In some embodiments, 10 the liposome has a diameter of 20 nm to 500 nm, or 20 nm to 200 nm, or any range therein between. In some embodiments, the liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lipid and a neutral 15 lipid. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimide. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and 20 HSPC. In addîtional embodiments, the liposome further comprises an oxidized phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that 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 five25 carbon sn-2 resîdue bearing omega-aldehyde or omega-carboxyl groups. In
107 some embodiments, the liposome comprises an OxPAPC selected from HOdiAPC, KOdiA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxyisoprostane-containing phospholipîd. In some embodiments, the liposome comprises an OxPAPC selected from l-palmitoyl-2-(5,6-epoxyisoprostane E2)sn-glycero-3-phosphocholine (5,6 PEIPC), l-palmitoyl-2-(epoxycyclopentenone)-sn-g!ycero-3 -phosphorylcholine (PECPC), 1 -palmitoyl-2-(epoxy-isoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), l-palmitoyl-2-glutar-oyl-snglycero-3-phosphocholine (PGPC); l-palmitoyl-2-(9'oxo-nonanoyl)-sn-glycero-3-phosphocho line; l-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholîne; l-palmitoyl-2-myristoyl-sn-glycero-3-phospho-choline; l-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; l-palmîtoyl-2-azelaoyl-sn-glycero-3phosphocholine; and l-palmitoyl-2-acetoyl-sn-glycero-3-phosphocho line. In some embodiments, the liposome comprises PGPC. In some embodiments, the OxPAPC within the liposome lipid bilayer is 0%-100% of total lipids, or any range therein between. In some embodiments, the liposome comprises a targeting moiety having a spécifie affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or an antigen binding fragment of an antibody. In some embodiments, the liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the liposome further comprises an immunostimulating agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabîlizer. In some embodiments, the steric stabilizer is polyethylene glycol (i.e., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In additional embodiments, the liposome is anionic or neutral. In
108 some embodiments, the liposome has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, -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 therein between. In other embodiments, the liposome is cationic. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that is more than zéro. In some embodiments, the liposome has a zêta potential that is 0.2 to 150 mV, 1 to 50 mV, 1 to 40 mV, I 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 therein between.
[0158] In some embodiments, the disclosure provides a pharmaceutical composition comprising trans-norbixin having the formula; Q-norbixin-Q
encapsulated by a liposome, wherein,
Q is a multivalent cation counterion.
In some embodiments, Q is a multivalent cation counterion. In some embodiments, Q is a multivalent métal cation. In further embodiments, Q is a multivalent transition métal cation. In some embodiments, Q is a divalent cation counterion. In further embodiments, Q is a divalent métal cation. In some embodiments, Q is at least one member selected from Ca2+, Mg2 1·, Zn2+, Cu2+, Co2+, and Fe2+. In further embodiments, Q is Ca2+ or Mg2+. In some embodiments, Q is Ca2+. In some embodiments, Q is Mg2+. In some embodiments, Q is a divalent organic counterion.
In some embodiments, Q is a monovalent cation counterion. In some embodiments, Q is a monovalent métal cation. In some embodiments, Q is at least one member selected from Na+, Li+, or K+. In some embodiments, Q is an
109 organic cation. In some embodiments, Q is a monovalent organic cation such as a protonated amine (e.g., a protonated diamine or a protonated polyamme). In some embodiments, Q is an organic cation such as NH4+, a protonated diamine or a protonated polyamine.
In some embodiments, the liposome contaîns 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, molécules of trans-norbixin. In some embodiments, the liposome contaîns 10 to 100,000, 100 to 10,000, or 1,000 to 5,000, molécules of transnorbixin, or any range therein between. In some embodiments, the transnorbîxin/lipid ratio of the liposomal composition is Ig/mol and about 1000 g/mol, or any range therein between. In some embodiments, the transnorbixin/lîpid 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 therein between. In some embodiments, the liposome comprises at least 0.1 % to 97% Q-norbixin-Q. In some embodiments, the liposome has a diameter of 20 nm to 500 nm, or 20 nm to 200 nm, or any range therein between. In some embodiments, the liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lîpid and a neutral lîpid. In further embodiments, the liposomal components comprise at least one seiected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholestérol-P EG; and cholesterol-maleimide. In further embodiments, the liposomal components comprise at least one seiected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and HSPC. In addîtional embodiments, the liposome further comprises an oxidized phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that 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
110 carbon sn-2 residue bearing omega-aldehyde or omega-carboxyl groups. In some embodiments, the liposome comprises an OxPAPC selected from HOdiAPC, KOdiA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxyîsoprostane-containing phospholipid. In some embodiments, the liposome comprises an OxPAPC selected from l-palmitoyl-2-(5,6-epoxyisoprostane E2)sn-glycero-3-phosphocholine (5,6 PEIPC), l-palmitoyl-2-(epoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), l-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), 1 -palmitoyl-2-glutar-oyl-snglycero-3-phosphocholine (PGPC); l-palmitoyl-2-(9'oxo-nonanoyl)-sn-glycero -3-phosphocholine; l-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-myristoyI-sn-glyc-ero-3-phosphocholine; 1 -palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and l-palm-itoyl-2-acet-oyl-sn-glycero-3-phosphocholine. In some embodiments, the liposome comprises PGPC. In some embodiments, the OxPAPC within the liposome lipid bilayer is 0%-100% of total lipids, or any range therein between. In some embodiments, the liposome comprises a targeting moiety having a spécifie affmity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or an antigen binding fragment of an antibody. In some embodiments, the liposome contains 1 to 1000, 50 to 750,100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the liposome further comprises an immunostimulating agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabilizer. In some embodiments, the steric stabilizer is polyethylene glycol (Le., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weight (Mn) of 200 to
111
5000 Daltons. In additional embodîments, the liposome is anionic or neutral. In some embodîments, the liposome has a zêta potential that is less than or equal to zéro. In some embodîments, the liposome has a zêta potential that is -150 to 0, 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 therein between. In other embodîments, the liposome is catîonic. In some embodîments, the liposomal composition comprises a liposome that has a zêta potential that is more than zéro. In some embodîments, the liposome has a zêta potential that 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 therein between.
[0159] In some embodîments, the disclosure provides a pharmaceutical composition comprising calcium trans-norbixin (CTN) encapsulated by a liposome. The CTN can exist in linear and/or cyclic form (shown below).
In some embodîments, the 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, molécules of trans-norbixin. In some embodîments, the liposome contains 10 to 100,000, 100 to 10,000, or 1,000 to 5,000, molécules of transnorbixin, or any range therein between. In some embodîments, the transnorbixin/Iipîd ratio of the liposomal composition is Ig/mol and about 1000 g/mol, or any range therein between. In some embodîments, the transnorbixin/lipîd 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 therein between. In some embodîments, the
112 liposome comprises at least 0.1 % to 97% trans-norbixin. In some embodiments.
the liposome has a diameter of 20 nm to 500 nm, or 20 nm to 200 nm, or any range therein between. In some embodiments, the liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimide. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and HSPC. In addîtional embodiments, the liposome further comprises an oxidized phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that is an oxidized phospholipid containing fragmented oxygenated sn-2 residues, an oxidized phospholipid containing ftill length oxygenated sn-2 residues, and/or an oxidized phospholipid containing a frvecarbon sn-2 resîdue bearing omega-aldehyde or omega-carboxyl groups. In some embodiments, the liposome comprises an OxPAPC selected from HOdiAPC, KOdiA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, the liposome comprises an OxPAPC selected from l-palmitoyl-2-(5,6-epoxyisoprostane E2)sn-glycero-3-phosphocholine (5,6 PEIPC), 1 -palmitoyl-2-(epoxycyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC), I -palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC), 1 -palmitoyl-2-glutaroylsn-gIycero-3-phosphocholine (PGPC); l-palmitoyl-2-(9'oxo-nonanoyl)-snglycero-3-phosphocholine; l-palmitoyl-2-arachinodoyl-sn-glycero-3-phosphocholine; 1 -palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2hexadecyl-sn-glycero-3-phosphocholine; l-palmîtoyl-2-azelaoyl-sn-giyc-ero-3phosphocholine; and l-palmitoyl-2-acetoyl-sn-gIycero-3-phosphochol-ine. In
113 some embodiments, the liposome comprises PGPC. In some embodiments, the
OxPAPC within the liposome lipid bilayer is 0%-100% of total lipids, or any range therein between. In some embodiments, the liposome comprises a targeting moiety having a spécifie affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or an antigen binding fragment of an antibody. In some embodiments, the liposome contains 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the liposome further comprises an immunostimulating agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabilizer. In some embodiments, the steric stabilizer is polyethylene glycol (i.e., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons. In additional embodiments, the liposome is anionic or neutral. In some embodiments, the liposome has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, -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 therein between. In other embodiments, the liposome is cationic. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that is more than zéro. In some embodiments, the liposome has a zêta potential that 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 therein between.
114
[01601 In some embodiments, the disclosure provides a pharmaceutical composition comprising magnésium trans-norbixin (MTN) encapsulatedby a liposome. The MTN can exist in linear and/or cyclic form (shown below).
In some embodiments, the liposome contaîns 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, molécules of trans-norbixin. In some embodiments, the liposome contaîns 10 to 100,000, 100 to 10,000, or 1,000 to 5,000, molécules of transnorbixin, or any range therein between. In some embodiments, the transnorbixîn/lîpid ratio of the liposomal composition is 1 g/mol and about 1000 g/mol, or any range therein between. In some embodiments, the transnorbixin/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 therein between. In some embodiments, the liposome comprises at least 0.1 % to 97% trans-norbixin. In some embodiments, the liposome has a diameter of 20 nm to 500 nm, or 20 nm to 200 nm, or any range therein between. In some embodiments, the liposome has a diameter of 80 nm to 120 nm, or any range therein between. In some embodiments, the liposome is formed from liposomal components. In further embodiments, the liposomal components comprise at least one of an anionic lipid and a neutral lipid. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimide. In further embodiments, the liposomal components comprise at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholestérol; and HSPC. In additional embodiments, the liposome further comprises an oxidized
115 phospholipid such as an OxPAPC. In some embodiments, the liposome comprises an OxPAPC that 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 fivecarbon sn-2 residue bearing omega-aldehyde or omega-carboxyl groups. In some embodiments, the liposome comprises an OxPAPC selected from HOdîAPC, KOdiA-PC, HOOA-PC and KOOA-PC, or the OxPAPC is an epoxyisoprostane-containing phospholipid. In some embodiments, the liposome comprises an OxPAPC selected from l-palmitoyl-2-(5,6-epoxyisoprostane E2)sn-glycero-3-phosphocholine (5,6 PEIPC), l-palmitoyl-2-(epoxycyclopentenone)-sn-glycero-3-phosphoryl-choline (PECPC),l-palmîtoyl-2-(epoxyîsoprostane E2)-sn-glycero-4-phospho-choline (PEIPC), 1 -palmitoyl-2-glutaroyl-snglycero-3-phosphocholine (PGPC); 1 -palmitoyl-2-(9'oxo-nonanoyl)-sn-glycero-3-phosphocholine; l-palmitoyl-2-arachinodoyI-sn-glycero-3-phosphocholine; 1 -palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1 -palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and l-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine. In some embodiments, the liposome comprises PGPC. In some embodiments, the OxPAPC within the liposome lipid bilayer is 0%-100% of total lipids, or any range therein between. In some embodiments, the liposome comprises a targeting moiety having a spécifie affinity for a surface antigen on a target cell of interest. In some embodiments, the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety îs attached to one or both of the PEG and the exterior of the liposome by a covalent bond. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is an antibody or an antigen bindîng fragment of an antibody. In some embodiments, the liposome contaîns 1 to 1000, 50 to 750, 100 to 500, or 30 to 200 targeting moieties, or any range therein between. In some embodiments, the liposome contaîns less than 500,000
116 or less than 200,000 molécules of trans-norbixîn. In some embodiments, the liposome contains between 10 to 100,000 molécules of trans-norbixîn, or any range therein between. In some embodiments, the liposome further comprises an immunostimulating agent (such as 1,6-beta glucan). In some embodiments, the liposome comprises a steric stabilizer. In some embodiments, the steric stabilizer is polyethylene glycol (i.e., the liposome is pegylated). In some embodiments, the PEG has a number average molecular weîght (Mn) of 200 to 5000 Daltons. In additional embodiments, the liposome is anionic or neutral. In some embodiments, the liposome has a zêta potential that is less than or equal to zéro. In some embodiments, the liposome has a zêta potential that is -150 to 0, -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 therein between. In other embodiments, the liposome is cationic. In some embodiments, the liposomal composition comprises a liposome that has a zêta potential that is more than zéro. In some embodiments, the liposome has a zêta potential that 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 therein between.
