WO2023288316A1 - Method of use for apoe peptides - Google Patents

Method of use for apoe peptides Download PDF

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Publication number
WO2023288316A1
WO2023288316A1 PCT/US2022/073797 US2022073797W WO2023288316A1 WO 2023288316 A1 WO2023288316 A1 WO 2023288316A1 US 2022073797 W US2022073797 W US 2022073797W WO 2023288316 A1 WO2023288316 A1 WO 2023288316A1
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Prior art keywords
lnp
lipid
cell
acid
amino
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PCT/US2022/073797
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French (fr)
Inventor
Brian K. Hubbard
Michael H. Serrano-Wu
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Anji Pharma (Us) Llc
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Publication of WO2023288316A1 publication Critical patent/WO2023288316A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides

Definitions

  • LNPs Lipid nanoparticles formulated with ionizable lipids can serve as cargo vehicles for delivery- of biologically active agents into cells.
  • LNPs lipid nanoparticles
  • ApoE apolipoprotein E
  • lipid nanoparticles comprising an apolipoprotein E (ApoE)-mimicking peptide and one or more lipids.
  • the LNPs benefit from enhanced association with target cells and delivery of an agent into the target cells, compared to LNPs without the ApoE-mimicking peptide.
  • Enhanced delivery of agents to target cells e.g., liver cells or splenic cells
  • a lipid nanoparticle comprises one or more lipids and an apolipoprotein E (ApoE)-mimicking peptide, wherein the ApoE-mimicking peptide comprises a receptor-binding domain of ApoE and a lipid-associating domain.
  • ApoE apolipoprotein E
  • the LNP is an ionizable LNP.
  • the one or more lipids comprise an ionizable lipid.
  • ionizable lipid comprises an amino group.
  • the amino group is an amine head group.
  • the ionizable lipid comprises two or more aliphatic tail groups, e.g., two to four aliphatic tail groups.
  • the aliphatic tail groups are hydrophobic tail groups, e.g., each independently alkyl or alkenyl groups.
  • the ionizable lipid is a cationic lipid.
  • the ionizable lipid is a neutral lipid.
  • the ionizable lipid is a zwitterionic lipid.
  • the ionizable lipid is protonated at physiological pH.
  • the one or more lipids comprise a structural lipid, optionally a steroid, such as a sterol, e.g., cholesterol.
  • the one or more lipids comprise a non-cationic helper lipid.
  • the non-cationic helper lipid is a phospholipid.
  • the non-cationic helper lipid comprises a diacylglyerol group.
  • the non-cationic helper lipid is a glycerophospholipid, such as a phosphatidylcholine.
  • the non-cationic helper lipid is not a phosphatidylcholine.
  • the phospholipid is distearoylphosphatidylcholine (DSPC).
  • the one or more lipids comprise a PEG-lipid comprising a lipid group covalently bonded to a PEG group.
  • the lipid group is a phospholipid.
  • the lipid group comprises a diacylglycerol, dialkylglycerol, or dialkylamine group.
  • the lipid group is a glycerophospholipid group.
  • the lipid group is dimyristoyl glycerol.
  • the lipid-associating domain comprises a class A amphipathic-helical domain, e.g., DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1), DWLRAFYDKVAEKLREAF (SEQ ID NO: 2), DWLRALYDKVAEKLREAL (SEQ ID NO: 3), DLLRALYDKVAEKLREAW (SEQ ID NO: 4), or FAEKLKEAVKDYFAKLWD (SEQ ID NO: 5), most preferably DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1).
  • DWLKAFYDKVAEKLKEAF SEQ ID NO: 1
  • DWLRAFYDKVAEKLREAF SEQ ID NO: 2
  • DWLRALYDKVAEKLREAL SEQ ID NO: 3
  • DLLRALYDKVAEKLREAW SEQ ID NO: 4
  • FAEKLKEAVKDYFAKLWD SEQ ID NO
  • the receptor-binding domain of ApoE is capable of binding to an LDL receptor.
  • the receptor-binding domain of ApoE is LRKLRKRLLR (SEQ ID NO: 6), LRRLRRRLLR (SEQ ID NO: 7), LRKMRKRLMR (SEQ ID NO: 8), or RLTRKRGLK (SEQ ID NO: 9).
