Patent Application
20210338823
One of the major challenges for applying protein and peptide biologies to clinical medicine is the lack of rational and efficient methods to circumvent epithelial and endothelial cell barriers separating large molecules from target tissues. In the case of epithelial cells lining mucosal surfaces, the pathway for absorption of large solutes (e.g., biologies) is by transcytosis—a process of transcellular endosome trafficking that connects one surface of the cell with the other.
The present disclosure, in some aspects, relates to using ceramides (e.g., naturally occurring ceramides and ceramide analogs or ceramide-like molecules) as delivery vehicles to deliver agents (e.g., therapeutic agents) into cells or across epithelial and/or endothelial barriers. The sorting of the agents via different endocytic pathways relate to the structure of the ceramide. In some embodiments, the ceramides are used to deliver agents to a targeted site, e.g., to treat a disease in a subject.
Some aspects of the present disclosure provide delivery vehicles comprising a ceramide and an agent to be delivered, wherein the ceramide: (a) does not contain a fatty acid; or (b) comprises a fatty acid of C1-C28; and wherein the agent is attached to the ceramide. In some embodiments, the ceramide is a ceramide analog. In some embodiments, the ceramide analog is selected from the group consisting of: 2-hydroxy-ceramide, diene-deoxy-ceramide, dihydroceramide, phytosphingosine, dihydroceramide phosphate, o-acyl-ceramide, ceramide phosphate, sphinganine, and methyl-sphingosine. In some embodiments, the ceramide analog comprises an unsaturated hydrocarbon chain attached to ornithine, tyrosine, glycine, leucine, praline, glutamine, or taurine.
In some embodiments, the ceramide is a glycoceramide. In some embodiments, the glycoceramide comprises a sugar selected from the group consisting of: glucose, galactose, fructose, and GalNac. In some embodiments, the agent to be delivered is attached to the sugar. In some embodiments, no sugar is attached to the ceramide. In some embodiments, the ceramide is a sphingosine. In some embodiments, the agent to be delivered is attached to the primary hydroxyl group of the ceramide. In some embodiments, the agent to be delivered is attached to the secondary hydroxyl group of the ceramide.
In some embodiments, the agent to be delivered is attached to the ceramide via a linker. In some embodiments, the linker is a pseudo-glycopeptide linker. In some embodiments, the pseudo-glycopeptide linker comprises at least one sugar attached to an amino acid backbone. In some embodiments, the at least one sugar is selected from the group consisting of: glucose, galactose, and N-Acetylgalactosamine. In some embodiments, the at least one sugar is attached to the amino acid backbone via a serine side chain.
In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker comprises an ester linkage. In some embodiments, the cleavable linker is a peptide linker comprising an ester linkage. In some embodiments, the cleavable linker comprises a cleavage motif for an endosomal protease. In some embodiments, the endosomal protease is furin or matriptase. In some embodiments, the linker is a disulfide linkage.
In some embodiments, the ceramide comprises a fatty acid of C1-C6. In some embodiments, the ceramide comprises a fatty acid of C4. In some embodiments, the ceramide comprises a fatty acid of C6. In some embodiments, the fatty acid has no double bonds between two carbon atoms. In some embodiments, the ceramide comprises a fatty acid of C7-C28. In some embodiments, the ceramide comprises a fatty acid of C8. In some embodiments, the fatty acid has at least one cis double bonds between two carbon atoms. In some embodiments, the at least one cis double bond is in C1-C18 region. In some embodiments, the fatty acid comprises a chemical moiety in C1-C18 region. In some embodiments, the ceramide does not comprise a fatty acid.
In some embodiments, the ceramide comprises a fatty acid of C1-C12.
In some embodiments, the agent to be delivered is selected from the group consisting of proteins, peptides, nucleic acids, polysaccharides and carbohydrates, lipids, glycoproteins, small molecules, synthetic organic and inorganic drugs exerting a biological effect when administered to a subject, and combinations thereof. In some embodiments, the agent to be delivered is a therapeutic agent. In some embodiments, the therapeutic agent is an anti-inflammatory agent, a vaccine antigen, a small molecule drug, an anti-cancer drug or chemotherapeutic drug, a clotting factor, a hormone, a steroid, a cytokine, an antibiotic, an antibody, a ScFv, a nanobody, a vaccine adjuvant, or a drug for the treatment of a cardiovascular disease, an infectious disease, an autoimmune disease, allergy, a blood disorder, a metabolic disorder, a skin disease, an eye disease, a lysosomal storage disease or a neurological disease.
In some embodiments, the agent to be delivered is a protein or a peptide. In some embodiments, the protein or peptide is a vaccine antigen. In some embodiments, the protein or peptide is an antibody, a ScFv, or a nanobody. In some embodiments, the protein or peptide is an enzyme. In some embodiments, the enzyme is a lysosomal replacement enzyme. In some embodiments, the protein or peptide is a hormone. In some embodiments, the protein or peptide is a neurotransmitter. In some embodiments, the protein or peptide is GLP-1, or a functional fragment thereof. In some embodiments, the protein or peptide is Exendin-4, or a functional fragment thereof. In some embodiments, the therapeutic agent comprises GLP-1 or a functional fragment thereof, and Exendin-4 or a functional fragment thereof. In some embodiments, the therapeutic agent comprises a ligand for a cell receptor. In some embodiments, the cell receptor is a growth factor receptor, a G-protein coupled receptor, or a toll-like receptor.
In some embodiments, the therapeutic agent is a nucleic acid.
Other aspects of the present disclosure provide ceramide-therapeutic agent complexes comprising a ceramide and an agent to be delivered, wherein the ceramide: (a) does not comprise a fatty acid; or (b) comprises a fatty acid of C1-C28; and wherein the agent is attached to the ceramide.
In some embodiments, the ceramide is a ceramide analog. In some embodiments, the ceramide analog is selected from the group consisting of: 2-hydroxy-ceramide, diene-deoxy-ceramide, dihydroceramide, phytosphingosine, dihydroceramide phosphate, o-acyl-ceramide, ceramide phosphate, sphinganine, and methyl-sphingosine. In some embodiments, the ceramide analog comprises an unsaturated hydrocarbon chain attached to ornithine, tyrosine, glycine, leucine, praline, glutamine, or taurine.
In some embodiments, the ceramide is a glycoceramide. In some embodiments, the glycoceramide comprises a sugar selected from the group consisting of: glucose, galactose, fructose, and GalNAc. In some embodiments, the agent to be delivered is attached to the sugar. In some embodiments, no sugar is attached to the ceramide. In some embodiments, the ceramide is a sphingosine. In some embodiments, the agent to be delivered is attached to the primary hydroxyl group of the ceramide. In some embodiments, the agent to be delivered is attached to the secondary hydroxyl group of the ceramide.
In some embodiments, the agent to be delivered is attached to the ceramide via a linker. In some embodiments, the linker is a pseudo-glycopeptide linker. In some embodiments, the pseudo-glycopeptide linker comprises at least one sugar attached to an amino acid backbone. In some embodiments, the at least one sugar is selected In some embodiments, the at least one sugar is attached to the amino acid backbone via a serine side chain.
In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker comprises an ester linkage. In some embodiments, the cleavable linker is a peptide linker comprising an ester linkage. In some embodiments, the cleavable linker comprises a cleavage motif for an endosomal protease. In some embodiments, the endosomal protease is furin or matriptase. In some embodiments, the linker is a disulfide linkage.
In some embodiments, the ceramide comprises a fatty acid of C1-C6. In some embodiments, the ceramide comprises a fatty acid of C4. In some embodiments, the ceramide comprises a fatty acid of C6. In some embodiments, the fatty acid has no double bonds between two carbon atoms. In some embodiments, the ceramide comprises a fatty acid of C7-C28. In some embodiments, the ceramide comprises a fatty acid of C8. In some embodiments, the fatty acid has at least one cis double bonds between two carbon atoms. In some embodiments, the at least one cis double bond is in C1-C18 region. In some embodiments, the fatty acid comprises a chemical moiety in C1-C18 region. In some embodiments, the ceramide does not comprise a fatty acid.
In some embodiments, the ceramide comprises a fatty acid of C1-C12.
In some embodiments, the therapeutic agent is selected from the group consisting of proteins, peptides, nucleic acids, polysaccharides and carbohydrates, lipids, glycoproteins, small molecules, synthetic organic and inorganic drugs exerting a biological effect when administered to a subject, and combinations thereof. In some embodiments, the therapeutic agent is an anti-inflammatory agent, a vaccine antigen, a small molecule drug, an anti-cancer drug or chemotherapeutic drug, a clotting factor, a hormone, a steroid, a cytokine, an antibiotic, an antibody, a ScFv, a nanobody, a vaccine adjuvant, or a drug for the treatment of cardiovascular disease, an infectious disease, an autoimmune disease, allergy, a blood disorder, a metabolic disorder, a skin disease, an eye disease, a lysosomal storage disease, or a neurological disease. In some embodiments, the therapeutic agent is a protein or a peptide. In some embodiments, the protein or peptide is a vaccine antigen. In some embodiments, the protein or peptide is an antibody, a ScFv, or a nanobody. In some embodiments, the protein or peptide is an enzyme. In some embodiments, the enzyme is a lysosomal storage enzyme. In some embodiments, the protein or peptide is a hormone. In some embodiments, the protein or peptide is a neurotransmitter. In some embodiments, the protein or peptide is GLP-1, or a functional fragment thereof. In some embodiments, the protein or peptide is Exendin-4, or a functional fragment thereof. In some embodiments, the therapeutic agent comprises GLP-1 or a functional fragment thereof, and Exendin-4 or a functional fragment thereof. In some embodiments, the therapeutic agent comprises a ligand for a cell receptor. In some embodiments, the cell receptor is a growth factor receptor, a G-protein coupled receptor, or a toll-like receptor.
In some embodiments, the therapeutic agent is a nucleic acid.
Further provided herein are compositions comprising the delivery vehicle, or the ceramide-therapeutic agent complex described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
Other aspects of the present disclosure provide methods of delivering an agent into a cell, across a mucosal surface, or across an endothelial barrier the method comprising contacting the delivery vehicle the ceramide-therapeutic complex with the cell, the mucosal surface, or the endothelial lumenal surface, under conditions appropriate for uptake of the delivery vehicle or the agent into the cell or absorption of the delivery vehicle or the agent across the mucosal surface or endothelial barrier.
Other aspects of the present disclosure provide methods of delivering an agent into a cell or across a mucosal or endothelial surface, the method comprising contacting the composition described herein with the cell, the mucosal surface, or the endothelial lumenal surface, under conditions appropriate for uptake of the composition or the agent into the cell or absorption of the composition or the agent across the mucosal surface or the endothelial barrier.
Other aspects of the present disclosure provide methods of delivering an agent into a cells, across a mucosal surface, or across an endothelial barrier in a subject, the method comprising administering to the subject the delivery vehicle, the ceramide-therapeutic agent, or the composition described herein.
Other aspects of the present disclosure provide methods of enhancing the half-life of an agent in a subject, the method comprising administering to the subject the delivery vehicle, the ceramide-therapeutic agent, or the composition described herein.
Other aspects of the present disclosure provide methods of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the delivery vehicle, the ceramide-therapeutic agent, or the composition described herein, wherein the effective amount is an amount sufficient to ameliorate/reduce the extent to which the disease or condition occurs in the subject. In some embodiments, the delivery vehicle, the ceramide-therapeutic agent complex, or the composition is administered parenterally. In some embodiments, the delivery vehicle, the ceramide-therapeutic agent complex, or the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intrathecally, intraperitoneally, intraarterially, intracardiacally, intraosseously, intraocularly, intravitreally, intranasally or intrapleurally. In some embodiments, the delivery vehicle, the ceramide-therapeutic agent complex, or the composition is administered nonparenterally. In some embodiments, the delivery vehicle, the ceramide-therapeutic agent complex, or the composition is administered orally, sublingually, topically, rectally, or via inhalation. For delivery across tight endothelial barriers, in some embodiments, the ceramide-therapeutic agent complex is delivered intravenously, intramuscularly, or subcutaneously.
The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
Delivery of biologically active molecules across tight mucosal epithelial barriers is a major challenge preventing application of most therapeutic peptides for oral drug delivery. The sorting endosome sorts cargo into four separate pathways: the lysosome pathway, the retrograde pathway to Golgi and ER, the recycling pathway back to the plasma membrane, and (in polarized cells like epithelial and endothelial cells) into the transcytotic pathway that connects one cell surface with the other, allowing for adsorption. The pathways are distinct and do not intersect with each other (e.g., as described in Saslowsky et al., J Biol Chem. Sep. 6; 288(36):25804-9, incorporated herein by reference). It is possible that lipid sorting into these different pathways may be regulated by the molecular shape of the lipid allowing them to move into highly-curved membrane buds and tubules that serve the recycling, retrograde and transcytotic pathways. Additionally, there can be specific sorting into the different pathways via distinct sorting mechanisms.