Formulation and Administration
[0161] The provided compositions can be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may include one or more nanoparticle compositions. For example, a pharmaceutical composition may include one or more nanoparticle compositions including one or more different therapeutic and/or prophylactics. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingrédients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are
117 available, for example, in Remington’s The Science and Practice of Pharmacy,
21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and accessory ingrédients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingrédient may be incompatible with one or more components of a nanoparticle composition. An excipient or accessory ingrédient may be incompatible with a component of a nanoparticle composition if its combination with the component may resuit in any undesirable biologîcal effect or otherwise deleterious effect.
[0162] In some embodiments, one or more excipients or accessory ingrédients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a nanoparticle composition. For example, the one or more excipients or accessory ingrédients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a 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, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
[0163] Standard methods for making liposomes înclude, but are not limited to methods reported in Liposomes: A Practical Approach, V. P. Torchilin, Volkmar Weissig Oxford University Press, 2003 and are well known in the art.
[0164] In some embodiments, the disclosure provides a liposome composition and a physiologically (i.e., pharmaceutically) acceptable carrier. As used herein, the term carrier refers to a typically inert substance used as a diluent or vehicle for a drug such as a therapeutîc agent. The term also encompasses a typically
118 înert substance that imparts cohesive qualifies to the composition. Typically, the physiologically acceptable carriers are présent in liquid form. Examples of hquid carriers inciude physiological saline, phosphate buffer, normal buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.), and the like. Sînce physiologically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition, there are a wide variety of suitable formulations of pharmaceutical compositions provided herein (See, e.g., Remington's Pharmaceutical Sciences, 17ώ ed., 1989).
[0165] The provided compositions may be sterilized by conventîonal, known sterilization techniques or may be produced under stérile conditions. Aqueous solutions can be packaged for use or filtered under aseptie conditions and lyophilized, the lyophilized préparation being combîned with a stérile aqueous solution prier to administration. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. Sugars can also be included for stabilizing the compositions, such as a stabilizer for lyophilized liposome compositions. . In some embodiments, the pharmaceutical composition comprises a tonicity agent at a concentration of greater than 0.1%, or 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 therein between. In some embodiments, the pharmaceutical composition comprises a tonicity agent such as dextrose, mannitol, glycerin, potassium chloride, or sodium chloride. In further embodiments, the pharmaceutical composition comprises dextrose, mannitol, glycerin, potassium chloride, or sodium chloride at a concentration of greater than 0.1%, or a concentration of 0.3% to 2.5%, 0.5%
119 to 2.0%, 0.5% to 1.5%, 0.5% to 1.5%, 0.6% to 1.1%, or any range thereîn between,
[0166] Formulations suitable for parentéral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and nonaqueous, isotonie stérile injection solutions, which can contaîn antioxidants, buffers, bacteriostats, and solutés that render the formulation isotonie with the blood of the intended récipient, and aqueous and non-aqueous stérile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from stérile powders, granules, and tablets. In some embodiments, the provided liposomal compositions are administered, for example, by intravenous infusion, topically, intraperitoneally, intravesically, or intrathecally. In particular embodiments, the liposome compositions are parentally or intravenously administered. Preferably, the pharmaceutical liposomal compositions are administered parentally, i.e. intraarticularly, intravenously, subcutaneously, or intramuscularly. In other embodiments, the pharmaceutical préparation may be administered topically.
[0167] In some embodiments, the provided pharmaceutical compositions (e.g., liposomal compositions are presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
[0168] In some embodiments, the pharmaceutical préparations are administered in unit dosage form. In such form the préparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., a liposome composition. The unit dosage form can be a packaged préparation, the package containing discrète quantities of préparation. The composition can, if desired, also contain other compatible therapeutic agents (e.g., as described herein).
[0169] In some embodiments, the liposome compositions including a therapeutic and/or diagnostic agent utilized in the pharmaceutical compositions
120 provided herein can be administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily. A daily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the liposome composition being employed. For example, dosages can be empirically determined considering the type and stage of the disease, disorder or condition diagnosed in a partîcular patient. The dose administered to a patient, in the context of the provided pharmaceutical compositions (e.g., liposome compositions) should be sufficient to affect a bénéficiai therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a partîcular liposome composition in a partîcular patient. Détermination of the proper dosage for a partîcular situation is within the skill of the practitioner. Generally, treatment is initîated with smaller dosages which are less than the optimum dose of the liposome composition. Thereafter, the dosage is increased by small incréments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
Liposome Loading
[0170] The provided carotenoid compositions can be loaded into liposomes using active or passive loading modalîties.
[0171] In some embodîments, the disclosure provides a method of preparîng a liposomal composition comprising an ionizable crocetin (e.g., of [l]-[97]) the; method comprising:
(a) forming a mixture comprising: liposomal components in solution;
(b) homogenizing the mixture to form liposomes in the solution; and
121 (c) Processing the mixture to form liposomes containing the ionizable carotenoid.
In some embodiments, the processing step includes one or more steps of: thin film hydration, extrusion, în-line mixing, éthanol injection technique, freezingand-thawing technique, reverse-phase évaporation, dynamic high pressure microfluidization, microfluidic mixing, double émulsion, freeze-dried double émulsion, 3D printing, membrane contactor method, and stirring. In some embodiments, the processing step includes one or more steps of modifying the size of the liposomes by one or more of steps of extrusion, high-pressure microfluidization, and/or sonication.
[0172] In some embodiments, the disclosure provides an active loading method to generate a carotenoid sait inside a liposome formulation using a soluble acetate métal salts gradient (calcium acetate or magnésium acetate).
[0173] Multivalent counterions used in accordance with the présent disclosure can be encapsulated in liposomes according to techniques described herein or otherwise known in the art. This includes the passive encapsulation techniques described below or otherwise known in the art.
[0174] In some embodiments, the disclosure provides a method of preparing a pharmaceutical composition comprising:
(a) preparing a liposomal solution containing liposomes in a weak acid sait of a multivalent métal;
(b) adding an ionizable carotenoid to the liposomal solution; and (c) maîntaining the ionizable carotenoid in the liposomal solution for sufficient time to load the carotenoid into liposomes.
In some embodiments, the ionizable carotenoid is an ionizable carotenoid in any of compositions [l]-[28] (e.g., trans-crocetin and trans-norbixin). In some embodiments, the carotenoid is a carotenoid disclosed in any of FIG. 1A-FIG. 1D. In some embodiments, the weak acid is selected from acetic acid, gluconic acid, tartaric acid, glutamic acid, citric acid, formic acid, and glycinic acid. In
122 some embodiments, the weak acid sait of a multivalent métal is used at a concentration from 0 mM to 2000 mM, or 50 mM to 500 mM, or any range therein between. In some embodiments, the multivalent métal is selected from Ca2+, Mg2', Zn2+, Cu2+, Co2+, Fe2+ and Fe3+. In some embodiments, the weak acid is acetic acid and the multivalent métal is Ca2+ (i.e., the weak acid sait of the multivalent métal is calcium acetate). In some embodiments, the weak acid is acetic acid and the multivalent métal is Mg2^ (i.e., the weak acid sait of the multivalent métal is magnésium acetate). Pharmaceutical compositions prepared according to the provided methods are also encompassed by the dîsclosure. The liposomal solution is preferably a buffered solution. However, it is apprecîated that any suitable solvent may be use to préparé and use the provided compositions. A preferred liposome solution has a pH at about physiological pH and comprises a buffer which has a buffering range to include physiological pH. A non-limiting example of a suitable buffer for the liposome solution is HEPES (e.g., 5 mM HEPES buffered saline pH 6.5). Pharmaceutical compositions prepared according to the method are also encompassed by the dîsclosure.
The multivalent metals used in accordance with the provided methods can be encapsulated in liposomes according to conventional techniques known in the art. These methods include, for example, passive encapsulation techniques described herein or otherwise known in the art. Loading of an ionizable carotenoid such as trans-crocetin may be established by maintaining the ionizable carotenoid in the liposomal solution for a suitable amount of tîme at a suitable température. Depending on the composition of the liposome, and the température, pH, and Chemical nature of the ionizable carotenoid, loading of the ionizable carotenoid may occur over a time period of minutes or hours. In some embodiments, loading is carried out at températures of, for example, 0° C to 95° C, or 20° C to 75° C, or any range therein between, preferably from about 40°C to about 80°C, or any range therein between.
123
Τη some embodiments, the disclosure further provides the step of (d) removing unencapsulated ionizabie carotenoid from the liposome préparation prepared according to (c). In some embodiment, the removal is carried out by passing the liposome préparation through a gel filtration column equilibrated with a second aqueous buffered solution, centrifugation, or dialysis, or related techniques. After removal of unencapsulated ionizabie carotenoid, the extent of ionizabie carotenoid loading may be determined by measurement of ionizabie carotenoid and lipid levels according to conventional techniques. Lîpid and drug concentrations may be determined using any suitable method known in the art, such as scintillation counting, spectrophotometric assays, and high performance liquid chromatography. Replacement of the liposome préparation solution to remove unencapsulated carotenoid and counterion, such as sodium acetate, can be accomplished using any of varions techniques, known in the art, including but not limited to chromatography of the liposome préparation through an extensive gel filtration column equilibrated with a second aqueous buffered solution, by centrifugation, extensive or repeated dialysis, exchange of the liposomal préparation, treating the liposomal préparation with chelating agents or by related techniques. Pharmaceutical compositions prepared according to the provided methods are also encompassed by the disclosure.
[0175] In some embodiments, the disclosure provides a method of preparîng a pharmaceutical composition comprising:
(a) preparîng a liposomal solution containing liposomes in a weak acid sait of a multivalent métal;
(b) adding trans-crocetin to the liposomal solution; and (c) maîntaining the ionizabie carotenoid in the liposomal solution for sufficient time to load the carotenoid into liposomes.
In some embodiments, the weak acid is seiected from acetic acid, gluconic acid, tartane acid, glutamic acid, citric acid, formic acid, and glycinîc acid. In some embodiments, the weak acid sait of a multivalent métal is used at a concentration
124 from 0 mM to 2000 mM, or 50 mM to 500 mM, or any range therein between.
In some embodiments, the multivalent métal is selected from Ca2+, Mg , Zn +, Cu2+, Co2+, Fe2 λ and Fe3+. In some embodiments, the weak acid is acetic acid and the multivalent métal is Ca2+ (Le., the weak acid sait of the multivalent métal is calcium acetate). In some embodiments, the weak acid is acetic acid and the multivalent métal is Mg2* (i.e., the weak acid sait of the multivalent métal is magnésium acetate). Pharmaceutical compositions prepared according to the method are also encompassed by the disclosure. The liposomal solution is preferably a buffered solution. However, it is appreciated that any suitable solvent may be utilized to practice the provided compositions and methods. A preferred liposome solution has a pH at about physiological pH and comprises a buffer which has a buffering range to include physiological pH. Non-limiting example of suitable buffers for the liposome solution is 5 mM HEPES buffered saline pH 6.5. Pharmaceutical compositions prepared according to the method are also encompassed by the disclosure.
Loading of trans-crocetin may be established by maintaining the trans-crocetin in the liposomal solution for a suitable amount of time at a suitable température. Depending on the composition of the liposome, and the température, pH, and Chemical nature of trans-crocetin, loading of the trans-crocetin may occur over a time period of minutes or hours. In some embodiments, loading is carried out at températures of, for example, 0° C to 95° C, or 20° C to 75° C, or any range therein, preferably from about 40°C to about 80°C.