  • the receptor-binding domain of ApoE is LRKLRKRLLR (SEQ ID NO: 6) or LRRLRRRLLR (SEQ ID NO: 7), most preferably LRRLRRRLLR (SEQ ID NO: 7).
  • the receptor-binding domain of ApoE is covalently bonded to the lipid-associating domain.
  • the C-terminus of the receptor- binding domain of ApoE is covalently bonded to the N-terminus of the lipid-associating domain.
  • an amino group is bonded to the C-terminus of the lipid- associating domain.
  • an acyl group is bonded to the N -terminus of the receptor-binding domain of ApoE.
  • the ApoE-mimicking peptide comprises a fatty acid moiety e.g., butanoyl, caproyl, octanoyl, decanoyl, lauroyl, myristoyl, myristoleoyl, palmitoyl, stearoyl, oleoyl, pahnitoleoyl, linoleoyl, linolenoyl, or arachidonoyl.
  • the fatty acid moiety is octanoyl, myristoyl, palmitoyl, or oleoyl.
  • the ApoE-mimicking peptide comprises a fatty acid moiety, and the fatty acid moiety is bonded to the N -terminus of the receptor-binding domain of ApoE.
  • the fatty' acid moiety comprises a chain of 4 to 20 carbon atoms.
  • the fatty acid moiety is a saturated fatty' acid moiety.
  • the fatty acid moiety comprises one, two, or three alkene groups.
  • the fatty acid moiety comprises an co-amino group.
  • the co-amino group comprises an acyl group.
  • the fetty acid moiety is 4-amino-butanoyl, 6-amino- caproyl, 8-amino-octanoyl, 10-amino-decanoyl, 12-amino-lauroyl, 14-amino-myristoyl, 14- amino-myristoleoyl, 16-amino-palmitoyl, 18 -amino -stearoyl, 18-amino-oleoyl, 16-amino- pahnitoleoyl, 18-amino-linoleoyl, 18-amino-linolenoyl, or 20-amino-arachidonoyl.
  • the fatty acid moiety is Ac-Aha.
  • the Apo-E mimicking peptide is Ac-Aha-hEl8A-NH 2 or Ac-Aha-[R]hE18A-NH 2 .
  • the ApoE-mimicking peptide is Ac- LRKLRKRLLRDWLKAFYDKVAEKLKEAF-NH 2 (SEQ ID NO: 10); octanoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH 2 (SEQ ID NO: 11); myristoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH 2 (SEQ ID NO: 12); palmitoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH 2 (SEQ ID NO: 13); or oleoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH 2 (SEQ ID NO: 14).
  • the LNP further comprises a payload, such as a drug, a peptide, a polypeptide, a protein, or a nucleic acid (e.g., RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR/Cas9, ssDNA, and DNA).
  • a payload such as a drug, a peptide, a polypeptide, a protein, or a nucleic acid (e.g., RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR/Cas9, ssDNA, and DNA).
  • the payload comprises an mRNA encoding a secreted protein, membrane-bound protein, intracellular protein, antibody molecule, or enzyme.
  • the LNP is formulated for systemic delivery.
  • the LNP is formulated for parenteral, e.g., intravenous, intramuscular, subcutaneous, intrathecal, or intradermal; or enteral, e.g., oral, rectal, or sublingual, delivery.
  • the LNP is not in an organism.
  • compositions that comprise an LNP as described herein and a pharmaceutically acceptable carrier.
  • compositions and methods described herein can be used to treat disease by administering an LNP as described herein to a subject.
  • the disease is coronary artery disease, rheumatoid arthritis, diabetes, neurodegenerative disease, Alzheimer’s disease, peripheral artery disease, cerebral vascular disease, diabetes-derived cardiovascular diseases, macular degeneration, congestive heart failure, systemic lupus, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, cardiovascular, renovascular diseases, metabolic diseases, immune disorders, fibrotic disease, inflammation, and/or infectious disease.
  • the LNP is systemically delivered to the subject.
  • the LNP is parenterally, e.g., intravenously, intramuscularly, subcutaneously, intrathecally, or intradermally; or enterally, e.g., orally, rectally, or sublingually, delivered to the subject.