Glycosphingolipids are present within the outer membrane leaflet of cell membranes. They contain a ligand-binding oligosaccharide domain that faces the extracellular space, and a ceramide domain that anchors the lipid in the membrane bilayer. Ceramides consist of a sphingosine chain (typically C18:1 or C20:1) coupled to a fatty acid that can have diverse structures. The oligosaccharide domain prevents lipid flip-flop between membrane leaflets, causing all the glycosphingolipids to be distributed among intracellular compartments only by vesicular trafficking. Sorting of proteins and certain sphingolipids to various intracellular compartments of eukaryotic cells depends on movement of membranes through the secretory and endocytic pathways by vesicular carriers. For proteins, this occurs according to multiple and hierarchically ordered sorting determinants structurally encoded within the protein itself or within the structure of an associated receptor or chaperone. Methods of using glycosphingolipids isoforms containing a ceramide that comprises fatty acids of different structures (e.g., different fatty acid chain length, with or without double bonds) to deliver an agent (e.g., a therapeutic agent) into a cell or across a mucosal barrier have been described (e.g., in U.S. Pat. No. 9,457,097, incorporated herein by reference).
Simplifying the lipid carrier may simplify their synthesis, amplify their activity in transport of biologies, and promote their clinical translation. It is known that ceramides alone (e.g., without the oligosaccharide group, or simply glycoceramides) can flip flop from one membrane leaflet to another (e.g., as described in Lopez-Montero et al., Biochim Biophys Acta. July; 1798(7): 1348-56, incorporated herein by reference). Provided herein, in some aspects, are the use of ceramides (e.g., ceramides alone or glycoceramides) for trafficking agents (e.g., therapeutic agents) intracellularly or across epithelial or endothelial barriers.
Accordingly, some aspects of the present disclosure provide delivery vehicles comprising a ceramide and an agent to be delivered, wherein the ceramide: (a) does not comprise a fatty acid; or (b) comprises a fatty acid of C1-C28; and wherein the agent is attached to the ceramide. A “delivery vehicle” refers to a molecule or system that delivers an agent (e.g., a therapeutic agent) to a desired location, e.g., without limitation, to enter a cell or to reach a desired cellular compartment (e.g., the endoplasmic reticulum), to reach a desired part in a subject (e.g., an organ), or to reach a diseased site in a subject (e.g., a tumor site). In some embodiments, the delivery vehicle includes the agent to be delivered. In some embodiments, the delivery vehicle is associated with (or attached to) the agent to be delivered. In these situations, complexes comprising the delivery vehicle and the agent to be delivered are formed and termed herein a “ceramide-agent complex.” In some embodiments, the agent is a therapeutic agent and the complex comprising the delivery vehicle and the therapeutic agent is herein termed a “ceramide-therapeutic agent complex.”
A “ceramide,” as used herein, refers to a molecule comprising a sphingosine core structure. A sphingosine is an amino alcohol with an unsaturated hydrocarbon chain that is typically 18-carbon or 20-carbon in length, which forms a primary part of sphingolipids (e.g., ceramides). The unsaturated hydrocarbon chain is attached to the amino acid serine to form the sphingosine. The term “ceramide” encompasses natural ceramides and ceramide analogs (e.g., synthetic or natural ceramide analogs). In some embodiments, the ceramide is a sphingolipid composed of sphingosine and a fatty acid. In some embodiments, the ceramide of the present disclosure is a ceramide analog. For example, the ceramide of the present disclosure may contain additional chemical moieties appended to a natural ceramide or contain modifications compared to a natural ceramide. Non-limiting examples of ceramide analogs that may be used in accordance with the present disclosure include: 2-hydroxy-ceramide, diene-deoxy-ceramide, dihydroceramide, dihydroceramide phosphate, o-acyl-ceramide, ceramide phosphate, sphinganine, and methyl-sphingosine.
In some embodiments, the ceramides of the present disclosure encompass ceramide analogs with the core structure built upon amino acids other than serine (i.e., having a core structure other than sphingosine). For example, in some embodiments, the ceramide described herein comprises amino acid backbones containing ornithine (e.g., N-acyloxyacyl-ornitine, and bacterial cerilipin, as described in Kawai et al., FEMS Immunol Med Microbiol 1999, 23, 67 and Tahara et al., Agric Biol Chem 1976, 40, 243, incorporated herein by reference), tyrosine (e.g., Brominated mololipids from sea sponge as described in Ross et al., J Nat Prod 2000, 63, 501, incorporated herein by reference), glycine (e.g., iso-3-hydroxy heptadecanoic acid-containing lipid from Cytophaga johnsonae, as described in Kawazoe et al., J Bacteriol 1991, 173, 5470, incorporated herein by reference), leucine (e.g., Lipstatin, which is an inhibitor of pancreatic protease, as described in Weibel et al., J Antibiot 1987, 1081, incorporated herein by reference), proline (e.g., N-stearoyl praline as described in Sivasamy et al., JAOCS 2001, 78, 897, incorporated herein by reference), glutamine (e.g., Volicitin, N-(17-hydroxylinolenoyl)-1-glutamine, as described in Pare et al., PNAS 1998, 95, 13971, incorporated herein by reference) or taurine (e.g., N-acyl taurine, as described in Saghatelian et al., Biochemistry 2006, 45, 9007, incorporated herein by reference). Non-limiting, exemplary structures of these ceramide analogs are provided in
A “fatty acid” is a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated. Fatty acids that have double bonds between backbone carbons are known as unsaturated. Fatty acids without double bonds between backbone carbons are known as saturated. The length of a fatty acid chain, is herein referred to using the number of backbone carbons atoms in the fatty acid chain. For example, a fatty acid chain with X number of backbone carbons is expressed as CX herein, wherein X is an integer. In some embodiments, X is 0, meaning the ceramide does not comprise a fatty acid. A ceramide that does not have a fatty acid is a sphingosine (also referred to as a “lyso-ceramide” herein). When a sphingosine is referred to herein, it also means the molecule does not have a sugar moiety. In some embodiments, X is an integer between 1-30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In some embodiments, the ceramide of the present disclosure comprises a fatty acid of C1-C28 (e.g., C1, C2, C3, C4, C5, C6, C1, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, or C28) in length. The number of double bonds between backbone carbons in a fatty acid chain with X number of backbone carbons is expressed as CX:Y, wherein Y is the number of double bonds between backbone carbons and is an integer. For example, a fatty acid chain with 20 backbone carbons and 1 double bond is expressed as “C20:1” herein. In some embodiments, Y is 0, meaning the fatty acid does not contain a double bond between backbone carbons (i.e., a saturated fatty acid). The ceramide chain of the present disclosure, in some embodiments, contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) double bonds between backbone carbons. In some embodiments, the double bond is a cis-double bond. A “cis-double bond” refers to an isoform of a double bond formed between two carbon atoms. In addition to the double bond, other chemical groups (e.g., —H, —CH3, —COOH) also form bonds with the carbon atom involved in the cis-double bond, and “cis” indicates that the chemical groups other than —H are on the same side of the carbon chain. One skilled in the art is familiar with these terms.
In some embodiments, the ceramide comprises a fatty acid of C1-C12 (e.g., for transcytosis applications). In some embodiments, the ceramide comprises a fatty acid of 03-C28 (e.g., for non-transcytosis applications).
In some embodiments, the ceramide comprises a fatty acid of C1-C6 (e.g., C1, C2, C3, C4, C5, or C6) in length. In some embodiments, the fatty acid of C1-C6 has no double bond between two carbon atoms (e.g., any of the backbone carbons). In some embodiments, the ceramide comprises a fatty acid chain of C4. In some embodiments, the ceramide comprises a fatty acid chain of C6. In some embodiments, the fatty acid of C1-C6 comprises at least one cis-double bond (e.g., 1, 2, or more cis double bond).
In some embodiments, the ceramide comprises a fatty acid of C7-C28 (e.g., C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, or C28) in length. In some embodiments, the ceramide comprises a fatty acid chain of C8. In some embodiments, the fatty acid of C7-C28 comprises at least one cis-double bonds between two carbon atoms (e.g., any of the backbone carbons). For example, the fatty acid of C7-C28 may comprise 1-10 cis-double bonds. In some embodiments, the fatty acid of C7-C28 comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-10, or 9-10 cis-double bonds. In some embodiments, the fatty acid of C7-C28 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cis-double bonds. In some embodiments, the fatty acid is C7-C16 and comprises at least one cis-double bonds. In some embodiments, the fatty acid is C17-C28 and comprises at least one cis-double bonds in C1-C18 region of the fatty acid. For example, the at least one cis-double bonds may be in C1-C18, C1-C17, C1-C16, C1-C15, C1-C14, C1-C13, C1-C12, C1-C11, C1-C10, C1-C9, C1-C8, C1-C7, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C18, C2-C17, C2-C16, C2-C15, C2-C14, C2-C13, C2-C12, C2-C11, C2-C10, C2-C9, C2-C8, C2-C7, C2-C6, C2-C5, C2-C4, C2-C3, C3-C18, C3-C17, C3-C16, C3-C15, C3-C14, C3-C13, C3-C12, C3-C11, C3-C10, C3-C9, C3-C8, C3-C7, C3-C6, C3-C5, C3-C4, C4-C18, C4-C17, C4-C16, C4-C15, C4-C14, C4-C13, C4-C12, C4-C11, C4-C10, C4-C9, C4-C8, C4-C7, C4-C6, C4-C5, C5-C18, C5-C17, C5-C16, C5-C15, C5-C14, C5-C13, C5-C12, C5-C11, C5-C10, C5-C9, C5-C8, C5-C7, C5-C6, C6-C18, C6-C17, C6-C16, C6-C15, C6-C14, C6-C13, C6-C12, C6-C11, C6-C10, C6-C9, C6-C8, C6-C7, C7-C18, C7-C17, C7-C16, C7-C15, C7-C14, C7-C13, C7-C12, C7-C11, C7-C10, C7-C9, C7-C8, C8-C18, C8-C17, C8-C16, C8-C15, C8-C14, C8-C13, C8-C12, C8-C11, C8-C10, C8-C9, C9-C18, C9-C17, C9-C16, C9-C15, C9-C14, C9-C13, C9-C12, C9-C11, C9-C10, C10-C18, C10-C17, C10-C16, C10-C15, C10-C14, C10-C13, C10-C12, C10-C11, C11-C18, C11-C17, C11-C16, C11-C15, C11-C14, C11-C13, C11-C12, C12-C18, C12-C17, C12-C16, C12-C15, C12-C14, C12-C13, C13-C18, C13-C17, C13-C16, C13-C15, C13-C14, C14-C18, C14-C17, C14-C16, C14-C15, C15-C18, C15-C17, C15-C16, C16-C18, C16-C17, or C17-C18 region of the fatty acid. In some embodiments, the fatty acid is C17-C28 and the at least one cis-double bonds are in C1-C16 region of the fatty acid. In some embodiments, the fatty acid is C17-C28 and the at least one cis-double bonds are in C1-C14 region of the fatty acid. In some embodiments, the fatty acid of C7-C28 has no double bond between two carbon atoms (e.g., any of the backbone carbons).