In some embodiments, the disclosure further provides the step of (d) removing unencapsulated trans-crocetin from the liposome préparation prepared according to (c). In some embodiment, the removal is carried out by passing the liposome préparation through a gel filtration column equilibrated with a second aqueous buffered solution, or by centrifugation, dialysis, or related techniques. After removal of unencapsulated trans-crocetin, the extent of trans-crocetin loading may be determined by measurement of trans-crocetin and lipid levels according
125 to conventional techniques. Lipid and drug concentrations may be determined by employing any suîtable method known in the art, such as scintillation countîng, spectrophotometric assays, and high performance liquid chromatography. Replacement of the liposome préparation solution to remove unencapsulated trans-crocetîn and counterion, such as sodium acetate, can be accomplished using any of varions techniques, known in the art, including but not limited to chromatography of the liposome préparation through an extensive gel filtration column equilibrated with a second aqueous buffered solution, centrifugation, extensive or repeated dialysis, exchange of the liposomal préparation, treating the liposomal préparation with chelating agents or by related techniques. Phannaceutical compositions prepared according to the provided methods are also encompassed by the disclosure.
[0176] Phannaceutical compositions comprising an ionizable carotenoid sait prepared according to the provided methods are also encompassed by the disclosure. In some embodiments, the ionizable carotenoid is an ionizable carotenoid in any of compositions [l]-[28] (e.g., trans-crocetin and transnorbixin). In some embodiments, the ionizable carotenoid is a carotenoid disclosed in any of FIGS. 1A-1D. In some embodiments, the disclosure provides a pharmaceutical composition comprising a liposome encapsulating an ionizable carotenoid, wherein the ionizable carotenoid is loaded into liposomes in the presence of intra-liposomal multivalent counterions (e.g., Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+, and Fe3+). In some embodiments, the multivalent counterions comprise Ca2+. In some embodiments, the multivalent counterions comprise Mg2+. In some embodiments, the multivalent counterions comprise Fe3+.
[0177] In some embodiments, the disclosure provides a pharmaceutical composition comprising a liposome encapsulating a trans-crocetin sait, wherein the trans-crocetin is loaded into liposomes in the presence of intra-liposomal multivalent counterions (e.g., Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+, and Fe3+).
126
In some embodiments, the multivalent counterions comprise Ca2+. In some embodiments, the multivalent counterions comprise Mg . In some embodiments, the multivalent counterions comprise Fe3+.
Methods of Treatment and Use
[0178] The provided phannaceutical compositions such as liposomal compositions, hâve uses that provide advances over prior treatments of diseases and disorders that include without limitation, infection and infectious diseases such as HIV/AIDS: human immunodeficiency virus-1 (HIV-1), tuberculosis, malaria and its complications such as cérébral malaria, severe anémia, acidosis, acute kidney failure and ARDS, sepsis, inflammation (e.g., chronic inflammatory diseases), ischemia, (încluding an ischémie condition such as ischémie stroke, coronary artery disease, peripheral vascular disease, cérébral vascular disease, ischemia associated rénal pathologies, and ischemia associated with wounds); shock (e.g., hémorrhagie shock), stroke, cardiovascular disease, rénal pathologies, wound healing, metabolic disease, hyperproliferative diseases such as cancer, and disorders of the immune System, cardiovascular System, digestive, nervous, respiratory, and endocrine System. In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition in a subject needing such treatment or prévention, the method comprising administering a phannaceutical composition provided herein (e.g., the phannaceutical composition of any of [l]-[79]) to the subject. Use of a phannaceutical composition provided herein (e.g., the phannaceutical composition of any of [ 1 ]-(79]), in the manufacture of a médicament for the treatment of a disease, disorder or condition in a subject is also provided herein. As are, phannaceutical compositions of any of [l]-[79] for use in a medical médicament.
[0179] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with endotoxemia in a subject needing such treatment or prévention, the method comprising
127 administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject.
[0180] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with sepsis in a subject needing such treatment or prévention, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the subject has a low grade endotoxemic disease.
[0181] In some embodiments, the disclosure provides a method for treating or preventing a subject at risk of developing sepsis, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the subject is immunocompromîsed or immunosuppressed. In some embodiments, the subject is critically ill. In some embodiments, the subject elderly or néonatal. In some embodiments, the subject has febrile neutropenia. In some embodiments, the subject has an infection.
[0182] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with bum injury in a subject that is a bum victim, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject.
In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with infection in a subject needing such treatment or prévention, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the infection is a bacterial infection (e.g., a P. aeruginosa infection, an S. aureus infection (e.g., MRSA), mycobacterium tuberculosis infection, an enterococcal infection (e.g., VRE), or a condition associated therewith. In some embodiments,
128 the infection is a fungal infection (e.g., a candidiasis infection such as invasive candidiasîs) or a condition associated therewith. In some embodiments, the infection is a parasitic infection (e.g., Schistosomiasis, and human African trypanosomiasis), or a condition associated therewith. In some embodiments, the 5 infection is malaria or a condition associated therewith, such as cérébral malaria, severe anémia, acidosîs, acute kidney failure and ARDS, In some embodiments, the infection is a viral infection (e.g., Ebola, Dengue and Marburg) or a condition associated therewith, such as influenza, measles, and a viral hémorrhagie fever.
[0183] In some embodiments, the disclosure provides a method for treating or 10 preventing a disease, disorder or condition associated with ischemia or hypoxia in a subject needing such treatment or prévention, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the disease or condition associated with ischemia or hypoxia is 15 associated with surgery or traumatic injury. In some embodiments, the disease or condition is ischemic-reperfiision injury, transient cérébral ischemia, cérébral ischemia-reperfusion, ischémie stroke, hémorrhagie stroke, traumatic brain injury, migraine (e.g., a chronic migraine or severe migraine disorder), gastrointestinal ischemia, kidney disease, pulmonary embolism, acute 20 respiratory failure, néonatal respiratory distress syndrome, obstetric emergencies to reduce périnatal comorbidity (such as, pre/eclampsia and conditions that lead to cérébral palsy), myocardîal infarction, acute limb or mesenteric ischemia, cardiac cirrhosis, chronic peripheral vascular disease, congestive heart failure, atherosclerotic stenosis, anémia, thrombosis, embolism, macular degeneration, 25 a neurodegenerative disease (e.g., Alzheimer’s disease, Parkinson’s disease, and
Amyotrophie Latéral Sclerosis (ALS)), sleep apnea, and surgery or traumatic injury. In some embodiments, the disease or condition associated with ischemia or hypoxia is myocardîal infarction, or congestive heart failure with or without cardiac cirrhosis. In some embodiments, the disease or condition is pulmonary
129 embolism, acute respiratory faîlure, chronîc peripheral vascular disease.
atherosclerotic stenosis, anémia, thrombosis, or embolism. In some embodiments, the disease or condition associated with îschemia or hypoxia is macular degeneration or an oncologie condition associated with hypoxia. In some embodiments, the disease or condition is kidney disease. In some embodiments, the disease or condition is lipopolysaccharide médication or toxin induced acute kidney injury (AKI) or end stage kidney disease.
[0184] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with shock in a subject needing such treatment or prévention, the method comprising adminîstering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the disease or condition is associated with cardiogenic shock. In some embodiments, the disease or condition is associated with, hypovolémie 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.
[0185] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with nitric oxide deficiency in a subject needing such treatment or prévention, the method comprising adminîstering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the disease or disorder is sickle cell disease, paroxysmal noctumal hemoglobinuria (PNH), a hemolytic anémia, a thalassemia, another red blood cell disorder, or a condition associated therewith. In some embodiments, the disease or disorder is a purpura such as thrombotic thrombocytic purpura (TTP), hemolytic urémie syndrome (HUS), idiopathic thrombocytopenia (ITP), or and another platelet disorder, or a condition associated therewith. In some
130 embodiment, the disease or disorder is a coagulation abnormality such as disseminated intravascular coagulopathy (DIC), purpura fulmînans, hepann induced thrombocytopenia (HIT), hyperleukocytosis, hyper viscosity syndrome, or a condition associated therewîth.
[0186] In some embodîments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with inflammation in a subject needing such treatment or prévention, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodîments, the disease or condition associated with inflammation is lowgrade inflammation. In some embodîments, the disease or condition associated with inflammation is systemic inflammation. In some embodîments, the disease or condition associated with inflammation is acute inflammation or a chronic inflammatory disease.
[0187] In some embodîments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with a cardiovascular disease or condition in a subject needing such treatment or prévention, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodîments, cardiovascular disease or condition is coronary artery disease. In some embodîments the cardiovascular disease or condition is myocardial infarction, sudden cardiac death, cardiorespiratory 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 (PCAS), chronic venous insufficiency, heart disease, congestive heart failure, or a chronic skîn ulcer.
[0188] In some embodîments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with a liver disease or
131 condition in a subject needing such treatment or prévention, the method comprisîng administering a pharmaceutical composition provided herem (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the liver disease or condition is cirrhosis. In some embodiments, the liver disease or condition is nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH). In some embodiments, the liver disease or condition is alcoholic liver disease. In some embodiments, the liver disease or condition is acute liver injury.
[0189] In some embodiments, the disclosure provides a method for treating or preventing a disease, dîsorder or condition associated with a lung disease or condition in a subject needing such treatment or prévention, the method comprisîng administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the lung disease or condition is acute respiratory distress syndrome (ARDS). In some embodiments, the lung disease or condition is chronic obstructive pulmonary disease. In some embodiments, the lung disease or condition is pulmonary fibrosis. In some embodiments, the lung disease or condition is pulmonary hemorrhage. In some embodiments, the lung disease or condition is asthma. In some embodiments, the lung disease or condition is lung injury. In some embodiments, the lung disease or condition is lung cancer. In some embodiments, the condition is cystic fibrosis.
[0190] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with a kidney disease or condition in a subject needing such treatment or prévention, the method comprisîng administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the kidney disease or condition is lipopolysaccharide-induced acute kidney injury (AK1). In some embodiments, the kidney disease or condition is chronic rénal failure with or without end stage kidney disease.
132
[0191] In some embodiments, the dîsclosure provides a method for treating or preventing a dîsease, disorder or condition associated with a vascular disease m a subject needing such treatment or prévention, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [ l]-[79]) to the subject. In some embodiments, the disease or condition is coronary artery disease. In some embodiments, the disease or condition is hypertension. In some embodiments, the disease or condition is atherosclerosîs. In some embodiments, the disease or condition is post-cardiac arrest syndrome (PCAS). In some embodiments, the disease or condition is occlusive arterial disease, peripheral vascular disease, chronic venous insufficiency, chrome skin ulcers, or Raynaud's disease. In some embodiments, the disease, disorder or condition associated with a vascular disease is heart disease. In further embodiments, the disease, disorder or condition is congestive heart failure. In some embodiments, the disease, disorder or condition associated with vascular disease is ischémie bowel disease.
[0192] In some embodiments, the dîsclosure provides a method for treating or preventing a disease, disorder or condition associated with a heart attack or stroke in a subject needing such treatment or prévention and/or at risk of having a heart attack or stroke, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of[1][79]) to the subject. In some embodiments, the disease, disorder or condition is ischémie stroke. In some embodiments, the disease, disorder or condition is hémorrhagie stroke.
[0193] In some embodiments, the dîsclosure provides a method for treating or preventing a disease, disorder or condition associated with nervous System in a subject needing such treatment or prévention, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the disease or condition is pain (e.g., chronic pain). In some
133 embodiments, the disease or condition is a neurodegeneratîve disease (e.g.,
Alzheimer’s disease or Parkinson’s disease). In some embodiments, the disease, disorder or condition associated with nervous System is neural injury.
[0194] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with înflammatory bowel disease in a subject needing such treatment or prévention, the method comprising administering a pharmaceuticai composition provided herein (e.g., the pharmaceuticai composition of any of [l]-[79]) to the subject. In some embodiments, the disease, disorder or condition is Crohn’s disease. In some embodiments, the disease, disorder or condition is ulcerative colitis.