  • a therapeutically effective dose of a LNP composition described herein is about 0.01 mg/kg to about 500 mg/kg, about 0.05 mg/kg to about 250 mg/kg, about 0.1 mg/kg to about 100 mg/kg, about 0.5 mg/kg to about 50 mg/kg, or about 1 mg/kg to about 25 mg/kg. In preferable embodiments, the therapeutically effective dose of a LNP composition described herein is about 0.01 mg/kg to about 100 mg/kg.
  • the LNP is internalized by a cell.
  • the LNP increases internalization by a cell compared to the same LNP without the ApoE- mimicking peptide.
  • the cell is a liver cell, e.g., a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a liver sinusoidal cell.
  • the cell is a brain cell.
  • the cell is a splenic cell, e.g., a splenocyte.
  • the cell is an ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell, or a skeletal muscle cell. In some embodiments, the cell is a non-immune cell. In other embodiments, the cell is a cancer cell. In yet other embodiments, the cell is a T-cell. In some embodiments, the cell is a stem cell, such as a hematopoietic cell. In other embodiments, the cell is a lung cell . In yet other embodiments, the cell is a kidney cell. [0034] In some embodiments, the total plasma concentration of cholesterol in the subject is lowered. In certain embodiments, the plasma LDL concentration, the plasma VLDL concentration, or both in the subject are lowered.
  • compositions and methods may be understood more readily by reference to the following detailed description of particular embodiments.
  • compositions and methods are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
  • lipid nanoparticles and methods for treating disease with lipid nanoparticles, e.g., by binding apolipoprotein E (ApoE) and inhibiting the downstream effects of ApoE.
  • lipid nanoparticles comprising one or more lipids and an apolipoprotein E (ApoE)-mimicking peptide, wherein the ApoE-mimicking peptide comprises a receptor-binding domain of ApoE and a lipid- associating domain.
  • pharmaceutical compositions are provided herein comprising an LNP disclosed herein.
  • methods of treating a disease comprising administering an LNP disclosed herein or a pharmaceutical composition disclosed herein.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • administering or “administration of’ a substance, a composition or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art.
  • a composition or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct).
  • a composition or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the composition or agent.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • a composition or an agent is administered orally, e.g., to a subject by ingestion.
  • the orally administered composition or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.
  • alkyl as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s- butyl, r-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like.
  • the alkyl group can be cyclic or acyclic.
  • the alkyl group can be branched or unbranched (i.e., linear).
  • the alkyl group can also be substituted or unsubstituted (preferably unsubstituted).
  • the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, carboxylate, or thiol, as described herein.
  • a "lower alkyl” group is an alkyl group containing from one to six (e.g. , from one to four) carbon atoms.
  • alkenyl refers to an aliphatic group containing at least one carbon-carbon double bond and is intended to include both "unsubstituted alkenyls" and “substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed below, except where stability is prohibitive.
  • an alkenyl group may be substituted by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
  • An “alkylene” group refers to a divalent alkyl radical, which may be branched or unbranched (i.e., linear). Any of the above mentioned monovalent alkyl groups may be converted to an alkylene by abstraction of a second hydrogen atom from the alkyl.
  • alkylenes include alkylene and C2-3 alkylene.
  • Typical alkylene groups include, but are not limited to CH 2 C(CH 3 ) 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, and the like.
  • the alkylene group can also be substituted or unsubstituted.
  • the alkylene group can be substituted with one or more groups including, but not limited to, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, sulfonamide, urea, amide, carbamate, ester, carboxylate, or thiol, as described herein.
  • alkenylene includes divalent, straight or branched, unsaturated, acyclic hydrocarbyl groups having at least one carbon-carbon double bond and, in some embodiments, no carbon-carbon triple bonds. Any of the above-mentioned monovalent alkenyl groups may be converted to an alkenylene by abstraction of a second hydrogen atom from the alkenyl.
  • Representative alkenylenes include
  • Cx-y when used in conjunction with a chemical moiety, such as alkyl or alkylene, is meant to include groups that contain from x to y carbons in the chain.
  • Cx-y alkyl refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain and branched-chain alkyd and alkylene groups that contain from x to y carbons in the chain.
  • C n As defined herein, “C n ,” where “n” is an integer, describes a hydrocarbon molecule or fragment (e.g., an alkyl group) wherein “n” denotes the number of carbon atoms in the fragment or molecule.
  • an “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • VH heavy chain variable region
  • the heavy chain constant region is comprised of three domains, CHI, CH 2 and CHS.