In some embodiments, the fatty acid of C7-C28 comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemical moieties (e.g., bulky chemical moieties) appended (e.g., covalently attached) to the fatty acid chain. Non-limiting examples of chemical moieties that may be appended to the C7-C28 fatty acid chain of the ceramide described herein include: branched methylation or acylation, bulky non-polar fluorophores such as BODIPY, aromatic rings, sterols, prenylation, halogenation (e.g., fluorination), and any compound that deviates from the linear structure of a fully saturated hydrocarbon chain. In some embodiments, the fatty acid is C7-C16 and comprises at least one chemical moieties (e.g., bulky chemical moieties) appended to the fatty acid chain. In some embodiments, the fatty acid is C17-C28 and comprises at least one chemical moieties (e.g., bulky chemical moieties) appended to the fatty acid chain in C1-C18 region of the fatty acid. For example, the at least one chemical moieties (e.g., bulky chemical moieties) may be appended in C1-C18, C1-C17, C1-C16, C1-C15, C1-C14, C1-C13, C1-C12, C1-C11, C1-C10, C1-C9, C1-C8, C1-C7, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C18, C2-C17, C2-C16, C2-C15, C2-C14, C2-C13, C2-C12, C2-C11, C2-C10, C2-C9, C2-C8, C2-C7, C2-C6, C2-C5, C2-C4, C2-C3, C3-C18, C3-C17, C3-C16, C3-C15, C3-C14, C3-C13, C3-C12, C3-C11, C3-C10, C3-C9, C3-C8, C3-C7, C3-C6, C3-C5, C3-C4, C4-C18, C4-C17, C4-C16, C4-C15, C4-C14, C4-C13, C4-C12, C4-C11, C4-C10, C4-C9, C4-C8, C4-C7, C4-C6, C4-C5, C5-C18, C5-C17, C5-C16, C5-C15, C5-C14, C5-C13, C5-C12, C5-C11, C5-C10, C5-C9, C5-C8, C5-C7, C5-C6, C6-C18, C6-C17, C6-C16, C6-C15, C6-C14, C6-C13, C6-C12, C6-C11, C6-C10, C6-C9, C6-C8, C6-C7, C7-C18, C7-C17, C7-C16, C7-C15, C7-C14, C7-C13, C7-C12, C7-C11, C7-C10, C7-C9, C7-C8, C8-C18, C8-C17, C8-C16, C8-C15, C8-C14, C8-C13, C8-C12, C8-C11, C8-C10, C8-C9, C9-C18, C9-C17, C9-C16, C9-C15, C9-C14, C9-C13, C9-C12, C9-C11, C9-C10, C10-C18, C10-C17, C10-C16, C10-C15, C10-C14, C10-C13, C10-C12, C10-C11, C11-C18, C11-C17, C11-C16, C11-C15, C11-C14, C11-C13, C11-C12, C12-C18, C12-C17, C12-C16, C12-C15, C12-C14, C12-C13, C13-C18, C13-C17, C13-C16, C13-C15, C13-C14, C14-C18, C14-C17, C14-C16, C14-C15, C15-C18, C15-C17, C15-C16, C16-C18, C16-C17, or C17-C18 region of the fatty acid. In some embodiments, the fatty acid is C17-C28 and the at least one chemical moieties (e.g., bulky chemical moieties) are appended in C1-C16 region of the fatty acid. In some embodiments, the fatty acid is C17-C28 and the at least one chemical moieties (e.g., bulky chemical moieties) are appended in C1-C14 region of the fatty acid.
In some embodiments, any of the ceramide of the present disclosure (e.g., a ceramide with or without a fatty acid chain) further comprises a sugar (e.g., a glycoceramide). In some embodiments, the ceramide comprises a fatty acid chain of C1-C6 and further comprises a sugar. In some embodiments, the ceramide comprises a fatty acid chain of C7-C28 and further comprises a sugar. In some embodiments, the ceramide does not comprise a fatty acid chain (i.e., a sphingosine or a lyso-ceramide) and further comprises a sugar. A ceramide that does not contain a fatty acid chain but comprises a sugar is also referred to herein as a “glycosphingosine” or a “lyso-glycoceramide.”
A “glycoceramide” refers to a ceramide comprising a sugar attached to its primary hydroxyl group (
In some embodiments, the ceramide of the present disclosure does not comprise a sugar. In some embodiments, the agent is attached to the ceramide (e.g., to the primary hydroxyl group of the ceramide, see
The agent may be attached to the ceramide by any methods known in the art. In some embodiments, the agent is attached non-covalently, e.g., without limitation, by van der Waals forces, hydrophobic interaction, hydrogen bond interaction, or ionic interactions.
In some embodiments, the agent is attached covalently. For example, in some embodiments, the ceramide (e.g., the primary hydroxyl group of the ceramide) or the sugar of the glycoceramide may be functionalized with a reactive chemical group. One example of such reactive group is a “click chemistry handle.” Click chemistry is a chemical approach introduced describes chemistry tailored to generate substances quickly and reliably by joining small units together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395). Exemplary coupling reactions (some of which may be classified as “Click chemistry”) include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgon cycloaddition; thiol-yne addition; imine formation; and Michael additions (e.g., maleimide addition). Non-limiting examples of a click chemistry handle include an azide handle, an alkyne handle, or an aziridine handle. Azide is the anion with the formula N3-. It is the conjugate base of hydrazoic acid (HN3). N3- is a linear anion that is isoelectronic with CO2, NCO—, N2O, NO2+ and NCF. Azide can be described by several resonance structures, an important one being —N═N+=N—. An alkyne is an unsaturated hydrocarbon containing at least one carbon-carbon triple bond. The simplest acyclic alkynes with only one triple bond and no other functional groups form a homologous series with the general chemical formula CnH2n-2. Alkynes are traditionally known as acetylenes, although the name acetylene also refers specifically to C2H2, known formally as ethyne using IUPAC nomenclature. Like other hydrocarbons, alkynes are generally hydrophobic but tend to be more reactive. Aziridines are organic compounds containing the aziridine functional group, a three-membered heterocycle with one amine group (—NH—) and two methylene bridges (˜CH2-). The parent compound is aziridine (or ethylene imine), with molecular formula C2H5N.
Other non-limiting, exemplary reactive groups include: acetals, ketals, hemiacetals, and hemiketals, carboxylic acids, strong non-oxidizing acids, strong oxidizing acids, weak acids, acrylates and acrylic acids, acyl halides, sulfonyl halides, chloroformates, alcohols and polyols, aldehydes, alkynes with or without acetylenic hydrogen amides and imides, amines, aromatic, amines, phosphines, pyridines, anhydrides, aryl halides, azo, diazo, azido, hydrazine, and azide compounds, strong bases, weak bases, carbamates, carbonate salts, chlorosilanes, conjugated dienes, cyanides, inorganic, diazonium salts, epoxides, esters, sulfate esters, phosphate esters, thiophosphate esters borate esters, ethers, soluble fluoride salts, fluorinated organic compounds, halogenated organic compounds, halogenating agents, aliphatic saturated hydrocarbons, aliphatic unsaturated hydrocarbons, hydrocarbons, aromatic, insufficient information for classification, isocyanates and isothiocyanates, ketones, metal hydrides, metal alkyls, metal aryls, and silanes, alkali metals, nitrate and nitrite compounds, inorganic, nitrides, phosphides, carbides, and silicides, nitriles, nitro, nitroso, nitrate, nitrite compounds, organic, non-redox-active inorganic compounds, organometallics, oximes, peroxides, organic, phenolic salts, phenols and cresols, polymerizable compounds, quaternary ammonium and phosphonium salts, strong reducing agents, weak reducing agents, acidic salts, basic salts, siloxanes, inorganic sulfides, organic sulfides, sulfite and thiosulfate salts, sulfonates, phosphonates, organic thiophosphonates, thiocarbamate esters and salts, and dithiocarbamate esters and salts. The agent to be attached to the ceramide (e.g., via the reactive chemical group) may contain a corresponding chemical group that reacts with the oligosaccharide or ceramide, thus resulting in covalent attachment. For example, an agent that is a protein or polypeptide can be coupled via its N- or C-terminus, or via an endogenous residue (e.g., lysine) by chemical cross-linking.
In some embodiments, the agent is attached to the ceramide via a linker. In some embodiments, the linker is a pseudo-glycopeptide linker (e.g., see
In some embodiments, the linker is a peptide linker. In some embodiments, the agent is a protein or a peptide. In such instances, one or more of the amino acids of the protein or peptide may be modified to include a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for attaching to the oligosaccharide.
In some embodiments, the linker is a cleavable linker. A “cleavable linker” refers to a linker that can be cleaved by a chemical agent or an enzyme and thus release the agent from the ceramide carrier. In some embodiments, the cleavable linker comprises an ester linkage (e.g., the linker can be a peptide linker comprising an ester linker). In some embodiments, the ester linkage can be cleaved by an esterase (e.g., leukocyte esterase whose level is elevated at sites of inflammation, or carboxylesterase hCE-2 that is specific to gastrointestinal endosomes).
In some embodiments, the cleavable linker comprises a cleavage motif for an endosomal protease. An “endosomal protease” refers to a protease that is present in endosomes. It is herein interchangeably referred to as a “lysosomal protease.” Endosomal proteases belong to the aspartic, cysteine, or serine proteinase families of hydrolytic enzymes. Endosomal proteases are expressed ubiquitously, and in a tissue- or cell type-specific manner and are usually detected within all vesicles of the endocytic pathway. Reference and classification of endosomal proteases is available in the art. For example, lists of known endosomal proteases can be found in the MERGES database (merops.sanger.ac.uk). In some embodiments, the endosomal protease is furin or matriptase. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Furin cleaves proteins downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg/Lys)-Arg′). Matriptase is a trypsin-like integral-membrane serine peptidase and cleaves substrates with Arg or Lys at the P1 position and prefers small side-chain amino acids, such as Ala and Gly, at the P2 position.
In some embodiments, the cleavable linker comprises a disulfide linkage. A “disulfide linkage” is also referred to as a disulfide bond or S—S bond, which is a covalent bond derived from two thiol groups. Disulfide bonds can be formed by oxidation of sulfhydryl groups and can be cleaved via reduction (e.g., via using reductants such as tris(2-carboxyethyl)phosphine (TCEP), 2-Mercaptoethanol (β-ME) or dithiothreitol (DTT)).
The ceramides described herein are able to act as delivery vehicles to deliver an agent across cell membrane or across mucosal barrier and direct intracellular trafficking of the agent. For example, in some embodiments, the ceramide-agent complex are directed by the ceramide to a desired intracellular location, e.g., the endoplasmic reticulum (ER). In some embodiments, the ceramide-agent complex is directed by the ceramide away from degradative pathways (e.g., lysosome). As such, in some embodiments, the cellular half-life of the agent is prolonged when the agent is part of the ceramide-agent complex, compared to when the agent is delivered into cells alone. In some embodiments, the cellular half-life of the agent is prolonged by at least 20% when the agent is part of the ceramide-agent complex, compared to when the agent is delivered into cells alone. In some embodiments, the cellular half-life of the agent is prolonged by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 500 fold, at least 1000 fold or more, when the agent is part of the ceramide-agent complex, compared to when the agent is delivered into cells alone. In some embodiments, the cellular half-life of the agent is prolonged by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 500 fold, 1000 fold or more, when the agent is part of the ceramide-agent complex, compared to when the agent is delivered into cells alone.
In some embodiments, the agent is delivered across an epithelial or endothelial barrier (e.g., a mucosal barrier) by transcytosis, when the agent is attached to the ceramide to form a ceramide-agent complex. Mucosal barrier is composed of compact epithelial cell lining (e.g., in the stomach or in the intestines). The intestinal mucosal barrier, also referred to as intestinal barrier, refers to the property of the intestinal mucosa that ensures adequate containment of undesirable luminal contents within the intestine while preserving the ability to absorb nutrients. The gastric mucosal barrier is the property of the stomach that allows it to safely contain the gastric acid required for digestion.
In some embodiments, the agent is not able to cross an epithelial or endothelial barrier (e.g., a mucosal barrier) alone and is able to cross mucosal barriers in complex with the ceramides described herein. In some embodiments, the delivery of the agent across an epithelial or endothelial barrier (e.g., a mucosal barrier) is enhanced (e.g., by at least 20%) when the agent is in complex with the ceramides described herein, compared to when the agent is delivered alone. For example, the delivery of the agent across an epithelial or endothelial barrier (e.g., a mucosal barrier) is enhanced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 500 fold, at least 1000 fold or more, when the agent is in complex with the ceramides described herein, compared to when the agent is delivered alone. In some embodiments, the delivery of the agent across an epithelial or endothelial barrier (e.g., a mucosal barrier) is enhanced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 500 fold, 1000 fold or more, when the agent is in complex with the ceramides described herein, compared to when the agent is delivered alone.
Other aspects of the present disclosure provide agents that may be delivered by the ceramides described herein. The agent may be any bioactive agent or therapeutic agent. A “therapeutic agent” refers to an agent that has therapeutic effects to a disease or disorder. The complex between the ceramide and the therapeutic agent is referred to herein as the “ceramide-therapeutic agent complex.” A therapeutic agent may be, without limitation, proteins, peptides, nucleic acids, small molecules drugs, polysaccharides and carbohydrates, lipids, glycoproteins, small molecules, synthetic organic and inorganic drugs exerting a biological effect when administered to a subject, and combinations thereof. In some embodiments, the therapeutic agent is an anti-inflammatory agent, a vaccine antigen, a vaccine adjuvant, an antibody, a ScFv, a nanobody, and enzyme, an anti-cancer drug or chemotherapeutic drug, a clotting factor, a hormone, a steroid, a cytokine, an antibiotic, or a drug for the treatment of cardiovascular disease, an infectious disease, an autoimmune disease, allergy, a blood disorder, a metabolic disorder, a skin disease, an eye disease, a lysosomal storage disease, or a neurological disease. In some embodiments, the therapeutic agent is a protein or a peptide. In some embodiments, the protein or peptide is glucagon-like peptide-1 (GLP-1), or a functional fragment thereof. In some embodiments, the protein or peptide is Exendin-4, or a functional fragment thereof.