[0195] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with type 2 diabètes or prédisposition for diabètes in a subject needing such treatment or prévention, the method comprising administering a pharmaceuticai composition provided herein (e.g., the pharmaceuticai composition of any of [l]-[79]) to the subject. In some embodiments, the disease, disorder or condition is metabolic disease. In some embodiments, the disease, disorder or condition is insulîn résistance. In some embodiments, the disease, disorder or condition is a diabetic vascular disease (e.g., 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 gangrené.
[0196] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with an autoimmune disorder in a subject needing such treatment or prévention, the method comprising administering a pharmaceuticai composition provided herein (e.g., the pharmaceuticai composition of any of [l]-[79]) to the subject. In some embodiments, the autoimmune disorder is psoriasis. In some embodiments, the
134 autoimmune disorder is cystic fibrosis. In some embodiments, the autoimmune disorder is rheumatoid arthritis.
[0197] In some embodiments, the disclosure provides a method for treating or preventing a disease, disorder or condition associated with sclerosis in a subject 5 needing such treatment or prévention, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject. In some embodiments, the disease, disorder or condition associated with sclerosis is systemic sclerosis.
[0198] In some embodiments, the disclosure provides a method for treating 10 endotoxemia in a subject needing such treatment, the method comprising administering a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [ 1 ]-[79]) to the subject. In some embodiments, the endotoxemia is associated with a condition such as periodontal disease (e.g., periodontitis or inflammation of the gums), chrome 15 alcoholism, chronic smoking, transplantation, or néonatal neçrotizing enterocolitis, or néonatal ear infection.
[0199] In some embodiments, the disclosure provides a method of reducîng systemic levels of LPS, endotoxin and/or another trigger of systemic inflammation in a subject in need thereof, the method comprising administering 20 a pharmaceutical composition provided herein (e.g., the pharmaceutical composition of any of [l]-[79]) to the subject.
Combination Therapy
[0200] The compositions provided herein can be administered alone or in combination therapy with one or more additional therapeutic agents. In some 25 embodiments, the composition is administered in combination therapy with another therapeutic agent. Combinations may be administered either concomitantly, e.g., combîned in the same delivery vehicle (e.g., liposome), as an admixture, separately but simultaneously or concurrently; or sequentially.
135
This includes présentations in which the combined therapeutic agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but sîmultaneously, e.g., as through separate intravenous lines into the same individual. Administration in combination further includes the separate administration of one of the therapeutic agents given first, followed by the second. Methods of treatment using the combination therapy are also provided.
[0201] In additional embodiments, a composition provided herein is administered in combination with another therapeutic agent. In some embodiments, a composition of any of [l]-[28] is administered in combination with another therapeutic agent. In some embodiments, a composition comprising a sait of a carotenoid provided in any of FIGs. 1A-1D herein, is administered in combination therapy with another therapeutic agent. In some embodiments, a composition comprising a multivalent sait (e.g., a divalent sait or a trivalent sait) of a carotenoid provided in any of FIGs. 1A-1D herein, is administered in combination therapy with another therapeutic agent. In partîcular embodiments, a composition comprising a multivalent sait of trans-crocetin (e.g., CTC or MTC) is administered in combination therapy with another therapeutic agent. In other partîcular embodiments, a composition comprising a multivalent sait of trans-norbixin (e.g., CTN or MTN) is administered in combination therapy with another therapeutic agent.
[0202] In some embodiments, a pharmaceutical composition comprising a sait of one or more ionizabie carotenoids is administered in combination therapy with a carotenoid comprising at least one polar group or monocyclic group. In some embodiments, the sait of the ionizabie carotenoid is a multivalent sait (e.g., sait containing divalent, trivalent or tetravalent counterion). In some embodiments the ionizabie carotenoid is a carotenoid of any of [l]-[28] and/or FIGs. 1A-1D. In one embodiment, the carotenoid comprising at least one polar group or monocyclic group polar group is symmetric. In another embodiment,
136 a divalent ionizable carotenoid sait composition is administered in combination therapy with at least one carotenoid selected from: zeanthin, astaxanthin, lutein, and xanthophyll. In another embodiment, the divalent ionizable carotenoid sait composition is administered in combination therapy with astaxanthin. In another embodiment, the carotenoid comprising at least one polar group or monocyclic group polar group is asymmetric. In another embodiment, a divalent ionizable carotenoid sait composition disclosed herein is administered in combination abscisic acid (ABA).
[0203] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is administered in combination therapy with a standard of care treatment for the disease, disorder, or condition to be treated. In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS, 1A-1D. In particular embodiments, the ionizable carotenoid is trans-crocetin (e.g., CTC and MTC), In other particular embodiments, the ionizable carotenoid is transnorbixîn (e.g., CTN and MTN).
[0204] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is administered in combination therapy with an antimicrobial agent. In some embodiments, the antimicrobial agent is an anti-bacterial agent. In some embodiments, the antibacterial agent is selected from, but not limited to, ertapenem, piperacillin-tazobactam, cefepime, aztreonam, metronidazole, meropenem, ceftriaxone, ciprofloxacin, vancomycin, linezolid, tobramycin, levofloxacin, azithromycin, cefazolin, and ampicillîn. In some embodiments, the antibacterial agent is selected from, but not limited to, ceftriaxone, levofloxacin, ciprofloxacin, cefazolin, piperacillin-tazobactam, meropenem, metronidazole, vancomycin, and ampicillîn. In other embodiments, the antimicrobial agent is an anti-fungal agent. In further embodiments, the antifungal agent is caspofungin or another antifungal drug. In other embodiments,
137 the antimicrobial agent is an anti-malarial agent. In further embodiments, the anti-malarial agent is selected from, but not limited to, artemisinin and its analogs, chloroquin and its analogs, atovaquone, a quinine dérivative, proguanil or another anti-malarial drug. In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS. 1A-1D. In particular embodiments, the ionizable carotenoid is transcrocetin (e.g., CTC and MTC). In other particular embodiments, the ionizable carotenoid is trans-norbixin (e.g., CTN and MTN).
[0205] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is administered in combination therapy with activated protein C (e.g., rhAPC), or drotrecogin alfa (actîvated) (DAA). In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS. 1A-1D. In particular embodiments, the ionizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other particular embodiments, the ionizable carotenoid is trans-norbixin (e.g., CTN and MTN).
[0206] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is administered in combination therapy with a corticosteroid (e.g., a glucocorticoid or mineralocorticoid such as fludrocortisonel). In some embodiments, the corticosteroid is a glucocorticoid. In further embodiments, the glucocorticoid is selected from cortisone, ethamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone and méthylprednisolone. In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS. 1A-1D. In particular embodiments, the ionizable carotenoid is trans20783
138 crocetin (e.g., CTC and MTC). In other particular embodiments, the ionizable carotenoid is trans-norbixin (e.g., CTN and MTN).
[0207] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is administered in combination therapy with intravenous administration of a vitamin. In some embodiments, the vitamin is vitamin C (ascorbic acid). In some embodiments, the vitamin is vitamin A. In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, tri valent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS. 1A-1D. In particular embodiments, the ionizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other particular embodiments, the ionizable carotenoid is trans-norbixin (e.g., CTN and MTN).
[0208] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is administered in combination therapy with a glucocorticoid and vitamin C (e.g., intravenous vitamin C administration). In some embodiments, the glucocorticoid is selected from cortisone, ethamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone and méthylprednisolone. In further embodiments, the glucocorticoid is hydrocortisone. In additional embodiments, at least one ionizable carotenoid composition provided herein (e.g., a divalent sait composition comprising an ionizable carotenoid disclosed in FIG. IA, FIG. IB, FIG. IC, and/or FIG. ID) is administered in combination therapy with a glucocorticoid, vitamin C, and thiamine. In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS. 1A-1D. In particular embodiments, the ionizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other particular embodiments, the ionizable carotenoid is transnorbixin (e.g., CTN and MTN).
139
In some embodiments, a pharmaceutical composition comprising an îonizable carotenoid sait provided herein is administered in combination therapy with a vasopressor agent. In some embodiments, the vasopressor therapeutic agent is norepinephrine or similar drugs, or angiotensin II (e.g.r GIAPREZA™). In some embodiments, the vasopressor therapeutic agent is epinephrine, phenylnephrîne, dopamine, or vasopressîn. In some embodiments, the vasopressor therapeutic agent îs ephedrine, milrinone, isoproterenol, dobutamine, isoproterenol, or dopamine.
[0209] In some embodiments, a pharmaceutical composition comprising an îonizable carotenoid sait provided herein is administered in combination therapy with a thrombolytic therapeutic agent. In some embodiments, the thrombolytic therapeutic agent tissue plasminogen activator (tPA), In some embodiments, the sait ofthe îonizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the îonizable carotenoid is a carotenoid of any of [ 1 ]-[28] and/or FIGS. 1A-1D. In particular embodiments, the îonizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other particular embodiments, the îonizable carotenoid is trans-norbixin (e.g., CTN and MTN).
[0210] In some embodiments, a pharmaceutical composition comprising an îonizable carotenoid sait provided herein is administered in combination therapy with a therapeutic agent. In some embodiments, a pharmaceutical composition of any of [ 1 ]-[28] îs administered in combination with a therapeutic agent. In some embodiments, a pharmaceutical composition comprising a multivalent sait of a carotenoid provided in any of FIGs. 1A-1D herein, is administered in combination therapy with a therapeutic agent. In some embodiments, the sait of the îonizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the îonizable carotenoid is a carotenoid of any of [1 ]-[28] and/or FIGS. 1A-1D. In particular embodiments, the îonizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other particular embodiments, the îonizable carotenoid îs trans-norbixin (e.g., CTN and MTN).
140
[0211] In some embodiments, a phannaceutical composition comprising an ionizable carotenoid sait 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 inhïbitor (statin). In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS. 1A-1D. In particular embodiments, the ionizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other particular embodiments, the ionizable carotenoid is trans10 norbîxin (e.g., CTN and MTN).
[0212] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is administered in combination therapy with an anti-inflammatory therapeutic agent. In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or 15 tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS. 1A-1D. In particular embodiments, the ionizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other particular embodiments, the ionizable carotenoid is trans-norbixin (e.g., CTN and MTN).
[0213] In some embodiments, a pharmaceutical composition comprising an 20 ionizable carotenoid sait provided herein is administered in combination therapy with oxygen and/or intravenous fluids to maintain/încrease blood oxygen levels and/or blood pressure or hyperbaric therapy. In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of 25 any of [1 ]-[28] and/or FIGS. 1A-1D. In particular embodiments, the ionizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other particular embodiments, the ionizable carotenoid is trans-norbixin (e.g., CTN and MTN).
[0214] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is administered in combination therapy
141 with a chemotherapeutîc agent (e.g., to enhance the effect of chemotherapy on cancer cells and mitigate the effects of chemotherapy-înduced myelosuppression and anémia). In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS. 1A-1D. In partîcular embodiments, the ionizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other partîcular embodiments, the ionizable carotenoid is transnorbixîn (e.g., CTN and MTN).
[0215] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is admînistered in combination therapy with immunotherapy. In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [l]-[28] and/or FIGS. 1A-1D. In partîcular embodiments, the ionizable carotenoid is transcrocetin (e.g., CTC and MTC). In other partîcular embodiments, the ionizable carotenoid is trans-norbixin (e.g., CTN and MTN).
[0216] In some embodiments, a pharmaceutical composition comprising an ionizable carotenoid sait provided herein is admînistered in combination therapy with radiotherapy. In some embodiments, the sait of the ionizable carotenoid is a multivalent sait (e.g., divalent, trivalent or tetravalent). In some embodiments the ionizable carotenoid is a carotenoid of any of [1 ]-[28] and/or FIGS. 1A-1D. In partîcular embodiments, the ionizable carotenoid is trans-crocetin (e.g., CTC and MTC). In other partîcular embodiments, the ionizable carotenoid is transnorbixin (e.g., CTN and MTN).