  • each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy - terminus in the following order: FR1, CDR1, FR2, CDR2, FRS, CDRS, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq)
  • ApoE mimetic is interchangeable with apolipoprotein-E mimicking peptide.
  • ApoE mimetics are peptides that are related to, characteristic of, or mimic ApoE.
  • ApoE mimetics include ApoE peptides (i.e., peptides derived from full length ApoE).
  • the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compositions such that the second composition is administered while the previously administered therapeutic composition is still effective in the body (e.g., the two compositions are simultaneously effective in the patient, which may include synergistic effects of the two compositions).
  • the different therapeutic compositions can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially.
  • the different therapeutic compositions can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another.
  • an individual who receives such treatment can benefit from a combined effect of different therapeutic compositions.
  • contacting means establishing a physical connection between two or more entities.
  • contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and a nanoparticle are made to share a physical connection.
  • Methods of contacting cells with external entities both in vivo and ex vivo are well known in the biological arts.
  • contacting a nanoparticle composition and a mammalian cell disposed within a mammal may be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve varied amounts of nanoparticle compositions.
  • routes of administration e.g., intravenous, intramuscular, intradermal, and subcutaneous
  • more than one mammalian cell may be contacted by a nanoparticle composition.
  • delivering means providing an entity to a destination.
  • delivering a therapeutic and/or prophylactic to a subject may involve administering a nanoparticle composition including the therapeutic and/or prophylactic to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route).
  • Administration of a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.
  • domain switched refers to the lipid-associating peptide is covalently linked to the receptor binding domain of apolipoprotein E such that the lipid-associating peptide is at the N-terminus of the synthetic apolipoprotein E-mimicking peptide.
  • the peptide 18A-hE is exemplary of a domain switched peptide.
  • fatty acid refers to a mono-carboxylic acid having an aliphatic chain (“tail”), wherein said aliphatic chain may be either saturated, mono- unsaturated (having one unsaturated bond anywhere on the aliphatic chain) or poly unsaturated (having at least two unsaturated bonds anywhere on the aliphatic chain).
  • An unsaturated bond on the aliphatic chain may be a double (in the cis and/or trans configuration) or a triple bond.
  • the length of the aliphatic chain (being either saturated, monounsaturated or polyunsaturated) of a fatty acid may vary from 8 to 32 carbon atoms.
  • Fatty acids may be derived from a natural source (either an animal or plant source), synthetic source or semi-synthetic source.
  • high-density lipoprotein refers to a class of lipoproteins, varying somewhat in their size (8-11 run in diameter), that can transport cholesterol.
  • HDL cholesterol is cholesterol that is associated with HDLs.
  • About one-fourth to one-third of blood cholesterol is carried by high-density lipoprotein (HDL).
  • HOL cholesterol is known as “good” cholesterol, because high levels of HDL seem to protect against heart attack.
  • Low levels of HDL (less than 40 mg/dL in men and less than 50 mg/dL in women) also increase the risk of heart disease.
  • LDL receptor refers to a receptor that mediates the endocytosis of cholesterol-rich low-density lipoprotein (LDL).
  • Lipid refers to structurally diverse group of organic compounds that are fatty acid derivatives or sterols or could be lipid like materials as in lipidoids (example C 12-200) or polymer conjugated lipids and are characterized by being insoluble in water but soluble in many organic solvents.
  • lipoprotein refers to a biochemical assembly that contains both proteins and lipids.
  • the lipids or their derivatives may be covalently or non- covalently bound to the proteins.
  • Many enzymes, transporters, structural proteins, antigens, adhesins, and toxins are lipoproteins. Examples include the high density and low density lipoproteins of the blood, the transmembrane proteins of the mitochondrion and the chloroplast, and bacterial lipoproteins.
  • liposome is a structure including a lipid containing membrane enclosing an aqueous interior. Liposomes may have one or more lipid membranes.
  • Liposomes include single-layered liposomes (also known in the art as unilamellar liposomes) and multi-layered liposomes (also known in the art as multilamellar liposomes).
  • low-density lipoprotein or “LDL” is a lipoprotein that varies in size (approx. 22 nm) and can contain a changing number of triglycerides and cholesteryl esters they actually have a mass and size distribution.