“Glucagon-like peptide-1 (GLP-1)” is a 30 amino acid long peptide hormone deriving from the tissue-specific posttranslational processing of the proglucagon gene. It is produced and secreted by intestinal enteroendocrine L-cells and certain neurons within the nucleus of the solitary tract in the brainstem upon food consumption. The initial product GLP-1(1-37) is susceptible to amidation and proteolytic cleavage which gives rise to the two truncated and equipotent biologically active forms, GLP-1 (7-36)amide and GLP-1(7-37). Active GLP-1 composes two α-helices from amino acid position 13-20 and 24-35 separated by a linker region. GLP-1 possesses several physiological properties that make it (and its functional analogs) a subject of intensive investigation as a potential treatment of diabetes mellitus. Further, GLP-1 is has the ability to decrease blood sugar levels in a glucose-dependent manner by enhancing the secretion of insulin. Thus, GLP-1 has been associated with numerous regulatory and protective effects. GLP-1-based treatment has been associated with weight loss and lower hypoglycemia risks, two very important aspects of a life with diabetes.
“Exendin-4” is a peptide agonist of the glucagon-like peptide (GLP) receptor that promotes insulin secretion. Exendin-4 binds to the intact human Glucagon-like peptide-1 receptor (GLP-1R) in a similar way to GLP-1 and bears a 50% amino acid homology to GLP-1. Exendin-4 facilitates glucose control via augmentation of pancreas response (i.e. increases insulin secretion) in response to eating meals, suppressing pancreatic release of glucagon in response to eating, reducing rate of gastric emptying, suppressing appetite, and reducing liver fat content. In some embodiments, the therapeutic agent is a fusion protein comprise GLP-1 or a functional fragment thereof and Exendin-4 or a functional fragment thereof.
In some embodiments, the therapeutic agent is a vaccine antigen. A “vaccine antigen” is a molecule or moiety that, when administered to a subject, activates or increases the production of antibodies that specifically bind the antigen. In some embodiments, an antigen is a protein or a polysaccharide. Antigens of pathogens are well known to those of skill in the art and include, but are not limited to parts (coats, capsules, cell walls, flagella, fimbriae, and toxins) of bacteria, viruses, and other microorganisms. A vaccine typically comprises an antigen, and is intentionally administered to a subject to induce an immune response in the recipient subject. The antigen may be from a pathogenic virus, bacteria, or fungi.
Examples of pathogenic virus include, without limitation: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses′); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).
Examples of pathogenic bacteria include, without limitation: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic spp.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, and Actinomyces israelii.
Examples of pathogenic fungi include, without limitation: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. Other infectious organisms (i.e., protists) include: Plasmodium falciparum and Toxoplasma gondii.
In some embodiments, the therapeutic agent is an agent that induces immunological tolerance. Immunologic tolerance is a state of immune unresponsiveness specific to a particular antigen or set of antigens induced by previous exposure to that antigen or set. In some embodiments, the immunologic tolerance is oral tolerance. Oral tolerance is the state of local and systemic immune unresponsiveness that is induced by oral administration of innocuous antigen such as food proteins. In some embodiments, the therapeutic agent is an agent for induce immunological tolerance for the treatment of allergy or autoimmune disease (e.g., multiple sclerosis).
Other non-limiting examples of agents that may be delivered using the ceramides described herein are provided.
Non-limiting, exemplary chemopharmaceutically compositions that may be used in the liposome drug delivery systems of the present disclosure include, Actinomycin, All-trans retinoic acid, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Tempo side, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, and Vinorelbine.
Examples of antineoplastic compounds include, without limitation: nitrosoureas, e.g., carmustine, lomustine, semustine, strepzotocin; Methylhydrazines, e.g., procarbazine, dacarbazine; steroid hormones, e.g., glucocorticoids, estrogens, progestins, androgens, tetrahydrodesoxycaricosterone, cytokines and growth factors; Asparaginase.
Examples of immunoactive compounds include, without limitation:: immunosuppressives, e.g., pyrimethamine, trimethopterin, penicillamine, cyclosporine, azathioprine; immunostimulants, e.g., levamisole, diethyl dithiocarbamate, enkephalins, endorphins.
Examples of antimicrobial compounds include, without limitation: antibiotics, e.g., beta lactam, penicillin, cephalosporins, carbapenims and monobactams, beta-lactamase inhibitors, aminoglycosides, macrolides, tetracyclins, spectinomycin; Antimalarials, Amebicides, Antiprotazoal, Antifungals, e.g., amphotericin beta or clotrimazole, antiviral, e.g., acyclovir, idoxuridine, ribavirin, trifluridine, vidarbine, gancyclovir.
Examples of parasiticides include, without limitation: antihalmintics, Radiopharmaceutics, gastrointestinal drugs.
Examples of hematologic compounds include, without limitation: immunoglobulins; blood clotting proteins; e.g., antihemophilic factor, factor IX complex; anticoagulants, e.g., dicumarol, heparin Na; fibrolysin inhibitors, tranexamic acid.
Examples of cardiovascular drugs include, without limitation: peripheral antiadrenergic drugs, centrally acting antihypertensive drugs, e.g., methyldopa, methyldopa HCl; antihypertensive direct vasodilators, e.g., diazoxide, hydralazine HCl; drugs affecting renin-angiotensin system; peripheral vasodilators, phentolamine; antianginal drugs; cardiac glycosides; inodilators; e.g., amrinone, milrinone, enoximone, fenoximone, imazodan, sulmazole; antidysrhythmic; calcium entry blockers; drugs affecting blood lipids; ranitidine, bosentan, rezulin.
Examples of respiratory drugs include, without limitation: sypathomimetic drugs: albuterol, bitolterol mesylate, dobutamine HCl, dopamine HCl, ephedrine SO, epinephrine, fenfluramine HCl, isoproterenol HCl, methoxamine HCl, norepinephrine bitartrate, phenylephrine HCl, ritodrine HCl; cholinomimetic drugs, e.g., acetylcholine Cl; anticholinesterases, e.g., edrophonium Cl; cholinesterase reactivators; adrenergic blocking drugs, e.g., acebutolol HCl, atenolol, esmolol HCl, labetalol HCl, metoprolol, nadolol, phentolamine mesylate, propanolol HCl; antimuscarinic drugs, e.g., anisotropine methylbromide, atropine SO4, clinidium Br, glycopyrrolate, ipratropium Br, scopolamine HBr.
Examples of neuromuscular blocking drugs include, without limitation: depolarizing, e.g., atracurium besylate, hexafluorenium Br, metocurine iodide, succinylcholine Cl, tubocurarine Cl, vecuronium Br; centrally acting muscle relaxants, e.g., baclofen.
Examples of neurotransmitters and neurotransmitter agents include, without limiation: acetylcholine, adenosine, adenosine triphosphate, amino acid neurotransmitters, e.g., excitatory amino acids, GABA, glycine; biogenic amine neurotransmitters, e.g., dopamine, epinephrine, histamine, norepinephrine, octopamine, serotonin, tyramine; neuropeptides, nitric oxide, K+ channel toxins,
Examples of antiparkinson drugs include, without limiation: amaltidine HCl, benztropine mesylate, e.g., carbidopa.
Examples of diuretic drugs include, without limitation: dichlorphenamide, methazolamide, bendroflumethiazide, polythiazide.
Examples of uterine, antimigraine drugs include, without limitation: carboprost tromethamine, mesylate, methysergide maleate.
Examples of hormones include, without limitation: pituitary hormones, e.g., chorionic gonadotropin, cosyntropin, menotropins, somatotropin, iorticotropin, protirelin, thyrotropin, vasopressin, lypressin; adrenal hormones, e.g., beclomethasone dipropionate, betamethasone, dexamethasone, triamcinolone; pancreatic hormones, e.g., glucagon, insulin; parathyroid hormone, e.g., dihydrochysterol; thyroid hormones, e.g., calcitonin etidronate disodium, levothyroxine Na, liothyronine Na, liotrix, thyroglobulin, teriparatide acetate; antithyroid drugs; estrogenic hormones; progestins and antagonists, hormonal contraceptives, testicular hormones; gastrointestinal hormones: cholecystokinin, enteroglycan, galanin, gastric inhibitory polypeptide, epidermal growth factor-urogastrone, gastric inhibitory polypeptide, gastrin-releasing peptide, gastrins, pentagastrin, tetragastrin, motilin, peptide YY, secretin, vasoactive intestinal peptide, sincalide.
Examples of enzymes include, without limitation: lysosomal storage enzymes, hyaluronidase, streptokinase, tissue plasminogen activator, urokinase, PGE-adenosine deaminase, oxidoreductases, transferases, polymerases, hydrolases, lyases, synthases, isomerases, and ligases, digestive enzymes (e.g., proteases, lipases, carbohydrases, and nucleases). In some embodiments, the enzyme is selected from the group consisting of lactase, beta-galactosidase, a pancreatic enzyme, an oil-degrading enzyme, mucinase, cellulase, isomaltase, alginase, digestive lipases (e.g., lingual lipase, pancreatic lipase, phospholipase), amylases, cellulases, lysozyme, proteases (e.g., pepsin, trypsin, chymotrypsin, carboxypeptidase, elastase), esterases (e.g. sterol esterase), disaccharidases (e.g., sucrase, lactase, beta-galactosidase, maltase, isomaltase), DNases, and RNases.
Examples of intravenous anesthetics include, without limitation: droperidol, etomidate, fetanyl citrate/droperidol, hexobarbital, ketamine HCl, methohexital Na, thiamylal Na, thiopental Na.
Examples of antiepileptics include, without limitation, carbamazepine, clonazepam, divalproex Na, ethosuximide, mephenytoin, paramethadione, phenytoin, primidone.
Examples of peptides and proteins that may be used as therapeutic agents include, without limiation: ankyrins, arrestins, bacterial membrane proteins, clathrin, connexins, dystrophin, endothelin receptor, spectrin, selectin, cytokines; chemokines; growth factors, insulin, erythropoietin (EPO), tumor necrosis factor (TNF), neuropeptides, neuropeptide Y, neurotensin, transforming growth factor alpha, transforming growth factor beta, interferon (IFN), and hormones, growth inhibitors, e.g., genistein, steroids etc; glycoproteins, e.g., ABC transporters, platelet glycoproteins, GPIb-IX complex, GPIIb-IIIa complex, vitronectin, thrombomodulin, CD4, CD55, CD58, CD59, CD44, lymphocye function-associated antigen, intercellular adhesion molecule, vascular cell adhesion molecule, Thy-1, antiporters, CA-15-3 antigen, fibronectins, laminin, myelin-associated glycoprotein, GAP, GAP-43, Exendin-4, and GLP-1.
Examples of cytokines and cytokine receptors include, without limitation: interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor, IL-9 receptor, IL-10 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-14 receptor, IL-15 receptor, IL-16 receptor, IL-17 receptor, IL-18 receptor, lymphokine inhibitory factor, macrophage colony stimulating factor, platelet derived growth factor, stem cell factor, tumor growth factor beta, tumor necrosis factor, lymphotoxin, Fas, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, interferon-alpha, interferon-beta, interferon-gamma.
Examples of growth factors and protein hormones include, without limitation: erythropoietin, angiogenin, hepatocyte growth factor, fibroblast growth factor, keratinocyte growth factor, nerve growth factor, tumor growth factor-alpha, thrombopoietin, thyroid stimulating factor, thyroid releasing hormone, neurotrophin, epidermal growth factor, VEGF, ciliary neurotrophic factor, LDL, somatomedin, insulin growth factor, insulin-like growth factor I and II.
Examples of chemokines include, without limitation: ENA-78, EEC, GRO-alpha, GRO-beta, GRO-gamma, HRG, LIE, IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP-1 alpha, MIP-1beta, MIG, MDC, NT-3, NT-4, SCF, LIE, leptin, RANTES, lymphotactin, eotaxin-1, eotaxin-2, TARC, TECK, WAP-1, WAP-2, GCP-1, GCP-2; alpha-chemokine receptors: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7; beta-chemokine receptors: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7.