Kits for Administration of Active Agents
[0217] In another embodiments, the disclosure provides a kit for administering a provided ionizable carotenoid composition to a subject for treating a disease, disorder, or condition. In some embodiments, the disclosure provides a kit for
142 delivering a therapeutic agent to a subject, the kit comprising: (a) a first composition comprising a disclosed ionizable carotenoid composition (e.g., a liposome comprising a multivalent trans-crocetin sait); and a (b) second composition containing for example, reagents, buffers, excipients, or another therapeutic agent that is stored separately prior to administration to the subject. Such kits typically include two or more components necessary for treating a disease State, such as hypoxia or inflammation related condition. In some embodiments, the kits include for example, a provided lipid compositions, reagents, buffers, containers and/or equipment. The liposome compositions and formulations can be in lyophilized form and then reconstituted prior to administration. In some embodiments, the kits include a packaging assembly that include one or more components used for treating the disease State of a patient. For example, a packaging assembly may include separate containers that house the therapeutic liposomes and other excipients or therapeutic agents that can be mixed with the 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- Production of Calcium Trans-crocetin Liposomes
[0218] Two different variants of trans-crocetin were used to produce transcrocetin liposomes, namely: trans-crocetin free acid (TC) and its sodium sait, sodium trans-crocetin (STC). Trans-crocetin was encapsulated in liposomes by the followîng procedures.
Multiple Bilayer (Multilamellaf Vesicle (MLV) Production:
[0219] First, the lipid components of the liposome lipid membrane were weighed out and combined as a concentrated solution in éthanol at a température of around 65°C. In one préparation, the lipids used were hydrogenated soy
143 phosphatidylcholine, cholestérol, and DSPE-PEG-2000 (1,2-distearoyl-snglycero-3-phosphoethan-olamine-N-[methoxy(polyethylene glycol)-2000]). The molar ratio of HSPC: cholestérol: PEG-DSPE was approximately 3:2:0.15. In another préparation, the lipids used were HSPC, cholestérol, PEG-DSPE2000, and l-palmitoyl-2-glutaryl-xn-glycero-3-phosphocholine (PGPC). The molar ratio of HSPC: cholestérol: PEG-DSPE:PGPC was approximately 2.7:2:0.15:0.3. Next, calcium acetate was dissolved in an aqueous buffer at a concentration of 125 mM, or 250 mM, with a pH of 7.0. The calcium acetate solution was heated up to 65°C.
[0220] The ethanolîc lipid solution was added into the calcium acetate solution using a pipet. During this step the solution was well stirred using a magnetic stirrer. The mixing was performed at an elevated température (63°C-72°C) to ensure that the lipids were in a liquid crystalline state (as opposed to the gel State that they would attain at températures below the lipid transition température (Tm = 5 1°C-54°C)). As a resuit, the lipids were hydrated and formed multiple bilayer (multilamellar) vesicles (MLVs) containing calcium acetate in the interior space. Downsizing of MLVs Using Filter Extrusion:
[0221] The MLVs were fragmented into unilamellar (single bilayer) vesicles of the desired size by high-pressure extrusion using two passes through stacked (track-etched 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 température was maintained above the Tm to ensure plasticity of the lipid membranes. As a result of the extrusion, large and heterogeneous in size and lamellarity MLVs were tumed into small, homogenous (100-120 nm) unilamellar vesicles (ULVs) that sequestered calcium acetate in their interior space. A Malvem Zetasizer Nano ZS instrument (Southborough, MA) with back scattering detector (90°) was used for measuring the hydrodynamic size (diameter) of the vesicles at 25 °C in a plastic
144 micro cuvette. The samples were diluted 50-fold in formulation matrix before analysis.
[0222] After ULVs containing calcium acetate had been produced, the extraliposomal calcium acetate was removed using SEC (size exclusion chromatography, with PD-10 columns) or TFF (tangential flow diafiltration). Tonicity reagent was added to the liposomes to balance the osmolality (final concentration: 5% dextrose for 125 mM calcium acetate liposomes and 10% dextrose in for 250 mM calcium acetate liposomes). Once the calcium acetate gradient was generated, the trans-crocetin loading procedure is preferably performed within 24 hours. The lipid content of the prepared liposome solution was determined by phosphate assay.
[0223] 1 mg/mL trans-crocetin solution was prepared in 10% dextrose (for 250 mM calcium acetate liposomes) and pH was adjusted to 8. The trans-crocetin solution was mixed with calcium acetate liposome solution at different Drug/Lipid ratios (100 g/mM, 80 g/mM, 60 g/mM or 40 g/mM). The mixture was then thoroughly stirred and heated to 65°C for 30 minutes, followed by quick cool down to room température using an ice water bath. This step can be replaced by stirring the mixture at room température ovemight.
[0224] The movement of trans-crocetin molécule (charge-free, neutral form) across the liposome lipid bîlayer was driven by the gradient generated with calcium acetate (in other words, acetic acid diffused out, trans-crocetin diffused în). Trans-crocetin was then trapped inside of the liposomes by ionizing and then forming a precipitate with calcium (as a calcium sait form (calcium transcrocetin, CTC)).
Purification of Liposomes:
[0225] The extra-liposomal trans-crocetin was removed using SEC (PD-10 columns) or TFF. In this example, the buffer used in SEC was HBS (HEPES
145 buffered saline, pH 6.5). Upon completion of purification, filter sterilîzation was perfbrmed using a 0.22 micron filter. A Malvem Zetasizer Nano ZS instrument (Southborough, MA) with back scattering detector (90°) was used for measuring the hydrodynamic size (diameter) of the vesicles at 25°C in a plastic 5 micro cuvette. The samples were diluted before analysis.
Table 1. Physical characteristics of représentative CTC loaded nanoparticles
Starting concentration Encapsulation efficiency Final concentration Drug/Lipid Ratio Diameter PDI Zêta potential
CTC LPs 1 mg/ml trans-crocetin disodium 96.9% 0.24 mg/ml 78.6 g/mM lipids 105.7nm 0.056 -2.88 mV
CTC Lps 0 75 mg/ml trans-crocetin disodium 98.32% 3.92 mg/ml 68.23 g/mM lipids 103.8 nm 0.041 -2.71 mV
CTC Lps 0.75 mg/mL trans-crocetin disodium 99.47% 3.90mg/mL 66.23 g/mM lipids 100.8 nm 0.031 - 3.67 mV
CTC Lps 0.75 mg/ml trans-crocetin disodium 92.59% 2.49mg/mL 34.74 g/mM lipids 101.9nm 0.038 -3.83 mV
PGPC CTC Lp2 0.75 mg/ml trans-crocetin disodium 98.30% 5.34 mg/mL 85.74 g/mM lipids 95.9 nm 0 043 - 3.66 mV
Example 2- Préparation of Calcium Acetate Liposomes with Nanoassemblr®
[0226] Calcium acetate loaded liposomes were prepared by the following 10 procedure. First, the lipid components of the liposome lipid membrane were weighed out and combined as a concentrated solution in éthanol at a température of around 65°C. In one example, the lipids used were hydrogenated soy phosphatidylcholine, cholestérol, and DSPE-PEG-2000 (1,2-distearoyl-snglycero-3-phosphoethanolamîne-N-[methoxy-(polyethylene glycol)-2000]).
[0227] The molar ratio of HSPC: cholestérol: PEG-DSPE was approximately
3:2:0.15. In another example, the lipids used were HSPC, cholestérol, PEGDSPE-2000, and 1 -palmitoyl-2-glutaryl-5,n-glycero-3-phosphocholîne (PGPC). The molar ratio of HSPC: cholestérol :PEG-DSPE ;PGPC was approximately 2.7:2:0.15:0.3.
146
[0228] Next, calcium acetate was dissolved in an aqueous buffer at a concentration of 125 or 250 mM, with a pH of 7.0. The calcium acetate solution was heated to 65°C. The ethanolîc lipid solution and the calcium acetate solution were separately transferred to syringes. Two solutions were injected into microfluidîc channel and mixed while flowing through it with Précision NanoSystems’ NanoAssemblr® device. The mixing was performed at an elevated température (63°C-72°C) to ensure that the lipids were in the liquid crystalline State (as opposedto the gel state that they would attain at températures below the lipid transition température (Tm = 51°C-54°C)). The size of liposome can be controlled by ratio between lipid solution and aqueous solution, as well as the mixing flow rate.
Example 3- MTC Liposome Génération and Characterization
Production of Trans-crocetin Liposomes with Magnésium Acetate Gradient:
[0229] To produce magnésium trans-crocetin liposomes, two different variants of the molécule can be used namely: trans-crocetin free acid (TC) and its sodium sait, sodium trans-crocetinate (STC).
[0230] Liposome with magnésium acetate is prepared by the following procedure. First, the lipid components of the liposome membrane were weighed out and combined as a concentrated solution in éthanol at a température of around 65°C. In one example, the lipids used were hydrogenated soy phosphatidylcholine, cholestérol, and DSPE-PEG-2000 (1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]). The molar ratio of HSPC: cholestérol: PEG-DSPE was approxîmately 3:2:0.15. In another example, the lipids used were HSPC, cholestérol, PEG-DSPE-2000, and l-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC). The molar ratio of HSPC: Cholestérol: PEG-DSPE:PGPC was approxîmately 2.7:2:0.15:0.3. Next, magnésium acetate was dissolved in an aqueous buffer at a concentration of 125 or 250 mM with a pH of 7.0. The magnésium acetate solution was heated
147 up to 65°C. The ethanolic lipid solution was added into the magnésium acetate solution using a pipette. During this step the solution was well stirred using a magnetic stirrer. The mixing was performed at an elevated température (63°C 72°C) to ensure that the lipids were in a liquid crystalline State (as opposed to the gel State that they attain at températures below the lipid transition température Tm = 5 rC-54°C)). As a resuit, the lipids were hydrated and form muitilamellar vesicles (MLVs) containing magnésium acetate in their interior space (internai solution).
Downsizing of MLVs Usine Filter Extrusion:
[0231] The MLVs are fragmented into unilamellar (single bilayer) vesicles of the desired size by high-pressure extrusion using two passes through stacked (track-etched polycarbonate) membranes. The stacked membranes bave two layers with a pore size of 200nm and six layers with a pore size of 100 nm. During extrusion, the température was maintained above the Tm. As a resuit of the extrusion, large and heterogeneous in size and lamellarity MLVs were tumed into small, homogenous (100-120 nm) unilamellar vesicles (ULVs) that sequestered the calcium acetate in their interior space. A Malvem Zetasizer Nano ZS instrument (Southborough, MA) with back scattering détecter (90°) was used for measuring the hydrodynamic size (diameter) of the vesicles at 25°C in a plastic micro cuvette. The samples were diluted 50-fold in formulation matrix before analysis.
Gradient Génération:
[0232] After ULVs containing magnésium acetate were produced, the extraliposomal magnésium acetate was removed using SEC (size exclusion chromatography, with PD-10 columns) or TFF (tangential flow diafiltration). Tonicity reagent solutions (such as 50% dextrose) were added to the liposomesto balance the osmolality (final concentration: 5% dextrose for 125 mM
148 magnésium acetate liposomes and 10% dextrose for 250 mM magnésium acetate liposomes). The lipid content of the prepared liposome solution was determined by phosphate assay.
Trans-crocetin Loading into Magnésium Acetate Liposomes:
[0233] 1 mg/mL trans-crocetin or trans-crocetin sodium solution was prepared in 10% dextrose (for 250 mM magnésium acetate liposomes) and pH was adjusted to 8-8.5 with sodium hydroxide. Trans-crocetin sodium solution was mixed with magnésium acetate liposome solution at different Drug/lipid ratio (100 g/mol, 80 g/mol, 60 g/mol or 40 g/mol). The mixture was then thoroughly stirred and heated up to 65°C for 30 minutes, followed by quick cool down to room température using an ice water bath. This step can be replaced by stirring the mixture at room température ovemight.
Purification of Liposomes:
[0234] The extra-liposomal trans-crocetin was removed using SEC (PD-10 columns) or TFF. In this example, the buffer used in SEC was HBS (HEPES buffered saline, pH 6.5). Upon completion of purification, filter sterilizatîon was performed using a 0.2-0.22 micron filter. A Malvem Zetasizer Nano ZS instrument (Southborough, MA) with back scattering detector (90°) was used for measuring the hydrodynamic sîze (diameter) at 25 °C in a plastic micro cuvette. The samples were diluted before analysis.