  • Each native LDL particle contains a single apolipoproteinB-100 molecule (Apo B-100, a protein with 4536 amino acid amino acid residues) and a phospholipid coat that circles the triglycerides and cholesteryl esters, keeping them soluble in the aqueous environment.
  • LDL is commonly referred to as bad cholesterol.
  • LDL cholesterol is cholesterol that is associated with LDLs.
  • LDL cholesterol When too much LDL cholesterol circulates in the blood, it can slowly build up in the inner walls of the arteries that feed the heart and brain. Together with other substances, it can form plaque, a thick, hard deposit that can narrow the arteries and make them less flexible. This condition is known as atherosclerosis. If a clot forms and blocks a narrowed artery, then heart attack or stroke can result.
  • an “mRNA” refers to a messenger ribonucleic acid.
  • An mRNA may be naturally or non-naturally occurring.
  • an mRNA may include modified and/or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers.
  • An mRNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and/or a polyadenylation signal.
  • An mRNA may have a nucleotide sequence encoding a polypeptide. Translation of an mRNA, for example, in vivo translation of an mRNA inside a mammalian cell, may produce a polypeptide.
  • nanoparticle refers to a particle having any one structural feature on a scale of less than about lOOOnm that exhibits novel properties as compared to a bulk sample of the same material.
  • nanoparticles have any one structural feature on a scale of less than about 500 nm, less than about 200 nm, or about 100 nm.
  • nanoparticles have any one structural feature on a scale of from about 50 nm to about 500 nm, from about 50 nm to about 200 nm or from about 70 to about 120 nm.
  • a nanoparticle is a particle having one or more dimensions of the order of about 1 - lOOOnm.
  • a nanoparticle is a particle having one or more dimensions of the order of about 10- 500 nm. In other exemplary- embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 50- 200 nm.
  • a spherical nanoparticle would have a diameter, for example, of between about 50-100 or 70- 120 nanometers. A nanoparticle most often behaves as a unit in terms of its transport and properties.
  • nanoparticles typically develop at a size scale of under 1000 nm, or at a size of about lOOnm, but nanoparticles can be of a larger size, for example, for particles that are oblong, tubular, and the like. Although the size of most molecules would fit into the above outline, individual molecules are usually not referred to as nanoparticles.
  • nucleic acid is used in its broadest sense and encompasses any compound and/or substance that includes a polymer of nucleotides. These polymers are often referred to as polynucleotides.
  • nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having ab-D-ribo configuration, a- LNA having an a-L-ribo configuration (a diastereomer of LNA), 2*-amino-LNA having a 2'- amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization) or hybrids thereof.
  • RNAs ribonucleic acids
  • a polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • phrases "pharmaceutically acceptable” is employed herein to refer 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.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • compositions may also include salts of one or more compounds.
  • Salts may be pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety to its salt form (e.g., by reacting a free base group with a suitable organic acid).
  • examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfide, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate,
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, tiimethylamine, triethylamine, ethylamine, and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.
  • a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains.
  • polypeptide refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, natural amino acids, etc.), polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-naturally occurring.
  • a therapeutic composition that “prevents” a disorder or condition refers to a composition that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • reverse oriented refers to a peptide, or a portion of the peptide, has a reverse amino acid sequence as compared to a non-reverse oriented peptide (i.e., the original sequence is read (or written) from right to left).
  • a non-reverse oriented peptide i.e., the original sequence is read (or written) from right to left.
  • reverse analog a peptide having its reverse sequence is as follows: EDCBA.
  • EDCBA In a dual domain peptide for example, Ac-hE-18A-NH2, either the hE sequence is read from right to left or the 18A sequence is read from right to left.
  • LRKLRKRLLR- DWLKAFYDKVAEKLKEAF (SEQ ID NO: 10) can be RLLRKRLKRL- DWLKAFYDKVAEKLKEAF (SEQ ID NO: 15) or LRKLRKRLLR- FAEKLKEAVKDYFAKLWD (SEQ ID NO: 16).
  • an “RNA” refers to a ribonucleic acid that may be naturally or non- naturally occurring.
  • an RNA may include modified and/or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers.
  • An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and/or a polyadenylation signal .
  • An RNA may have a nucleotide sequence encoding a polypeptide of interest.
  • an RNA may be a messenger RNA (mRNA).