In some embodiments, antibodies that may be delivered using the delivery vehicle described herein target antigens including, without limitation: (a) anti-cluster of differentiation antigen CD-1 through CD-166 and the ligands or counter receptors for these molecules; (b) anti-cytokine antibodies, e.g., anti-IL-1 through anti-IL-18 and the receptors for these molecules; (c) anti-immune receptor antibodies, antibodies against T cell receptors, major histocompatibility complexes I and II, B cell receptors, selectin killer inhibitory receptors, killer activating receptors, OX-40, MadCAM-1, Gly-CAM1, integrins, cadherens, sialoadherens, Fas, CTLA-4, Fc.gamma.-receptors, Fcalpha-receptors, Fc.epsilon.-receptors, Fc.mu.-receptors, and their ligands; (d) anti-metalloproteinase antibodies, e.g., collagenase, MMP-1 through MMP-8, TIMP-1, TIMP-2; anti-cell lysis/proinflammatory molecules, e.g., perforin, complement components, prostanoids, nitron oxide, thromboxanes; and (e) anti-adhesion molecules, e.g., carcioembryonic antigens, lamins, fibronectins.
Non-limiting, exemplary antibodies and fragments thereof include: bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), alemtuzumab (CAMPATH®, indicated for B cell chronic lymphocytic leukemia), gemtuzumab (MYLOTARG®, hP67.6, anti-CD33, indicated for leukemia such as acute myeloid leukemia), rituximab (RITUXAN®), tositumomab (BEXXAR®, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that binds simultaneously to HER-2/neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI)), oregovomab (OVAREX®, indicated for ovarian cancer), edrecolomab (PANOREX®), daclizumab (ZENAPAX®), palivizumab (SYNAGIS®, indicated for respiratory conditions such as RSV infection), ibritumomab tiuxetan (ZEVALIN®, indicated for Non-Hodgkin's lymphoma), cetuximah (ERBITUX®), MDX-447, MDX-22, MDX-220 (anti-TAG-72), IOR-C5, IOR-T6 (anti-CD1), IOR EGF/R3, celogovab (ONCOSCINT® OV103), epratuzumab (LYMPHOCIDE®), pemtumomab (THERAGYN®) and Gliomab-H (indicated for brain cancer, melanoma). Other antibodies and antibody fragments are contemplated and may be used in accordance with the disclosure. In some embodiments, the therapeutic agent is a nanobody. A “nanobody” is a therapeutic protein based on single-domain antibody fragments that contain the unique structural and functional properties of naturally-occurring heavy chain only antibodies. In some embodiments, the nanobody is anti-inflammatory. In some embodiments, the nanobody is against pathogenic agents (e.g., anthrax).
In some embodiments, the therapeutic agent is a ligand for a cell receptor (e.g., without limitation, a growth factor receptor, a G-protein coupled receptor, or a toll-like receptor).
A regulatory protein may be, in some embodiments, a transcription factor or a immunoregulatory protein. Non-limiting, exemplary transcriptional factors include: those of the NFkB family, such as Rel-A, c-Rel, Rel-B, p50 and p52; those of the AP-1 family, such as Fos, FosB, Fra-1, Fra-2, Jun, JunB and JunD; ATF; CREB; STAT-1, -2, -3, -4, -5 and -6; NFAT-1, -2 and -4; MAE; Thyroid Factor; IRE; Oct-1 and -2; NF-Y; Egr-1; and USF-43, EGR1, Sp1, and E2F1.
Examples of antiviral agents include, without limitation: reverse transcriptase inhibitors and nucleoside analogs, e.g. ddI, ddC, 3TC, ddA, AZT; protease inhibitors, e.g., Invirase, ABT-538; inhibitors of in RNA processing, e.g., ribavirin.
Other non-limiting examples of known therapeutics which may be delivered by coupling to a ceramide described herein include:
(a) Capoten, Monopril, Pravachol, Avapro, Plavix, Cefzil, Duricef/Ultracef, Azactam, Videx, Zerit, Maxipime, VePesid, Paraplatin, Platinol, Taxol, UFT, Buspar, Serzone, Stadol NS, Estrace, Glucophage (Bristol-Myers Squibb);
(b) Ceclor, Lorabid, Dynabac, Prozac, Darvon, Permax, Zyprexa, Humalog, Axid, Gemzar, Evista (Eli Lily);
(c) Vasotec/Vaseretic, Mevacor, Zocor, Prinivil/Prinizide, Plendil, Cozaar/Hyzaar, Pepcid, Prilosec, Primaxin, Noroxin, Recombivax HB, Varivax, Timoptic/XE, Trusopt, Proscar, Fosamax, Sinemet, Crixivan, Propecia, Vioxx, Singulair, Maxalt, Ivermectin (Merck & Co.);
(d) Diflucan, Unasyn, Sulperazon, Zithromax, Trovan, Procardia XL, Cardura, Norvasc, Dofetilide, Feldene, Zoloft, Zeldox, Glucotrol XL, Zyrtec, Eletriptan, Viagra, Droloxifene, Aricept, Lipitor (Pfizer);
(e) Vantin, Rescriptor, Vistide, Genotropin, Micronase/Glyn./Glyb., Fragmin, Total Medrol, Xanax/alprazolam, Sermion, Halcion/triazolam, Freedox, Dostinex, Edronax, Mirapex, Pharmorubicin, Adriamycin, Camptosar, Remisar, Depo-Provera, Caverject, Detrusitol, Estring, Healon, Xalatan, Rogaine (Pharmacia & Upjohn);
(f) Lopid, Accrupil, Dilantin, Cognex, Neurontin, Loestrin, Dilzem, Fempatch, Estrostep, Rezulin, Lipitor, Omnicef, FemHRT, Suramin, Clinafloxacin (Warner Lambert).
Non-limiting examples of therapeutic agents for eye diseases include: Anti-infective drugs (e.g., Aciclovir, Chloramphenicol, Ciprofloxacin, Gentamicin, Neomycin, Polymyxin B); Anti-inflammatory drugs (e.g., Betamethasone, Dexamethasone, Emedastine, Nedocromil sodium, Prednisolone, Sodium cromoglicate); Artificial tears (e.g., Carmellose, Hydroxyethylcellulose, Hypromellose, Polyvinyl alcohol); and Mydriatics (e.g., Atropine, cyclopentolate, Phenylephrine).
Further non-limiting examples of therapeutic agents which may be delivered by the ceramide-therapeutic agent complex of the present invention may be found in: Goodman and Gilman's The Pharmacological Basis of Therapeutics. 9th ed. McGraw-Hill 1996, incorporated herein by reference.
The delivery vehicle comprising a ceramide and an agent to be delivered, or a ceramide-agent complex (e.g., a ceramide-therapeutic agent) complex may be formulated into pharmaceutical compositions. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. “Pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. A “pharmaceutically acceptable carrier” may be a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the patient (e.g., physiologically compatible, sterile, physiologic pH, etc.). The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
The formulation of the pharmaceutical composition may dependent upon the route of administration. Injectable preparations suitable for parenteral administration or intratumoral, peritumoral, intralesional or perilesional administration include, for example, sterile injectable aqueous or oleaginous suspensions and may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 propanediol or 1,3 butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the anti-inflammatory agent, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the anti-inflammatory agent is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term release, are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
In some embodiments, the pharmaceutical compositions used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. The pharmaceutical composition ordinarily will be stored in lyophilized form or as an aqueous solution if it is highly stable to thermal and oxidative denaturation. The pH of the preparations typically will be about from 6 to 8, although higher or lower pH values can also be appropriate in certain instances.
Other aspects of the present disclosure provide methods of delivering an agent (e.g., a therapeutic agent) into a cell or across a mucosal surface or an endothelial barrier, the method comprising contacting the delivery vehicle, the ceramide-agent complex (e.g., the ceramide-therapeutic agent complex), or the pharmaceutical composition comprising the delivery vehicle or the ceramide-agent complex (e.g., the ceramide-therapeutic agent complex) with the cell, the mucosal surface, or the endothelial lumenal surface, under conditions appropriate for uptake of the delivery vehicle or the agent into the cell or absorption of the delivery vehicle or the agent across the mucosal surface or the endothelial barrier (e.g., via transcytosis). In some embodiments, the delivery vehicle, the ceramide-agent complex, or the pharmaceutical composition comprising the delivery vehicle or the ceramide-agent complex (e.g., the ceramide-therapeutic agent complex) are administered to a subject.
Methods of delivering agents (e.g., therapeutic agents) to different organs via intravenous infusion of the delivery vehicle or the ceramide-agent complex are also provided. Such organs may be, for example, without limitation, skeleton, joints, muscles, tendons, various types of glands, esophagus, stomach, small intestine, large intestine, liver, pancreas, pharynx, larynx, trachea, bronchi, lungs, diaphragm, kidneys, ureters, bladder, urethra, ovaries, uterus, prostate, heart, lymph nodes, bone marrow, thymus, spleen, brain, and spinal cord. In some embodiments, the delivery vehicle or the ceramide-agent complex is delivered across an endothelial barrier (e.g., the endothelial barrier in the heart or brain) when it reaches the organ after intravenous infusion. In some embodiments, the intravenously infused delivery vehicle or the ceramide-agent complex are delivered to different organs and are sorted into intracellular compartments such as the lysosome (e.g., for lysosomal replacement therapies) or ER (e.g., for addressing protein folding problems). Delivery to liver after intravenous infusion of the delivery vehicle or the ceramide-agent complex greatly enhances (e.g., by at least 20%) the delivery of the agent to the liver, compared to delivering the agent alone.
Methods of enhancing the half-life of an agent in a subject are provided, the method comprising administering to the subject the delivery vehicle, the ceramide-therapeutic agent complex, or the composition described herein.
Methods of treating a disease or condition in a subject in need thereof are provided, the method comprising administering to the subject an effective amount of the delivery vehicle, the ceramide-therapeutic agent complex, or the composition described herein, wherein the effective amount is an amount sufficient to ameliorate/reduce the extent to which the disease or condition occurs in the subject. The disease may be any disease that can be treated by the agents described herein. In some embodiments, the disease is infection, allergy, autoimmune diseases (e.g., multiple sclerosis), liver diseases, lung diseases, neurological diseases, eye diseases or cancer.
An “effective amount” is the amount necessary or sufficient to have a desired effect in a subject. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and other factors within the knowledge and expertise of the health care practitioner. For example, an effective amount could be that amount necessary to eliminate a tumor, cancer, or bacterial, viral or fungal infection. This amount will vary from individual to individual and can be determined empirically using known methods by one of ordinary skill in the art.
The delivery vehicle, the ceramide-agent complex, or the pharmaceutical composition comprising the delivery vehicle or the ceramide-agent complex (e.g., the ceramide-therapeutic agent complex) may be administered by any route. Routes of administration include enteral routes, such as oral and any other means by which the gastrointestinal tract is involved, and parenteral routes, such as by injection (subcutaneous, intravenous, intramuscular injection) or infusion (typically by intravenous route). In some embodiments, the administration is done intravenously, intramuscularly, intradermally, subcutaneously, intrathecally, intraperitoneally, intraarterially, intracardiacally, intraosseously, intraocularly, intravitreally, intrapleurally, intranasally, or injection into the joint. The injection can be in a bolus or a continuous infusion. In some embodiments, the administration is done nonparenterally (e.g., done orally, sublingually, topically, rectally, via inhalation, nasally, as eye drops, as eye patches, to the cervix, or to the skin). For delivery across tight endothelial barriers, in some embodiments, the ceramide-therapeutic agent complex is delivered intravenously, intramuscularly, or subcutaneously.
Methods of treating a disease or disorder are also provided. The delivery vehicle, the ceramide-agent complex, or the pharmaceutical composition comprising the delivery vehicle or the ceramide-agent complex (e.g., the ceramide-therapeutic agent complex) may be administered to a subject who has, has had or is susceptible to developing one or more conditions/diseases that require or would benefit from treatment. For example, the compositions described herein may be used to treat, prevent or ameliorate immune system deficiencies, infectious diseases (viral, fungal, bacterial or parasitic), autoimmune diseases, diabetes, blood disorders, cancers, metabolic disorders, allergies, inflammatory bowel disease and skin disorders. In addition, gangliosides attached to antigen can be administered to stimulate a subject's response to a vaccine. The antigen is selected from the group consisting of: an antigen that is characteristic of a pathogen, an antigen that is characteristic of an autoimmune disease, an antigen that is characteristic of an allergen and an antigen that is characteristic of a tumor. In some embodiments, the disease or disorder to be treated is diabetes. In some embodiments, the disease or disorder is infection, e.g., by a pathogenic virus, bacteria, or fungi. In some embodiments, the disease or disorder is cancer.