Table 2. Physical characteristics of représentative MTC loaded nanoparticles
149
Starting concentration Encapsulation efficîency Final concentration Drug/Lipid Ratio Diameter PDI Zêta potential
MTC LP (D/L-80) 0.75 mg/mL Trans-crocetin disodium 99.98% 5.03 mg/ml 77.22 g/mol lipids 102.1 nm 0.046 -2.32 mV
MTC LP (D/L-60) 0.75 mg/mL Trans-crocetin disodium 98.82% 4.00 mg/mL 58.83 g/mol lipids 103,4 nm 0.034 -3.23 mV
MTC LP (D/L-40) 0.75 mg/mL Trans-crocetin disodium 98.90% 2.25 mg/mL 35.13 g/mol lipids 103.7 nm 0.039 -3.23 mV
150
Table 3: Liposomal CTC and MTC PK resuit summary
Test article Tm (h) AUC (mg/ml*h) Cm ht (nig/ml) Plasma Exposure (fold increase compared to free drug STC) NCA analysis
STC free drug | 0.35 0.21 0.36 NA
STC free drug 0.47 0.26 NA NA
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
Fluorescent Dye Labeled Liposome 12.2 NA NA NA
[0235] Balb/c mice (3 mice / group) were treated with a single dose of STC free drug, CTC/MTC-LPs (D/L ratio 80, 60, 40), and fluorescent dye labeled liposome via a slow intravenous bolus in order to collect serial blood samples at 5 varions time points over a 24 hour period (typîcally, 5 min, 1 hr., 2 hr., 4 hr., 8 hr., and 24 hr.).
[0236] 5 pL of each plasma sample was mixed with 395 pL methanol containing
1% formic acid. Sample mixtures were well mixed by vortexing. Samples were incubated at -20°C for 1 hr. and then equilibrated at room température for 15 10 min. Samples were vortexed and then centriftiged at 10000 RPM for 10 min at room température. 200 pL of supematant was removed from each sample wîthout disturbing pellet and analyzed by HPLC. If the amount of plasma permitted, this analysis was duplicated.
[0237] The concentration of STC in the plasma samples was quantified by 15 standard curve constructed by analyzing plasma samples containing known amount of STC. PK profiles were analyzed.
151
Table 4: CTC liposome stability
Test article Analysis date Particle size (nm) PDI Zêta potential (mV) Lipid Conc. (mM) Crocetin Conc. (mg/mL) Hesulting D/L
CTC-LP-80 lst Initial 101.1 0.039 -3.09 63.88 5.01 78.47
CTC-LP-80 lst 1 Month 100.3 0.046 -1.56 1.64 0.13 77.73
CTC-LP-80 lst 2 Months 99.33 0.049 -3.44 1,25 0.10 76.46
CTC-LP-80 lst 6 Months 99.56 0.046 -2.49 2.03 0.16 78.37
CTC-LP-80 lst 6 Months 103.3 0.06 -2.21 1.73 0.14 78.66
CTC-LP-80 2nd Initial 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 Initial 102.3 0.042 -0.80 70.57 5.48 77.64
CTC-LP-80 3rd 2 Months 102.9 0.038 -2.11 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 Moths 104.2 0.090 -2.17 1.12 0.09 77.10
CTC-LP-80 4th Initial 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-80 4th 3 Months 102.8 0.073 -5.01 1.36 0.11 79.06
CTC-LP-60 Initial 100.8 0.0 -3.7 58.91 3.90 66.23
CTC-LP-60 4 Months 104.0 0.0 -1.9 2.12 0.14 68.12
CTC-LP-60 5 Months 103.2 0.037 -2.63 1.70 0.12 68.89
CTC-LP-40 Initial 101.9 0.0 -3.8 71.54 2.49 34.74
CTC-LP-40 4 Months 106.1 0.0 -2.1 2.54 0.09 36.58
CTC-LP-40 5 Months 1 103.1__ 0.038 -2.28 2.21 0.08 36.41
[0238] CTC liposome stability was further assessed by characterizing liposome solution after the liposomes were purified from potentially leached our drug by size exclusion column after certain storage duration (up to 6 months). The characterization methods were same as previously described.
[0239] The CTC liposomes showed almost the same drug/lipid ratio within error range. Therefore, negligible drug leaching over 6 months at the storage condition (4°C) was confirmed.
152
Table 5: Evaluation of liposome batch reproducibility and stability
D/L of BC sampies Mar Apr May Jun Jul Aug Sep Oct
CTC-LP-80 (Ist) 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
[0240] Liposome batch reproducibility and stability were evaluated by characterizing the D/L.
[0241] CTC liposomes showed negligible change in this évaluation. Thus, CTC 5 liposomes showed stability at least 6 months.
Table 6: MTC liposome stability
Test article Analysis date Particle size (nm) PDJ Zêta Potential (mV) Lipid Conc. (mM) Drug Conc. (mg/mL) DI (g/mol)
MTC-LP-80 Initial 102.1 0.046 -2.32 65.2 5.03 77.22
MTC-LP-80 1 Month 104.4 0.038 -2.84 19.70 1.53 77.72
MTC-LP-80 2 Months 105.5 0.051 -4.78 18.29 1.41 77.22
MTC-LP-60 Initial 103.4 0.034 -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 0.056 -4.45 23.31 1.39 59.85
MTC-LP-40 Initial 103.7 0.039 -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 0.058 -5.74 23,21 0.84 36.27
[0242] Détermination procedures were the same as previously described.
[0243] MTC liposomes showed almost the same drug/lipid ratio within error range. Therefore, liposome is stable at least 2 months period at storage condition (4°C) was confirmed
Example 4- Liposomal CTC Efficacy Study Protocol and Résulte
Animais and Husbandry:
[0244] Male and/or female C57BL/6 mice ordered from Envigo Laboratories or the Jackson Lab (Bar Harbor, Maine) were acclimated to housing conditions and handled in accordance with Animal Use Protocol (AUP) number TP-05. The
153 animais were acclimated for approximately 1 week prior to study start. Only animais deemed healthy were included in this study. Animais were fed irradiated Teklad Global Rodent Diet 2918 and water ad libitum, Mice were group housed 5/cage in static cages with irradiated Teklad 1/8” corn cob bedding 7902 inside bioBubble® Clean Rooms that provide H.E.P. A filtered air into the bubble environment at 100 complété air changes per hour. The environment was controlled to a température range of 74°±5°F and a humidîty range of 30-70%. Treatment groups were identified by cage card. Individual mice were identified by indelible marker on the base of the tail. Ail procedures carried out in this experiment were conducted in compliance with the laws, régulations, and guidelines of the National Institutes of Health and with the approval of the TransPharm Animal Care and Use Committee.
Cecal Ligation Puncture and Post-operative Procedure:
[0245] On Day -1, male and/or female mice were anesthetîzed through use of isoflurane and brought to a surgical plane. The lower quadrants of the abdomen were shaved using an electric trimmer. On Day 0, mice were anesthetîzed through use of isoflurane and brought to a surgical plane. The shaved area was disinfected withthree altemating scrubs of chlorhexidine surgical scrub and 70% isopropanol. An abdominal longitudinal skin midline incision was made with iris scissors, without penetrating the peritoneal cavity. After the initial incision, small scissors were used to extend the incision 1.5-2 cm in order to gain entry to the peritoneal cavity. The midline white fascia of the abdominal musculature was identified and dissected for intermuscular incision and incision of fascia and peritoneal layers. The cecum was exteriorized using blunt anatomîcal forceps, leavîng the remainder of the small and large bowel within the peritoneal cavity and avoiding breach or damage to the mesenteric blood vessels. The cecum was ligated with a stérile 9.5 mm stainless Steel surgical clip below the ileocecal valve at the designated position (approximately 70% of the cecum will be ligated). Care was taken not to occlude the bowel. Before cecal perforation, the
154 cecal contents were gently pushed toward the distal cecum. The cecum was then perforated using a 16-gauge needle for severe grade sepsis. A single throughand-through puncture midway between the ligation and the tip of the cecum in a mesenteric-to-antimesenteric direction was performed. After removing the needle, the cecum was relocated into the abdominal cavity without spreading feces from the cecum onto the abdominal wall wound margins, and a small droplet of feces was extruded from both the mesenteric and antîmesenteric pénétration holes. Droplet size was as consistent as possible. The peritoneum, fasciae, and abdominal musculature were closed by applying simple running sutures (4-0 PDS or chromic gut surgical sutures) and the skin incision was closed with 9 mm autoclips or surgical glue. Immediately following surgery, mice were administered a subcutaneous (SC) injection of 0.5 mL of room température 0.9% saline. Animais were then allowed to recover following surgery in a clean cage placed on a warm, re-circulating heating pad, with free access to water and food pellets on the floor. The heating pad remained in place with the cage half on/half off the pad, to allow the animais the opportunity to move to a cooler part of the cage if desired. The mice remained in this environment until fully conscious and mobile. The heating pad was removed once the animal was stable.
[0246] Animais were monitored continually post-surgery, at least once every 2-3 minutes for approximately 30 minutes, until the animais had recovered and were able to move about on their own. Thereafter, the animais were observed every hour for at least 6 hours post-surgery. Animais were also closely monitored (every hour from 7 am-6 pm daily) throughout the study period, with additional observations at 10 pm and 2 am on Days 1 -3. A Rodent Postoperative Record was maintained (1 record per animal) during the study. Abnormal clinical signs were recorded if observed. Any animal exhibîtîng signs of
155 impending mortalîty was humanely euthanized. If an animal was euthanized, the time and date was recorded on the postoperative record.
Formulation and Dosing:
[0247] Mice were admînistered test article via IP injection beginning at 2 hours 5 post-surgery and contînuing once daily through Day 4 (5 days total dosing). Mice in Groups 1-3 were dosed with a volume of ~10 pL test article per gram of mouse body weight (per Table 7; these admînistered doses represent a dose of 50 mg/kg per mouse daily for 5 days). Mice in Group 4 received once daily administration of 0,9% saline via IP injection in a volume of 0.3 mL from Day 0-4. Mice were weighed daily and dose volumes were admînistered per Table 7.
Table 7: Dose values
Groups 1,2 and 3 (10 gL/g dose)
Body Weight Range Dose Volume
20-23.9 g 0,2 inL
24-26.9 g 0.25 mL
27-30.9 g 0.3 mL
Endpoint Analysis:
[0248] Efficacy of test articles was assessed by énumération of test animal mortality over 5 days following CLP surgery. Animais which remained surviving on Day 5 were humanely euthanized via CO2 overexposure.
Study Results:
[0249] Table 8 describes four CLP studies used to test different formulations of liposomal CTC. Studies 1 and 2 examined the CLP model in male mice. Studies 3 and 4 examined the CLP model in female mice. Test articles and results from each study are described below.
Table 8: Exemplary CLP studies of the efficacy of liposomal CTC
Study 1TP-936 Study 2TP-967 Study 3TP-983
Type Efficacy Efficacy Efficacy
156
Sex of Mouse Male Female Female
Study Size 30 40 50
Groups 3 4 5
Studv 1 (TP-936) Results:
[0250] Ail surgical and dosing procedures were performed as detailed in the study protocol (above). Sham animais demonstrated 100% survîval. Mice which underwent CLP and were treated with saline and îmipenem showed 50% 5 mortality. Animais treated with test article 1 and îmipenem or test article 2 and îmipenem demonstrated 30% and 10% death, respectively. Five of the nine deaths during the study were a resuit of euthanasia due to déhydration and latéral recumbency (FIG. 5).
[0251] Together, these data demonstrate that cecal ligatîon and puncture using 10 a 16-gauge needle causes mortality in C57B1/6J mice. Although both test articles (in combination with îmipenem) demonstrated a trending réduction in mortality when compared to the imipenem-treated control group.
Studv 2 (TP-967) Results:
[0252] Animais treated with saline vehicle and îmipenem (Group 4) 15 demonstrated 70% death (FIG. 6). One of the seven deaths was a result of euthanasia. Mice treated with PGPC-LP and îmipenem (Group 1) showed 60% mortality, with one of the six deaths attributed to euthanasia. Group 2, which was administered CTC-LP-80 and îmipenem, demonstrated 30% death (FIG. 6). Two of the three deaths were due to euthanasia. Mice which received PGPC20 CTC-LP-80 and îmipenem (Group 3) had 70% mortality (FIG. 6). Two of the seven deaths were due to euthanasia. None of the treatment groups showed a statistically significant différence in mortality when compared to the vehicle control group, but a strong trend of improvement in survival was observed.