  • RNAs may be selected from the non- liming group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, long non-coding RNA (IncRNA) and mixtures thereof.
  • siRNA small interfering RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • dsRNA Dicer-substrate RNA
  • shRNA small hairpin RNA
  • IncRNA long non-coding RNA
  • “scrambled” “scrambled version”, or “scrambled peptide” is refers to the composition of the amino acid sequence is the same as the unscrambled peptide, however the sequence of the amino acids is altered thus rendering the peptide unable to form either an a-amphipathic helix or does not possess lipid associating (or HSPG associating) properties. However, in some cases, as described in this invention, the scrambled peptide remains able to form a different helical structure, such as a n-helix. For example, if one peptide has the amino acid sequence ABCDE, the scrambled version of the peptide could have the amino acid sequence DEABC.
  • Scrambled peptides are often denoted as having a “Sc” prior to the portion of the peptide that is scrambled.
  • Sc-hE-18A denoted that the hE portion of the peptide is scrambled.
  • transfection refers to the introduction of a species (e.g., an RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.
  • a species e.g., an RNA
  • the term “treating” includes prophylactic and/or therapeutic treatments.
  • the term “prophylactic or therapeutic” treatment is art-recognized and includes administration to a subject of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
  • the unwanted condition e.g., disease or other unwanted state of the host animal
  • VLDL very Low Density Lipoproteins
  • LDL low density lipoprotein
  • VLDL particles have a diameter of 30-80 tun.
  • VLDL transports endogenous products where chylomicrons transport exogenous (dietary) products.
  • “Zwitterionic lipid” refers to a lipid molecule having both a positive and a negative charge.
  • Lipid Nanopartide (LNP) Compositions
  • the present disclosure provides lipid-based compositions, specifically lipid nanoparticles (LNPs), that comprise an apolipoprotein E (ApoE)-mimicking peptide and one or more lipids.
  • LNPs comprising an agent (e.g., a protein or a nucleic acid molecule) for delivery- to a target cell or population of target cells, methods for enhancing delivery- of an agent (e.g., a protein or a nucleic acid molecule) to a target cell or population of target cells, methods of delivering such LNPs to subjects that would benefit from modulation of target cell activity, and methods of treating diseases or disorders in such subjects.
  • LNP compositions comprising an apolipoprotein E (ApoE)-mimicking peptide and one or more lipids.
  • ApoE apolipoprotein E
  • lipid particles which are assemblies comprising lipid components and cargo.
  • Lipid particles may be, for example, liposomes, lipid micelles, solid lipid particles, lipoplexes, lipid nanoparticles (LNPs), or lipid-stabilized polymeric particles, comprised of one or a mixture of different biocompatible lipids.
  • LNPs lipid nanoparticles
  • the structural organization of these lipid particles may lead to an aqueous interior with a minimum bilayer as in liposomes or the lipid particles may have a solid interior as in solid nucleic acid lipid nanoparticles.
  • the lipid particles provided herein are lipid nanoparticles (LNPs).
  • the LNPs comprise ionizable lipids, structural lipids, non- cationic helper lipids, and/or PEG lipids.
  • the LNPs comprise other lipid components that are described below.
  • the one or more lipids comprise an ionizable lipid.
  • Ionizable lipids of the disclosure may comprise a central amine moiety and at least one biodegradable group.
  • the ionizable lipids described herein may be advantageously used in lipid nanoparticles of the disclosure for the delivery- of cargo to mammalian cells or organs.
  • the ionizable lipid ranges from about 20 mol % to about 80 mol % of the LNP composition.
  • the ionizable lipid ranges from about 30 mol % to about 70 mol %, from about 35 mol % to about 65 mol %, or from about 40 mol % to about 60 mol % of the LNP composition.
  • the ionizable lipids is Compound A, or a derivative or analog thereof:
  • the ionizable lipid is Compound B, or a derivative or analog thereof:
  • the ionizable lipid is Compound C, or a derivative or analog thereof:
  • the ionizable lipid is Compound D, or a derivative or analog thereof:
  • the ionizable lipid is Compound E, or a derivative or analog thereof:
  • the ionizable lipid is Compound F, or a derivative or analog thereof:
  • the ionizable lipid is Compound G, or a derivative or analog thereof:
  • the ionizable lipid is Compound H, or a derivative or analog thereof:
  • the ionizable lipids include, but are not limited to: [0094] and any combination thereof.