Immune system deficiencies include any disease or disorder in which a subject's immune system is not functioning normally or in which it would be useful to boost the subject's immune response, for example to eliminate a tumor or cancer (e.g. tumors of the brain, lung (e.g. small cell and non-small cell), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or an infection in a subject.
Examples of autoimmune diseases include, without limitation: Addison's disease, diabetes mellitus (type 1), Graves' disease, interstitial cystitis, lupus erythematous, multiple sclerosis and Hashimoto's thyroiditis. Allergic conditions include eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions.
Non-limiting, exemplary cancers include: neoplasms, malignant tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth such that it would be considered cancerous. The cancer may be a primary or metastatic cancer. Cancers include, but are not limited to, adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, cancer of the appendix, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, biliary tract cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma, malignant glioma, glioblastoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoid tumor, carcinoma of unknown origin, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute lymphocytic and myelogenous leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell tumors, Kaposi sarcoma, kidney cancer, renal cell cancer, laryngeal cancer, lip and oral cavity cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenstrom macroglobulinema, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous neck cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndromes, myeloproliferative disorders, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary cancer, plasma cell neoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous neck cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, choriocarcinoma, hematological neoplasm, adult T-cell leukemia, lymphoma, lymphocytic lymphoma, stromal tumors and germ cell tumors, or Wilms tumor. In some embodiments, the cancer is lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, brain and central nervous system cancer, skin cancer, ovarian cancer, leukemia, endometrial cancer, bone, cartilage and soft tissue sarcoma, lymphoma, neuroblastoma, nephroblastoma, retinoblastoma, or gonadal germ cell tumor.
As used herein, the term “treating” refers to the application or administration of the delivery vehicle, the ceramide-therapeutic agent complex, or the composition comprising such to a subject in need thereof. “A subject in need thereof”, refers to an individual who has a disease, a symptom of the disease, or a predisposition toward the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease.
A “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In some embodiments, the non-human animal is a mammal (e.g., rodent (e.g., mouse or rat), primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal.
In some embodiments, the subject is a companion animal (a pet). “A companion animal,” as used herein, refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a research animal. Non-limiting examples of research animals include: rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.
In some embodiments, a “subject in need thereof” refers to a subject that needs treatment of a disease described herein.
Alleviating a disease includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
“Development” or “progression” of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a disease includes initial onset and/or recurrence.
Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the isolated polypeptide or pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the Examples below. The Examples are intended to illustrate some of the benefits of the present disclosure and to describe particular embodiments, but are not intended to exemplify the full scope of the disclosure and, accordingly, do not limit the scope of the disclosure.
Mucosal surfaces represent vast areas where host tissues are separated from the environment only by a delicate but highly effective single layer of columnar epithelial cells, joined by tight junctions that are impermeable to proteins and even small peptides. Proteins non-specific ally taken up directly into the epithelial cell by endocytosis are generally transported to lysosomes for degradation. So far, the lack of rational and efficient methods to circumvent this barrier has prevented the application of most therapeutic proteins and peptides for mucosal drug delivery.
Endothelial cells also form vast and highly restrictive single cell thick barriers that separate most tissues from the blood stream. Except for the vasculature in the intestine, glomerulus, and liver, the healthy non-inflamed endothelial barrier strongly limits the permeability of large molecules; thus preventing access of many protein-based biologies to cells of many tissues—even when the therapeutic proteins are applied intravenously.
The pathway for large solutes (e.g., protein and peptide biologies) to cross simple epithelial and endothelial barriers with highly resistant intercellular tight junctions is by transcytosis—a process of transcellular membrane trafficking that connects one surface of polarized cells with the other (
It was found in non-polarized cells that GM1-ceramides containing “kinked” cis-unsaturated C18:1 or C16:1 fatty acids, or non-native “short chain” C12:0 to C4:0 fatty acids, enter the sorting/recycling endosome of epithelial cells for transport to various intracellular destinations: including the recycling pathway and retrograde pathway to Golgi and ER. These lipids do not traffic into the late endosome-lysosome pathway. In contrast, the GM1-sphingolipids with long saturated fatty acid chains (C16:0 or longer) sort efficiently into late endosomes and lysosomes (
In polarized epithelial cells, another sorting event emerges from the sorting endosome and leads into the transcytotic pathway. This pathway allows for the short- or unsaturated GM1-ceramides to cross the cell to the opposite cell surface, thus breeching the epithelial barrier by transcytosis (
Based on these results, the idea that the glycosphingolipids may be harnessed as trafficking vehicles for biologic drug delivery was tested. A all D-isomer amino acid reporter-peptide carrying a reactive aminooxy C-terminal residue for coupling to sialic acid of the glycoceramide GM1, a biotin group for rapid and high affinity isolation, and an alkyne group for coupling to cargo (e.g., for detection via Alexa Fluor 488) was developed (
Structure-function studies showed that certain novel short-chain ceramide species allowed for transcellular transport (transcytosis) of the pep tide-GM1 fusion molecule across epithelial monolayers in vitro as assessed biochemically (
When applied orally (by gastric gavage) to mice in vivo, the pep tide-GM1 fusion molecules were absorbed across the intestine into the circulation as assessed in blood and in liver tissue (
Consistent with the in vivo studies using the reporter-pep tide-GM1 fusion molecules, it was found that when applied to mice via gastric gavage, GLP-1-GM1 fusion molecules were absorbed and had effects over glucose metabolism, evidenced by reduced time to normalize blood sugar in glucose tolerance tests (
Another structural feature of GM1 that might play a role in trafficking is the extracellular oligosaccharide head group. Further described herein are studies designed to test if the oligosaccharide domain of GM1 is essential for intracellular trafficking and cargo transport. Simplifying the lipid carrier may be important to clinical translation. It is tested herein if a simple glycoceramide easily harvested from buttermilk can be used in place of GM1, and if the oligosaccharide domain of GM1 can be eliminated completely, thus defining a simplified core delivery molecule that can be fully and pragmatically synthesized.
Simplifying the glycoceramide structure would have two major significances. First, this would allow for total chemical synthesis of the molecules and permit easier translation to the clinic. Second, structural elements of the oligosaccharide head group that affect trafficking may be identified, enhancing the understanding of the structure-activity relationship for the glycosphingolipids, and aiding in design of new peptide linkers that recapture the lost functionalities.
Some functionality of the sugar head groups are anticipated. At minimum, the oligosaccharide domain of GM1 is expected to function by trapping the ceramide in the outer membrane leaflet, preventing flip-flop and rendering the molecule dependent on membrane trafficking for distribution within and across cells, or by shaping the molecule. Such functions, however, might be reproduced simply by attachment of peptide or protein cargo to the ceramide domain. Still, the oligosaccharide head group of GM1 might also participate in sorting reactions that drive GM1 trafficking. If so, it may be possible to design peptide linkers that recover such trafficking functions. The testing of the oligosaccharide domain and accommodation to its loss are the topics of this aim.
Initially, two simplified structural analogs of GM1 are tested: glucosylceramide (Glc-Cer—one sugar residue), and ceramide alone (no sugars). These lipids are commercially available with different ceramide structures (Avanti Polar Lipids). Because ceramide (Cer) and Glc-Cer lack a sialic acid, this necessitates an alternative chemical strategy to link peptides onto the ceramide headgroup. All lipids are coupled to the reporter peptide using copper-catalyzed “click” chemistry. Because Cer and Glc-Cer lack a sialic acid, this necessitates an alternative chemical strategy to link peptides onto the ceramide headgroup. Details of the chemical reactions are shown in
The transcytosis of Glc-Cer and Cer are tested in vitro using polarized cell lines T84, Caco2 and MDCK. Testing the new peptide-ceramide fusion molecules for transcytosis using the same methods described in
If reporter peptide fusions to ceramide alone mimic the GM1-trafficking platform, it can be concluded that the sugar groups are dispensable, except perhaps for blocking the sphingolipid from membrane flip-flop to the inner leaflet, a function presumably accommodated by the peptide-linker and cargo. If the oligosaccharide domain contributes decisively to trafficking function (and thus are required), peptide linkers that would replace the functionality of the oligosaccharide domains in trafficking can be designed. Due to the inherent difficulty in synthesizing complex O-linked oligosaccharides onto lipids, pseudo-glycopeptide linkers are synthesized using backbone amino acids coupled to glucose, galactose or galactose-N-acetyl residues via serine side chains (44). FMoc-protected building block amino acids containing monosaccharides are commercially available and will be used for solid phase peptide synthesis of short linear peptides containing up to 4-5 sugar residues each (
If glycoceramide (single sugar) mimics results with the GM1-lipids, but ceramide alone does not, it can be concluded that the sugar groups contribute to glycosphingolipid sorting primarily by anchoring the molecules in the outer-leaflet of the membrane. In both cases, it is possible that simplified sphingolipids can substitute for GM1 (or with redesigned peptide-sugar linkers).
The ability of ceramide alone or Glc-Cer to function as the GM1-lipid platform allow more rapid translation to clinical application, as these molecules can be synthesized or readily purified from milk fats.
Described herein is the design of a delivery vehicle that releases the cargo after transport. For instance, fusion of GM1 to the smaller peptide hormone GLP-1 causes a 3 to 8-fold loss of function. While still highly potent (pM activity) and physiologically relevant, the GLP-1-GM1 fusion molecule is still less potent than the native peptide. Here, two approaches to design the peptide linker so that it can release cargo from GM1 after uptake into the cell or after arrival in the basolateral endosome were tested. One approach involved the incorporation of an ester linkage in the peptide linker (as target for esterases increased at sites of inflammation, e.g., leukocyte esterase, or carboxylesterase hCE-2 that is specific to gastrointestinal endosomes) (1, 45); and the other involved incorporation of a cleavage motif for the endosomal protease furin (2-4).
Different furin cleavage motifs were tested for activity within the context of short peptide linker. The assay was designed as high-throughput using fluorescence energy transfer (FRET) by appending paired fluorophores at each end of the peptide to be tested.
The FRET 7 furin cleavage motif or a cleavable ester linkage are incorporated into the reporter-peptide linker, and also with GLP-1 incorporated as cargo. The GM1-C12:0 species are used as a fusion partner because this lipid does not readily release from membranes and will provide a more sensitive approach to test this technology. The efficiency for cleavage are first tested in vitro using recombinant esterases or recombinant furin and analyzed by HPLC. If cleavage is confirmed, the fusion molecules are tested for efficiency of cargo transport across highly resistant T84 epithelial barriers (as described in
The ceramide structure plays a decisive role in determining the cellular fate of glycosphingolipids after endocytosis-explained by their propensity to sort into highly curved tubules and buds emerging from the sorting endosome. It was tested herein whether glycosphingolipid sorting depends on molecular shape or on assembly into nano-domains (or both). GM1 species with saturated long chain fatty acids might a higher propensity to self-assemble with cholesterol into nano-domains. These domains might be recognized by cellular factors and sorted into the degradative pathway; preventing entering the recycling endosomal tubules.
To investigate the role of double bond positioning & hydrocarbon chain length of the ceramide fatty acid in lipid packing and subsequent differential endosomal sorting, GM1 isoforms with identical oligosaccharide head groups but ceramides of different endogenous structure were synthesized, by systematically increasing length and double bond position (C12:0 to C26:1).
The GM1 species with different ceramide structures were functionalized with a D-amino acid reporter peptide, containing a biotin and fluorophore group. Alternatively, a fluorophore was directly onto the sialic acid of the sugar head group. Lipids were purified by HPLC and structures validated by LC/MS (
Surface GM1 at steady state depends on lipid-sorting into highly curved tubules of the recycling pathway and this can be detected using fluorescent avidin probes. GM1 with saturated longer chain (>C14:0) fatty acid or long chain unsaturated fatty acid (>C24:1) are depleted from the plasma membrane, presumably sorted to the lysosome, suggesting inefficient entry into sorting tubules. GM1 containing a ceramide with C14:0 or C24:1 fatty acid have intermediate phenotype (
Fluorescently-labeled GM1 in sorting tubules emerging from early endosomes (labeled with dextran and TFnR) was measured directly in A431 cells and quantified. GM1 with short (<C14:0) or unsaturated (<C24:1) fatty acids escaped the degradative pathway and enter recycling endosomal tubules. Depletion of cellular cholesterol by MbCD releases GM1 species with long (>C14:0, ≥C24:1) fatty acids into recycling endosomal tubules (
Two additional endocytic pathways depend upon sorting into highly curved tubules-transcytosis and retrograde, providing other quantitative measures for GM1 entry into sorting tubules. Transcytosis of individual GM1 species were measured in polarized MDCK cells detecting apical to basolateral transport of GM1 with a fluorescent avidin probe. Retrograde trafficking of individual GM1 species was measured using cAMP after intoxification by cholera toxin as read out in GM1 negative cell lines. The toxin must traffic retrograde to the ER to induce cAMP. GM1 with longer chain fatty acids (≥C14:0) or ceramides containing a mono-unsaturated fatty acid (≥C22:1) inefficiently enter sorting tubules and (presumably) are retained in the lysosomal pathway (
A short reporter peptide (all-D isomer) was synthesized and chemically linked onto the headgroup of ceramide lipid containing a C2:0 fatty acid tail. The chemistry to do this was very difficult due to the nature of the components and insolubility on standard solvents. Many different approaches had been tried a strategy that worked well was found. The 3-step synthetic approach is described herein.