Study 3 (TP-986) Results:
[0253] Mice treated with CTC-LP-80 (50 mg/kg) and îmipenem demonstrated
70% death (FIG. 8). Three of the seven deaths were due to euthanasia. Mice
157 which received CTC-LP-80 (25 mg/kg) and imipenem had 40% mortality (FIG.
7). Two of the four deaths were due to euthanasia. Mice treated with CTC-LP80 (5 mg/kg) and imipenem had 20% death (FIG. 7). None of the deaths were due to euthanasia. This treatment demonstrated a statistically significant decrease in mortality when compared to the vehicle control group (P=0.0321). Mice which received CTC-LP-80 (1 mg/kg) and imipenem had 60% mortality (FIG. 7). None of the deaths were due to euthanasia. FIG. 7.
[0254] Together, these data demonstrate that cecal ligation and puncture using a 16-gauge needle causes mortality in C57B1/6 mice. Treatment with test article CTC-LP-80 (5 mg/kg) and imipenem demonstrated a statistically significant réduction in mortality when compared to the saline-treated control group.
Example 5 - Production STC Liposomes by Passive Loading
Passive Loading of Sodium Trans-crocetin with Extrader:
[0255] Trans-crocetin sodium was dissolved in an aqueous phase at its maximum solubility in the given aqueous media, for example 0.7 mg/ml in 5% dextrose. The ethanolic lipid solution containing HSPC, cholestérol, PEGDSPE, with/without PGPC was added into the aqueous solution using a pipet. During this step the solution was well stirred using a magnetic stirrer. The mixing was performed at an elevated température (63°C-72°C) to ensure that the lipids are in the liquid crystalline State (as opposed to the gel state they attain at températures below the lipid transition température (Tm = 51°C-54°C). As a resuit, the lipids are hydrated and formed multiple bilayer (multilamellar) vesicles (MLV) containing trans-crocetin sodium in the aqueous core. The MLVs were then downsized by extrusion as described previously.
Passive loading of trans-crocetin with Nanoassemblr®:
[0256] Trans-crocetin sodium was dissolved in an aqueous phase at its maximum solubility in the given aqueous media, for example 0.7 mg/ml in 5% dextrose. The ethanolic lipid solution containing HSPC, cholestérol, PEGDSPE, with/without PGPC and the trans-crocetin sodium aqueous solution were
158 separately transferred to syringes. Two solutions were injected into a microfluidic channel and mixed while flowing through it with Précision NanoSystems’ NanoAssemblr® device. The mixing was performed at an elevated température (63°C-72°C) to ensure that the lipids were in a liquid crystalline State (as opposed to the gel State that they attain at températures below the lipid transition température (Tm = 51°C-54°C). The liposome size can be controlled by varying the ratio between lipid solution and aqueous solution, as well as the mixing flow rate.
Passive Loadîng of Trans-crocetin bv Ethanol Injection Method:
[0257] Trans-crocetin (free acid) was dissolved in ethanolic lipid mixture at its maximum solubility. Then, an ethanolic lipid mixture containing trans-crocetin was either mixed with aqueous solution (e.g., buffers, buffered saline, or dextrose solution) and downsized by extrusion method or mixed with aqueous solution through microfluidic channel by NanoAssemblr® device.
Passive loading of trans-crocetin by thin film rehydration method:
[0258] Trans-crocetin (free acid) was dissolved in a volatile organic solvent (e.g., éthanol, methanol, chloroform, dichloromethane, etc.) along with other lipids: HSPC, cholestérol, PEG-DSPE, with/without PGPC. The organic solvent in trans-crocetin-lipid mixture was completely dried using the rotary evaporator by elevated température (e.g., 65 °C) in water bath and vacuum. While drying, the flask was rotated and thin film of dried trans-crocetin-lipid was formed on the wall of round bottom flask. An aqueous solution was added in to the thin film and rotated/agitated at elevated température (e.g., 65 °C). Rehydration of the thin film in aqueous solution forms multiple bilayer (multilamellar) vesicles (MLV) containing trans-crocetin in the lipid bilayer of MLVs. MLVs was then downsized by extrusion to desired small unilamellar vesicles (SUVs).
159
Example 6 - Production of Targeted Trans-Crocetin Liposomes: Post Insertion
[0259] Antibody or its fragments, such as Fab or scFv, which contains a cysteine residue at the C-terminal will be conjugated and incorporated înto the trans5 crocetin liposome through a “post insertion” method. Micelles of thiol-reactive lipopolymer (such as DSPE-PEG-maleîmide) will be prepared by dissolving in an aqueous solution at 10 mg/ml. Antibody (or its fragment) with a cysteine tail will be dissolved and reduced by a 10-20 mM reducing reagent (such as 2mercaptoethylamine, cysteine, or dithioerythritol) at pH < 7. The excess 10 reducing reagent will be removed thoroughly by SEC (size exclusion chromatography) or dialysis. The purified and reduced antibody (or its fragment) will be then incubated with the micelles of thiol-reactive lipopolymers at a molar ratio of 1:4. At the end of the reaction, the excess maleimide groups will be quenched by a small amount of cysteine (1 mM) or mercaptoethanol.
Unconjugated antibody (or its fragment) will be removed by SEC. Purified conjugated micelles will be then incubated with liposome at 37°C or elevated température at different Antibody/Lipid ratios (this ratio is antibody dépendent).
[0260] While the disclosed methods hâve been described in connection with what is presently considered to be the most practical and preferred embodiments, 20 it is to be understood that the methods encompassed by the dîsclosure are not to be limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications and équivalent arrangements included within the spirit and scope of the appended daims.
[0261] Ail publications, patents, patent applications, internet sites, and 25 accession numbers/database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference herein în their entîrety for ail purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession
160 number/database sequence were specifically and individually indicated to be so incorporated by reference.

Claims (62)

1% to 50% dextrose;
1% to 50% trehaiose;
1. A pharmaceutical composition comprising a liposome encapsulating an îonizable carotenoid sait, having the formula: Q- Polyene Carotenoid -Q, wherein, the Polyene Carotenoid comprises (a) 6, 7, 8, 9, or 6-8, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 2, 3, or more than 3, îonizable groups; and
Q is a multivalent counterion.
2. A pharmaceutical composition comprising a liposome encapsulating an îonizable carotenoid sait havîng the formula: Q- Ri-Polyene Carotenoid-Rz -Q, wherein the Polyene Carotenoid comprises (a) 6, 7, 8, 9, or 6-8, conjugated double bonds, (b) methyl or low alkyl (C2-C3) substitutions, and (c) 2, 3, or more than 3, îonizable groups;
Ri and R- are îonizable groups (e.g., the sanie îonizable group or different îonizable groups; and Q is a multivalent counterion.
3. The pharmaceutical composition according to claîm 1 or 2, wherein the Polyene Carotenoid comprises anionic îonizable groups or cationic îonizable groups (e.g., a primary, secondary, or tertiary amine group, a quatemary ammonium group, a choline group, a guanidine group, or an imidazoie group).
4. The pharmaceutical composition of claîm 3, wherein the Polyene Carotenoid comprises at least one anionic îonizable group selected from: a carboxylic group, a sulfonate group, a sulfate group, a phosphonate, a phosphate group, and a hydroxamate group.
5% dextrose suspended in an HEPES buffered solution; or
165
P a total concentration of sodium acetate and calcium acetate of between 50 mM to 500 mM.
5. A pharmaceutical composition comprising a liposome encapsulating an îonizable carotenoid sait having the formula: Q- trans-crocetin -Q, wherein Q is a multivalent cation counterion.
162
6. A pharmaceutical composition comprising a liposome encapsulatîng an ionizable carotenoid sait having the formula: Q- trans-norbixin -Q, wherein Q is a multivalent cation counterion.
7. The pharmaceutical composition according to any of daims 1-6, wherein Q is a divalent métal cation or a divalent organic cation such as, at least one divalent cation selected from Ca2+, Mg2+, Zn2+, Cu2+, Co2+, and Fe2+, a divalent organic cation such as protonated diamine, or a trivalent cation such as Fe3+.
8. The pharmaceutical composition according to claim 5, which comprises magnésium trans-crocetinate (MTC) or calcium trans-crocetinate (CTC).
9. The pharmaceutical composition according to claim 6, which comprises magnésium trans-norbixinate (MTN) or calcium trans-norbixinate (CTN).
10 The pharmaceutical composition according to any of daims 1-9, wherein the ionizable carotenoid/lipid ratio is about 1 toi000 g/mol, about 10 to 150 g/mol, about 20 to 100 g/mol, or any range therein between, or wherein the liposomes comprise at least 0.1% to 97% weîght by weight (w/w) ionizable carotenoid, or any range therein between.
11 The pharmaceutical composition according to daim 8, wherein the ionizable carotenoid/lipid ratio is about 1 tolOOO g/mol, or any range therein between.
12. The pharmaceutical composition according to any of daims 1-11, wherein the liposome has a diameter of 20 nm to 500 nm, 20 nm to 200 nm, or 80 nm to 120 nm, or any range therein between.
13. The pharmaceutical composition of daim 8, wherein the liposome has a diameter of 20 nm to 200 nm or 80 nm to 120 nm, or any range therein between.
14. The pharmaceutical composition according to any of daims 1-13, wherein the liposome is formed from liposomal components comprising:
at least one of a cationic lipid, an anionic lipid and a neutral lipid; or at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimîde.
163
15. The pharmaceutical composition of claim 13, wherein the liposome is formed from liposomal components comprising:
at least one of a cationic lipid, an anionic lipid and a neutral lipid; or at least one selected from: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholestérol; cholesterol-PEG; and cholesterol-maleimide.
16. The pharmaceutical composition according to any of daims 1-15, wherein the liposome comprises an oxidized phospholipid such as an OxPAPC.
17. The pharmaceutical composition according to claim 16, wherein the OxPAPC 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 fivecarbon sn-2 residue bearing omega-aldehyde or omega-carboxyl groups; or wherein the OxPAPC selected from HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC, l-palmitoyl-2-(5,6epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6 PEIPC), l-palmîtoyl-2-(epoxy-cyclopentenone)-sn-glycero-3-phosphorylcholine (PECPC),1 -palmitoyl-2-(epoxy-isoprostane E2)-snglycero-4-phosphocholîne (PEIPC), 1 -palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC); l-palmitoyl-2-(9'oxo-nonanoyl)-sn-glycero-3-phosphocholine; 1 -palmitoyl-2arachinodoyl-sn-glycero-3-phosphocholîne; l-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; l-paImitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-azelaoyl-sn-glycero-3phosphocholine; and 1 -palmitoyl-2-acetoyl-sn-glycero-3 -phospho-choline; PGPC; or the OxPAPC is an epoxyisoprostane-containing phospholipid.
18. The pharmaceutical composition according to any of daims 1-17, wherein the liposome comprises 0% to 100%, 0.1% to 30%, 1% to 25%, 5% to 20%, or 7% to 15% OxPAPC (e.g., about 10% OxPAPC), or any range therein between.
19. The pharmaceutical composition according to any of daims 1-18, wherein the liposome comprises HSPE, cholestérol, PEG-DSPE-2000, and OxPAPC at a molar ratio of 2 to 5, 1 to 4, 0.01 to 0.3, or 0.05 to 1.5.
20. The pharmaceutical composition according to any of daims 1-19, wherein the liposome is pegylated.
164
21. The pharmaceuticai composition according to any of daims 1 -20, wherein one or more liposomal components further comprises at least one steric stabilizer selected from: polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazolîne); poly(2-ethyl-2oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic poly-N-vinylpyrrolidones; L amino-acîd-based polymer; oligoglycerol, copolymer containing polyethylene glycol and polypropylene oxide, Poloxamer 188, and polyvinyl alcohol.
22. The pharmaceuticai composition according to daim 21, wherein the steric stabilizer is PEG and the PEG has a number average molecular weight (Mn) of 200 to 5000 Daltons.
23. The pharmaceuticai composition according to any of daims 1-22, wherein the liposome is anionic, neutral, or cationic.
24. The pharmaceuticai composition according to any of daims 1-23, wherein the liposome has a zêta potential of -150 to 150 mV, or -50 to 50 mV, or any range therein between.
25. The pharmaceuticai composition according to any of daims 1-24, wherein the liposome has a zêta potential that is less than or equal to zéro (e.g., -150 to 0, or -50 to 0 , or any range therein between).
26. The pharmaceuticai composition of daim 13, wherein the liposome has a zêta potential of -50 to 0, or any range therein between and wherein the liposome is pegylated.
27. The pharmaceuticai composition according to any of daims 1-26, which further comprises a pharmaceutically acceptable carrier.
28. The pharmaceuticai composition of daim 27, wherein the pharmaceutically acceptable carrier comprises;
a tonicity agent such as, dextrose, mannitol, glycérine, potassium chloride, sodium chloride, at a concentration of greater than 1%;
29. The pharmaceutical composition according to any of daims 1 -28, which comprises a buffer such as HEPES Buffered Saline (HBS) or similar, at a concentration of 1 to 200 mM and a pH of 2 to 8, or any range therein between.
5
30. The pharmaceutical composition according to any of daims 1 -29, which has a pH of 5-8, or a pH of 6-7, or any range therein between.
31. The pharmaceutical composition according to any of daims 1-30, wherein the liposome comprises 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, molécules, or between 10 to 100,000, 100 to 10 10,000, or 500 to 5,000, molécules of ionizable carotenoid, or any range therein between.
32. The pharmaceutical composition of daim 26, wherein the liposome comprises between 100 to 10,000, or 500 to 5,000, molécules of ionizable carotenoid, or any range therein between.
33. The pharmaceutical composition according to any of daims 1-32, wherein the 15 liposome further comprises a targeting moiety and wherein the targeting moiety has a spécifie affinity for a surface antigen on a target cell of interest.
34. The pharmaceutical composition of daim 33, wherein the targeting moiety is attached to one or both of a PEG and the exterior of the liposome, optionally wherein the targeting moiety is attached to one or both of the PEG and the exterior of the liposome by a covalent bond.
20
35. The pharmaceutical composition of daim 33 or 34, wherein the targeting moiety is a polypeptide, an antibody or an antigen binding fragment of an antibody.
36. The pharmaceutical composition according to any of daims 33-35, wherein the targeting moiety binds the surface antigen with an equilibrium dissociation constant (Kd) in a range of 50 x 10“12 to 10 x 10’6 as determined using BIACORE® analysis.
166
37. The pharmaceutical composition according to any of daims 33-36, wherein the targeting moiety specifically binds one or more folate receptors selected from: folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-Ô).
38. The pharmaceutical composition according to any of daims 33-37, wherein the targeting moiety comprises one or more selected from: 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.
39. The pharmaceutical composition according to any of daims 33-38, wherein the liposome is pegylated and comprises 1 to 1000, 50 to 750, or 100 to 500, or 30 to 200 targeting moieties, or any range thereîn between.
40. The pharmaceutical composition according to any of daims 1-39, further comprising one or more of an FABP, an inrmunostimulatory agent, an immunosuppressing agent, a détectable marker and a maleimîde, wherein the FABP, the immunostimulatory agent, the immunosuppressing agent, the détectable marker or the maleimîde is attached to said PEG or the exterior of the liposome.
41. The pharmaceutical composition of daim 40, wherein the immunostimulatory agent is at least one selected from: a fluorescein; a fluorescein isothiocyanate (FITC); a DNP; a beta glucan; a beta-l,3-glucan; a beta-l,6-glucan; a resolvin (e.g., a resolvin D such as Dn-6DPA or Dn-3DPA, a Resolvin E, or a T sériés resolvin); and a Toll-like receptor (TLR) modulating agent such as, an oxidized low-density lipoprotein (e.g., OXPAC, PGPC),or an eritoran-like lipid (e.g., E5564).
42. The pharmaceutical composition according to any of daims 1-41, which further comprises at least one cryoprotectant selected from consisting of mannitol, trehalose, sorbitol, and sucrose.
43. The pharmaceutical composition according to any of daims 1-42, for use in the treatment of disease or a condition in a subject.
167
44. Use of the pharmaceutical composition according to any of daims 1-43, in the manufacture of a médicament for the treatment of disease or a condition in a subject.
45. The pharmaceutical composition of claim 42 or 43 or use of claim 44, wherein the disease or condition is sepsis.
46. The pharmaceutical composition of claim 42 or 43 or use of claim 44, wherein the disease or condition is a lung disease or condition (e.g., acute respiratory di stress syndrome (ARDS), pulmonary fibrosîs, pulmonary hemorrhage, lung injury, lung cancer, chronic obstructive pulmonary disease (COPD), and other respiratory disorders).
47. The pharmaceutical composition of claim 42 or 43 or use of claim 44, wherein the disease or condition is characterized by ischemia or hypoxia (e.g., ischemic-reperfusion injury, transient cérébral ischemia, cérébral ischemia-reperfusion, ischémie stroke, hémorrhagie stroke, traumatic brain injury, migraine (e.g., a chronic migraine or severe migraine disorder), gastrointestinal ischemia, kidney disease. pulmonary embolism, acute respiratory failure, néonatal respiratory distress syndrome, obstetric emergencies to reduce périnatal comorbidity (such as, pre/eclampsia and conditions that lead to cérébral palsy), myocardial infarction, and acute limb or mesenteric ischemia, cardiac cirrhosis, chronic peripheral vascular disease, congestive heart failure, atherosclerotic stenosis, anémia, thrombosis, embolism, macular degeneration, a neurodegenerative disease (such as Alzheimer’s disease, Parkinson’s disease, or Amyotrophie Latéral Sclerosis (ALS)), sleep apnea, and surgery or traumatic injury).
48. The pharmaceutical composition of claim 42 or 43 or use of claim 44, wherein the disease or condition is an infection (e.g., a bacterial infection such as an P. aeruginosa infection, an S. aureus infection (e.g., MRS A) or a condition associated therewith, or an enterococcal infection (e.g., VRE), a fungal infection (e.g., a candidiasîs infection (e.g., invasive candidiasîs) or a condition associated therewith, or a parasitic infection or a condition associated therewith, such as malaria (or an associated condition such as cérébral malaria, severe anémia, acîdosis, acute kidney failure and ARDS), Schistosomiasis, and human African trypanosomiasis, and conditions associated therewith; a viral infection or a condition associated therewith such as Ebola, Dengue and Marburg (or an associated condition such as influenza, measles, and a viral hémorrhagie fever); bacteremîa; or endotoxemia, such as a low grade endotoxemic disease or endotoxemia
168 associated with conditions like periodontal disease (e.g., periodontitis or inflammation of the gums),
49. The pharmaceutical composition of claim 42 or 43 or use of claim 44, wherein the disease or condition is inflammation (e.g., systemîc inflammation, low-grade inflammation, acute inflammation, and chronic inflammatory disease); or inflammatory bowel disease (e.g., Crohn’s disease and ulcerative colitis);
50. The pharmaceutical composition of claim 42 or 43 or use of claim 44, wherein the disease or condition is heart attack or stroke (e.g., ischémie and hémorrhagie stroke), or shock (e.g., cardiogenic shock, hypovolémie shock, septîc shock, neurogenîc shock, and anaphylactîc shock).
51. The pharmaceutical composition of claim 42 or 43 or use of claim 44, wherein the disease or condition is cardiovascular disease (e.g., coronary artery disease such as myocardial infarction, sudden cardiac death, cardiorespiratory 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 (PCAS), chronic venons insufficiency, heart disease, congestive heart failure, chronic skin ulcers);
52. The pharmaceutical composition of claim 42 or 43 or use of claim 44, wherein the disease or condition is:
a liver disease or condition (e.g., cirrhosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH); alcoholic liver disease, acute liver injury, and cirrhosis of the liver);
a kidney disease (e.g., lipopolysaccharide médication or toxin induced acute kidney injury (AKI) and end stage kidney disease);
an autoimmune disorder (e.g., psoriasis, cystîc fïbrosis, and rheumatoid arthritis);
sclerosis (e.g., systemic sclerosis);
a metabolic disease, insulin résistance, or diabètes (e.g., type 2 diabètes) or an associated conditions such as gangrené, diabetîc necrosis, diabetic neuropathy, and diabetic vascular disease (e.g., microvascular disease such as retinopathy and nephropathy and diabetic ulcers);
169 a disease or condition associated with nitric oxide defîciency (e.g., sickle cell disease, paroxysmal noctumal hemoglobinuria (PNH), a hemolytic anémia, a thalassemia, another red blood cell disorder, a purpura such as thrombotîc thrombocytic purpura (TTP), hemolytic urémie syndrome (HUS), idiopathic thrombocytopenia (ITP), another platelet disorder, a coagulation abnormality such as dîsseminated intravascular coagulopathy (DIC), purpura fulminans, heparin induced thrombocytopenia (HIT), hyperleukocytosis, and hyper viscosity syndrome, or a condition associated therewith; or chronic alcoholism, chronic smoking, transplantation, néonatal necrotîzîng enterocolitis, or a néonatal ear infection.
53. The pharmaceutical composition of claim 42 or 43 or use of claim 44, wherein the subject: is a bum victim, is a critically ill patient, is at risk of developing sepsis, is immunocompromised, receives chemotherapy and/or is immunosuppressed (e.g., febrile neutropénie patients).
54. The pharmaceutical composition of claim 42 or 43 for use in reducing systemic levels of LPS, endotoxin and/or another trigger of systemic inflammation in a subject.
55. The use according to any of daims 42-52, wherein the pharmaceutical composition is admînistered in combination therapy with another therapeutîc agent.
56. A method of preparing a liposomal composition according to any of daims 1-43, the method comprising: forming a mixture comprising: liposomal components in solution; homogenizing the mixture to form liposomes in the solution; and processing the mixture to form liposomes containîng an ionizable carotenoid, wherein the processing step includes one or more steps of: thin film hydration, extrusion, in-line mixing, éthanol injection technique, freezing-andthawing technique, reverse-phase évaporation, dynamic high pressure microfluidization, microfluidic mixing, double émulsion, freeze-dried double émulsion, 3D printing, membrane contactor method, and stirring or one or more steps of modifying the size of the liposomes by one or more of steps of extrusion, high-pressure microfluidization, and/or sonication.
57. A method of preparing a pharmaceutical composition comprising:
170 preparing a liposomal solution containing liposomes in a weak acid sait of a multivalent métal or multivalent organic cations such as protonated amine;
(a) adding an ionizable carotenoid according to any of daims 1 -42 or 43 to the liposomal solution; and (b) maintaining the ionizable carotenoid in the liposomal solution for sufficient time to load the carotenoid into liposomes.
58. A method of preparing pharmaceutical composition comprising a liposome encapsulating trans-crocetin, the method comprising:
(a) preparing a liposomal solution comprising liposomes and a solution containing a weak acid sait of a multivalent métal;
(b) adding trans-crocetin to the liposomal solution; and (c) maintaining the trans-crocetin in the liposomal solution for sufficient time to load transcrocetin into liposomes.
59. The method of claim 57 or 58, wherein the weak acid is an organic acid (e.g., acetic acid, gluconic acid, tartaric acid, glutamic acid, citric acid, formic acid, and glycinic acid).
60. The method according to any of daims 57-59, wherein the multivalent métal is a divalent métal (e.g., a divalent métal selected from Ca2+, Mg2*, Zn2+, Cu2+, Co2+, and Fe2+), or a trivalent métal such as Fe3+.
61. The method according to any of daims 57-60, wherein the weak acid is acetic acid and the multivalent métal is Ca2+ or Mg2 '.
62. A pharmaceutical composition prepared according to the method according to any of daims 57-61.
OA1202000394 2018-05-03 2019-05-03 Carotenoid compositions and uses thereof OA20783A (en)

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US62/809,123 2019-02-22

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