A method called “Parikh-Doering” oxidation was used to oxidize the headgroup of ceramide to a reactive aldehyde in organic solvent. Reaction mixture contains: (1) SO3Py: 190-400 mg/mL in dry DMSO; (2) C8 glucosylceramide: 0.2-0.6M solution in dry DMSO with Triethylamine; and (3) Triethylamine: 7-17 equivalents. The resulting product was purified by solvent extraction and dried in a speed vac (
Step 2: Reaction of peptide containing aminooxy to the aldehyde group The “LC9” (D-isomer) linker peptide was coupled using oxime-mediated reductive amination (
The resulting crude peptide-ceramide conjugates or purified conjugates (60% yield) were analyzed by HPLC (
Alexa Fluorophore containing an azide reactive group was reacted to the peptide-ceramide conjugate via the N-terminal alkyne using Huisgen copper-catalyzed cycloaddition chemistry (
The in vitro transport (transcytosis) of the pep tide-GM1 conjugates across a monolayer of MDCK cells, and in vivo transport across an intestinal or nasal barrier into the blood are assessed. The peptide alone are used as negative controls, and the GM1 analogs as positive controls. C6, and C12 fatty acid analogs of ceramide were also included in the experiments as direct comparisons to the GM1 and GM3 species.
MDCK cells were seeded in 0.33 cm2 inserts 3 days prior in media containing HBSS and 100 mM defatted BSA. Electrical resistance was checked on day of experiment.
Make: 27 mL T84-SF PEGS 1% dfBSA (add 270 mg dfBSA to 27 mL)
Make: 500 uL×50 samples=25 mL T84-SF PLUS 0.1% dfBSA (add 25 mg to 25 mL)
The bioavailability of the peptide-GM1-C6:0 fusion molecule when applied nasally to mice and the pharmacokinetics are shown in
Next, the bioavailability and pharmacokinetics of a peptide fused to GM-C4:0 was tested in vivo in rat. The fusion molecule was validated in mice before the experiment (
A panel of ceramides and ceramide analogs were synthesized (
Cer-C6 was tested along with a 4° C. temperature block to demonstrate evidence for the mechanism of transport by transcytosis. The ceramide analog Diene C6 along (structure shown in
Next, a ceramide with a C2 fatty acid chain and without a sugar moiety (Cer-C2) was tested. Peptide fused to Cer-C2 was able to transport across monolayers of MDCKII cells in a transcytosis assay (
A repeat experiment of transcytosis in MDCK cells is shown in
Further, whether a ceramide can be absorbed via the nose is tested. A reporter peptide was conjugated to a ceramide-C6 (Cer-C6) was administered to C57BL/6J mice (n=1, from Jackson Labs) at a dose of 2 nmol/kg. GM1-C6 conjugated to the same reported peptide was used as control. A total of 5 μl per nostril was administered over 5 minutes under isoflurane. Blood was collected via cardiac puncture 15 minutes post dose and the amount of the conjugate was evaluated by pulling down with magnetic streptavidin beads followed by elution and plate fluorescence measurement. The data showed that ceramide-C6-reporter peptide conjugate can be absorbed via nose in mice, and that the peptide conjugated to Cer-C6 is absorbed more efficiently than peptide conjugated to GM1-C6 (
Diene-analogs (due to dehydration) and dihydro-analogs of ceramide have been underlined for clarity in this disclosure. Oximes may be a mix of cis and trans geometric isomers. Oximes have been arbitrarily drawn as cis-isomers in
(2S,3R,4E)-1-(O-Triphenylmethyl)-2-(N-hexanoylamino)-4-octadecene-1,3-diol [1-trityl-C6-ceramide 2 (R═—C5H11)]. To a vigorously stirred solution of C6-ceramide 1 (R═—C5H11) (30.3 mg, 7.62×10−5 mol), ethyldiisopropylamine (18 μL, 13 mg, 1.0×10−4 mol), and DMAP (1.1 mg, 9.0×10−6 mol) in 500 μL of dry CH2Cl2, was added trityl chloride (23.4 mg, 8.4×10−5 mol) in 200 μL of CH2Cl2 at room temperature over 5 min. The reaction mixture was stirred for 3 days. The mixture was concentrated and chromatographed to give 1-trityl-C6-ceramide 2 (R═—C5H11) (32.0 mg, 5.00×10−5 mol, 66%) as a clear and colorless viscous liquid that was homogeneous by TLC (90:10:0.1 CHCl3/EtOAc/TEA Rf 0.42): 1H NMR (DMSO-d6).
(2S,3R,4E)-3-O-Benzoyl-1-(O-triphenylmethyl)-2-(N-hexanoylamino)-4-octadecene-1,3-diol [1-trityl-3-benzoyl-C6-ceramide 3 (R═—C5H11)]. To a vigorously stirred solution of 1-trityl-C6-ceramide 2 (R═—C5H11) (9.5 mg, 1.5×10−5 mol), ethyldiisopropylamine (13.1 μL, 9.7 mg, 7.5×10−5 mol, 500 mol %), and catalytic DMAP in 500 μL of dry toluene, was added benzoyl chloride (2.5 mg, 1.8×10−5 mol). After 1 day, additional ethyldiisopropylamine (13.1 μL) and benzoyl chloride (2.5 mg) were added. The reaction was filtered through a plug of 0.2 g silica gel eluting with CHCl3/EtOAc/TEA (97:3:0.1 CHCl3/EtOAc/TEA). The filtrate containing product was chromatographed (97:3:0.1 CHCl3/EtOAc/TEA) to give 1-trityl-3-benzoyl-C6-ceramide 3 (R═—C5H11) (10.0 mg, 1.34×10−5 mol, 89%) as a clear and colorless viscous liquid that was homogeneous by TEC (97:3:0.1 CHCl3/EtOAc/TEA Rf 0.45): 1H NMR (DMSO-d6).
(2S,3R,4E)-3-O-Benzoyl-2-(N-hexanoylamino)-4-octadecene-1,3-diol [3-benzoyl-C6-ceramide 4a (R═—C5H11)]. To a stirred solution of l-trityl-3-benzoyl-C6-ceramide 3 (R═—C5H11) (10. mg, 1.3×10−5 mol) in 2 mL of CH2Cl2/MeOH (1:1) was added a solution of p-toluenesulfonic acid monohydrate (3.8 mg, 2.0×10−5 mol) in 0.5 mL of MeOH. After 3 days, CH2Cl2 was added and the solution washed twice with 1 mL portions of 8.5 mg/mL aqueous NaHCO3. The organic phase was then washed with H2O, dried over Na2SO4, and chromatographed (96:4 CH2Cl2/MeOH) to give 3-benzoyl-C6-ceramide 4a (R═—C5H11) (6.5 mg, 1.3×10−5 mol, 100%) as a white solid that was nearly homogeneous by TEC (96:4 CH2Cl2/MeOH, Rf 0.20): 1H NMR (DMSO-d6); mass spectrum C31H52NO4+: m/z calculated 502.4, observed 502.5.
(2S,3R,4E)-3-O-Benzoyl-1-O-(2-(2-(2-(4-formylbenzamido)ethoxy)ethoxy)ethan-1-carbamoyl)-2-(N-hexanoylamino)-4-octadecene-1,3-diol [1-(Ald-PEG2-carbamoyl)-C6-ceramide-3-benzoate 6a (R═—C5H11)]. To a stirred solution of triphosgene (5.9 mg, 2.0×10−5 mol, 20 eq) in 0.2 mL of anhydrous CH2Cl2 was added dropwise over 2 min a solution of 3-benzoyl-C6-ceramide 4a (R═—C5H11) (1.5 mg, 3.0×10−5 mol, 1 eq) in 0.2 mL of anhydrous CH2Cl2 containing ethydiisopropylamine (22 μL, 16 mg, 3.0×10−5 mol, 43 eq). After 2 h, the reaction mixture was distilled using a stream of nitrogen gas blowing over the reaction mixture and out through a solution of aqueous ammonium hydroxide/H2O/i-PrOH (1:1:1). The residue was redissolved in 0.1 mL of anhydrous CH2Cl2 to give a light yellow solution. To this solution of crude chloroformate 5a (R═—C5H11) was added rapidly dropwise a freshly prepared solution of Ald-PEG2-ammonium trifluoracetate (3.9 mg, 9.9×10−6 mol, 3.3 eq, Broadpharm, CAS 2055013-56-2) in 0.1 mL of dry CH2Cl2 containing ethydiisopropylamine (12 μL, 8.9 mg, 6.9×10−5 mol, 7 eq). After 1 h 15 min, the solvent was removed to give a yellow semi-solid that was chromatographed (96:4:0.1 CHCl3/EtOAc/TEA) to give a nearly quantitative yield of 1-(Ald-PEG2-carbamoyl)-C6-ceramide-3-benzoate 6a (R═—C5H11) as a faint yellow semisolid that was nearly homogeneous by TLC (96:4:0.1 CHCl3/EtOAc/TEA, Rf 0.18): 1H NMR (DMSO-d6).
The linker already has aldehyde function.
Coupling with LC9 to Give the Oxime.
LC9-oxime-PEG2-carbamoyl-C6-ceramide-3-benzoate 8a (R═—C5H11). To a solution of 1-(Ald-PEG2-carbamoyl)-C6-ceramide-3-benzoate 6a (R═—C5H11) (1.5 mg, 1.9×10−6 mol) and LC9 (2.7 mg, 1.9×10−6 mol) in 800 μL of DMF was added 1 μL of aniline and the reaction stirred overnight. HPLC purification of the reaction mixture gave LC9-oxime-PEG2-carbamoyl-C6-ceramide-3-benzoate 8a (R═—C5H11); mass spectrum C106H163N24O4S+: m/z calculated for (M+2H)+2 1118.6, observed 1118.9.
The “click” reaction with AF488-azide which is a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction) to give the corresponding predominantly 1,4-substituted-1,2,3-triazole (as drawn in
To a solution of C6-ceramide-3-benzoate 4a (R═—C5H11) (1 mg, 2×10−6 mol) in 0.2 mL of dry CH2Cl2, was added a 0.30 M solution of Dess-Martin periodinane (13 μL, 4×10−6 mol) dropwise over 1 min during which time the solution remained homogeneous. After 10 min, a vortex-mixed solution of H2O (0.04 μL, 0.04 mg, 2×10−6 mol) in 40 μL of CH2Cl2 was added dropwise over 10 min during which time the reaction mixture became cloudy. The reaction mixture was vigorously stirred for an additional 1.5 h. The reaction mixture was then washed with 0.2 mL of 1:1 saturated aqueous NaHCO3/15% Na2S2O3, dried over Na2SO4, filtered through a 0.45 μm GHP filtration cartridge, and the solvent removed to give a light yellow viscous oil that contained the corresponding aldehyde by TLC (90:10:0.1 CHCl3/EtOAc/TEA, Rf 0.66) and was used immediately.
Coupling with LC9 to Give the Oxime.
LC9-oxime-C6-ceramide-diene 10 (R═—C5H11). To a solution of the crude aldehyde (0.5 mg, 1×10−6 mol) and LC9 (1.7 mg, 1.2×10−6 mol) in 1 mL of DMF was added 1 μL of aniline and the reaction stirred overnight. HPLC purification of the reaction mixture gave LC9-oxime-C6-ceramide-diene analog 10 (R═—C5H11) (0.4 mg, 2×10−7 mol): mass spectrum C84H137N22O20S+: m/z calculated for (M+2H)+2 903.5, observed 903.8, calculated for (M+3H)+3 602.7, observed 603.1.
The “click” reaction with AF488-azide which is a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction) to give the corresponding predominantly 1,4-substituted-1,2,3-triazole (as drawn in
(2S,3R,4E)-3-O-Triethylsilyl-2-(N-hexanoylamino)-4-octadecene-1,3-diol [3-TES-C6-ceramide 4b (R═—C5H11)]. To a stirred solution of C6-ceramide 1 (R═—C5H11) (30.3 mg, 7.62×10−5 mol), ethyldiisopropylamine (150 μL, 111 mg, 8.5×10−4 mol), and DMAP (9.5 mg, 7.8×10−5 mol) in 1.5 mL of dry CH2Cl2, was added triethylsilyl chloride (12 μL, 11 mg, 7.3×10−5 mol) dropwise at room temperature over 2 min. The reaction mixture was stirred for 1.5 h. The mixture was concentrated and chromatographed (90:10:0.1 CHCl3/EtOAc/TEA) first eluting 1,3-diTES-C6-ceramide R═—C5H11) followed by 1-TES-C6-ceramide (R═—C5H11), then the desired 3-TES-C6-ceramide 4b (R═—C5H11) as the minor product (3.7 mg, 7.2×10−6 mol, 9.4%) as a clear and colorless viscous liquid that was homogeneous by TLC (90:10:0.1 CHCl3/EtOAc/TEA Rf 0.15): 1H NMR (DMSO-d6); mass spectrum C30H62NO3Si+: m/z calculated 512.5, observed 512.4.
Step One is oxidation of primary alcohol to aldehyde. To a solution of C6-ceramide-3-TES 4b (R═—C5H11) (2.2 mg, 4.3×10−6 mol) in 0.2 mL of dry CH2Cl2, was added a 0.30 M solution of Dess-Martin periodinane (22 μL, 5.7×10−6 mol, 1.5 eq) dropwise over 1 min during which time the solution remained homogeneous. After 10 min, a vortex-mixed solution of H2O (0.13 μL, 0.13 mg, 7.2×10−6 mol) in 130 μL of CH2Cl2 was added dropwise over 10 min during which time the reaction mixture became cloudy. The reaction mixture was vigorously stirred for an additional 15 min. The reaction mixture was then washed with 0.2 mL of 1:1 saturated aqueous NaHCO3/15% Na2S2O3, dried over Na2SO4, filtered through a 0.45 μm GHP filtration cartridge, and the solvent removed to give a light yellow viscous oil that contained the corresponding aldehyde by TEC (90:10:0.1 CHCl3/EtOAc/TEA, Rf 0.62) and was used immediately.
Step Two is the coupling with LC9 to give the oxime. LC9-oxime-C6-ceramide-3-TES 11a (R═—C5H11). To a solution of the crude aldehyde (2 mg, 4×10−6 mol) and LC9 (6.2 mg, 4.3×10−6 mol) in 1 mL of DMF was added 1 μL of aniline and the reaction stirred overnight. HPLC purification of the reaction mixture gave LC9-oxime-C6-ceramide-3-TES 11a (R═—C5H11); mass spectrum C90H153N22O21SSi+: m/z calculated for (M+2H)+2 969.6, observed 970.5, calculated for (M+3H)+3 646.7, observed 647.2.
Step Three is removal of the TES. LC9-oxime-C6-ceramide lib (R═—C5H11). A solution of LC9-oxime-C6-ceramide-3-TES 11a (R═—C5H11) in 80:20 acetic acid/H2O was stirred for two hours. Solvents were removed by lyophylization. HPLC purification of the reaction mixture gave LC9-oxime-C6-ceramide lib (R═—C5H11) (1.0 mg, 5.5×10−7 mol); mass spectrum C84H139N22O21S+: m/z calculated for (M+2H)+2 912.5, observed 912.7, calculated for (M+3H)+3 608.7, observed 609.1.
Step Four is the “click” reaction with AF488-azide which is a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction) to give the corresponding predominantly 1,4-substituted-1,2,3-triazole (as drawn in
AF488-LC9-oxime-C6-ceramide 14b (R═—C5H11). A solution of LC9-oxime-C6-ceramide lib (R═—C5H11) (1.0 mg, 5.5×10−7 mol), Alexa Fluor® 488 (0.71 mg, 8.2×10−7 mol), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (6×10−8 mol), tris(2-carboxyethyl)phosphine (lx 10−6 mol), ascorbic acid (lx 10−4 mol), Cu(II)SO4 (5×10−6 mol), in 1 mL of 8:2 DMSO/Tris buffer pH 8, was stirred overnight protected from light. The reaction mixture was centrifuged, and the colored precipitate was purified by HPLC to give AF488-LC9-oxime-C6-ceramide 14a (R═—C5H11) (100 □g, 6.2 10−8 mol), mass spectrum C111H165N28O31S3+: m/z calculated for (M+2H)+2 1241.1, observed 1241.6, calculated for (M+3H)+3 827.7, observed 828.2.
(2S,3R,4E)-3-O-Triethylsilyl-2-(N-octadecanoylamino)-4-octadecene-1,3-diol [3-TES-C18-ceramide 4b (R═—C17H35)]. To a stirred solution of C18-ceramide 1 (R═—C17H35) (41.2 mg, 7.28×10−5 mol), ethyldiisopropylamine (150 μL, 111 mg, 8.5×10−4 mol), and DMAP (12.7 mg, 1.04×10−5 mol) in 1.5 mL of dry CH2Cl2, was added triethylsilyl chloride (14 μL, 13 mg, 8.6×105 mol) dropwise at room temperature over 2 min. The reaction mixture was stirred for 1.5 h. The mixture was concentrated and chromatographed (90:10:0.1 CHCl3/EtOAc/TEA) first eluting 1,3-diTES-C18-ceramide R═—C17H35) followed by 1-TES-C18-ceramide (R═—C17H35), then the desired 3-TES-C18-ceramide 4b (R═—C17H35) as the minor product (2.5 mg, 3.7×10−6 mol, 5.1%) as an amorphous white solid that was homogeneous by TLC (90:10:0.1 CHCl3/EtOAc/TEA Rf 0.15): NMR (DMSO-d6).
Step One is oxidation of primary alcohol to aldehyde. To a solution of 3-TES-C18-ceramide 4b (R═—C17H35) (2.5 mg, 3.7×10−6 mol) in 0.2 mL of dry CH2Cl2, was added a 0.30 M solution of Dess-Martin periodinane (19 μL, 5.7×10−6 mol, 1.5 eq) dropwise over 1 min during which time the solution became cloudy. After 10 min, a vortex mixed solution of H2O (0.11 μL, 0.11 mg, 6.1×10−6 mol) in 110 μL of CH2Cl2 was added dropwise over 10 min. The reaction mixture was vigorously stirred for an additional 15 min. The reaction mixture was then washed with 0.2 mL of 1:1 saturated aqueous NaHCO3/15% Na2S2O3, dried over Na2SO4, filtered through a 0.45 μm GHP filtration cartridge, and the solvent removed to give yellow viscous oil that was highly homogeneous aldehyde by TLC (90:10:0.1 CHCl3/EtOAc/TEA, Rf 0.59) and was used immediately.
Step Two is the coupling with LC9 to give the oxime with protecting group removal. LC9-oxime-C18-ceramide 11e (R═—C17H35). To a solution of the crude aldehyde (2.5 mg, 3.7×10−6 mol) and LC9 (5.3 mg, 3.7×10−6 mol) in 1 mL of DMF was added 1 μL of aniline and the reaction stirred overnight. HPLC purification of the reaction mixture gave only a small amount of LC9-oxime-C18-ceramide-3-TES 11d (R═—C17H35), but most of the TES had fallen off and gave LC9-oxime-C18-ceramide 11e (R═—C17H35) (3.9 mg, 2.0×10−6 mol, 54% yield) as a white solid: 1H NMR (DMSO-d6); mass spectrum C96H163N22O21S+: m/z calculated for (M+2H)+2 996.6, observed 996.9, calculated for (M+3H)+3 664.7, observed 665.1.
Finally, the “click” reaction with AF488-azide which is a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction) to give the corresponding predominantly 1,4-substituted-1,2,3-triazole (as drawn in
AF488-LC9-oxime-C18-ceramide 14b (R═—C17H35). A solution of LC9-oxime-C18-ceramide 11e (R═—C17H35) (0.64 mg, 5.5×10−7 mol), Alexa Fluor® 488 (0.71 mg, 4.9×10−7 mol), tris[(1-benzyl-177-1,2,3-triazol-4-yl)methyl]amine (6×10−8 mol), tris(2-carboxyethyl)phosphine (1×10−6 mol), ascorbic acid (lx 10−4 mol), Cu(II)SO4 (5×10−6 mol), in 1 mL of 8:2 DMSO/Tris buffer pH 8, was stirred overnight protected from light. The reaction mixture was centrifuged, and the colored precipitate purified by HPLC to give AF488-LC9-oxime-C18-ceramide 14b (R═—C17H35); mass spectrum C123H189N28O31S3+: m/z calculated for (M+2H)+2 1325.7, observed 1325.6, calculated for (M+3H)+3 884.1, observed 884.8.
(2S,3R)-3-O-Triethylsilyl-2-(N-hexanoylamino)-octadecane-1,3-diol [3-TES-C6-dihydroceramide 7b (R═—C5H11)]. To a stirred solution of C6-dihydroceramide 7a (R═—C5H11) (27.8 mg, 6.96×10−5 mol), ethyldiisopropylamine (280 μL, 208 mg, 1.61×10−3 mol), and DMAP (12 mg, 9.8×10−5 mol) in 1.5 mL of dry CH2Cl2, was added triethylsilyl chloride (14 μL, 13 mg, 8.6×10−5 mol) dropwise at room temperature over 2 min. The reaction mixture was stirred for 1.5 h. The mixture was concentrated and chromatographed (90:10:0.1 CHCl3/EtOAc/TEA) first eluting 1,3-diTES-C6-dihydroceramide (R═—C5H11) followed by 1-TES-C6-dihydroceramide (R═—C5H11), then the desired 3-TES-C6-dihydroceramide 7b (R═—C5H11) as the minor product (2.7 mg, 5.2×10−6 mol, 7.5%) as a white waxy solid that was homogeneous by TEC (90:10:0.1 CHCl3/EtOAc/TEA Rf 0.14): NMR (DMSO-d6).
Step One is oxidation of primary alcohol to aldehyde. To a solution of C6-dihydroceramide-3-TES 7b (R═—C5H11) (2 mg, 4×10−6 mol) in 0.2 mL of dry CH2Cl2, was added a 0.30 M solution of Dess-Martin periodinane (13 μL, 3.9×10−6 mol, 1.5 eq) dropwise over 1 min during which time the solution remained homogeneous. After 10 min, a vortex-mixed solution of H2O (0.079 μL, 0.079 mg, 4.4×10−6 mol) in 10 μL of CH2Cl2 was added dropwise over 10 min during which time the reaction mixture became cloudy. The reaction mixture was vigorously stirred for an additional 30 min. The reaction mixture was then washed with 0.2 mL of 1:1 saturated aqueous NaHCO3/15% Na2S2O3, dried over Na2SO4, filtered through a 0.45 μm GHP filtration cartridge, and the solvent removed to give a light yellow viscous oil that contained the corresponding aldehyde by TEC (90:10:0.1 CHCl3/EtOAc/TEA, Rf 0.71) and was used immediately.
Step Two is the coupling with LC9 to give the oxime with protecting group removal. LC9-oxime-C6-dihydroceramide 12b (R═—C5H11). To a solution of the crude aldehyde (2 mg, 4×10−6 mol) and LC9 (5.6 mg, 3.9×10−6 mol) in 1 mL of DMF was added 1 μL of aniline and the reaction stirred overnight. HPLC purification of the reaction mixture gave only a small amount of protecting group removed LC9-oxime-C6-dihydroceramide 12b (R═—C5H11); mass spectrum C84H141N22O21S+: m/z calculated for (M+2H)+2 913.5, observed 913.7, calculated for (M+3H)+3 609.4, observed 609.7.
All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.
Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
It is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
Where websites are provided, URL addresses are provided as non-browser-executable codes, with periods of the respective web address in parentheses. The actual web addresses do not contain the parentheses.
In addition, it is to be understood that any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.
This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/656,474, filed Apr. 12, 2018, and entitled “CERAMIDE-LIKE LIPID-BASED DELIVERY VEHICLES AND USES THEREOF,” the entire contents of which are incorporated herein by reference.
This invention was made with government support under grants Nos. R37 DK048106, RO1 DK104868, R21 DK090603, and P30 DK034854, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/027281 | 4/12/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 20210030880 A1 | Feb 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62656474 | Apr 2018 | US |