  • Additional ionizable lipids of the invention can be selected from the non-limiting group consisting of 3- ⁇ didodecylamino)-Nl,Nl,4-tridodecyl-l-piperazineethanamine (KL10), N 1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4-tridodecyl- 1 ,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19
  • the ionizable lipids are one or more of the compounds described in PCT Publication Nos. WO 2015/074805, WO 2015/199952, WO 2017/112865, WO 2017/075531, and WO 2021/026358 hereby incorporated by reference in their entireties.
  • Other ionizable lipids suitable for formulating a composition of the present disclosure can include those described in US 2015/0239834, hereby incorporated by reference in its entirety.
  • ionizable lipid comprises an amino group.
  • the amino group is an amine head group.
  • the ionizable lipid comprises two or more aliphatic tail groups, e.g., two to four aliphatic tail groups. In certain embodiments, the aliphatic tail groups are hydrophobic tail groups, e.g., each independently alkyl or alkenyl groups. [0099] In some embodiments, the ionizable lipid is a cationic lipid. In other embodiments, the ionizable lipid is a neutral lipid. In some embodiments, the ionizable lipid is a zwitterionic lipid.
  • the ionizable lipid is protonated at physiological pH.
  • the one or more lipids comprise a structural lipid. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle.
  • Structural lipids can include, but are not limited to, cholesterol, fecosterol, ergosterol, bassicasterol, tomatidine, tomatine, ursolic, alpha-tocopherol, and mixtures thereof.
  • the structural lipid ranges from about 5 mol % to about 65 mol % of the LNP composition. In some embodiments, the structural lipid ranges from about about 20 mol % to about 60 mol %, from about 25 mol % to about 55 mol %, or from about 30 mol % to about 50 mol % of the LNP composition.
  • the structural lipid is cholesterol.
  • the structural lipid includes cholesterol and a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.
  • the structural lipid is a sterol.
  • “sterols” are steroid alcohols.
  • the structural lipid is a steroid.
  • the structural lipid is cholesterol.
  • the structural lipid is an analog of cholesterol.
  • the structural lipid is alpha- tocopherol.
  • the structural lipid is apytosterol.
  • the phytosterol is a sitosterol, a stigmasterol, a campesterol, a sitostanol, a campestanol, a brassicasterol, a fucosterol, beta-sitosterol, stigmastanol, beta-sitostanol, ergosterol, lupeol, cycloartol, D5-avenaserol, D7-avenaserol or a D7-stigmasterol, including analogs, salts or esters thereof, alone or in combination.
  • the phytosterol component of a LNP of the disclosure is a single phytosterol. In some embodiments, the phytosterol component of a LNP of the disclosure is a mixture of different phytosterols (e.g.2, 3, 4, 5 or 6 different phytosterols). In some embodiments, the phytosterol component of an LNP of the disclosure is a blend of one or more phytosterols and one or more zoosterols, such as a blend of a phytosterol (e.g., a sitosterol, such as beta-sitosterol) and cholesterol.
  • a phytosterol e.g., a sitosterol, such as beta-sitosterol
  • the one or more lipids comprise a structural lipid, optionally a steroid, such as a sterol, e.g., cholesterol.
  • a steroid such as a sterol, e.g., cholesterol.
  • the one or more lipids may comprise non-cationic helper lipids.
  • the non-cationic helper lipid ranges from about 5 mol % to about 50 mol % of the LNP composition. In some embodiments, the non-cationic helper lipid ranges from about 5 mol % to about 50 mol %, about 10 mol % to about 40 mol %, or about 10 mol % to about 30 mol % of the LNP composition. In some embodiments, the non- cationic helper lipid ranges from about 5 mol % to about 25 mol % of the LNP composition.
  • the non-cationic helper lipid is a phospholipid. In some embodiments, the non-cationic helper lipid is a phospholipid substitute or replacement. In some embodiments, the “non-cationic helper lipid” is a lipid comprising at least one fatty acid chain of at least 8 carbons in length and at least one polar head group moiety. In other embodiments, the helper lipid is not a phosphatidyl choline (PC). In certain embodiments, the non- cationic helper lipid is a phospholipid or a phospholipid substitute.
  • PC phosphatidyl choline
  • the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof.
  • phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. In some embodiments, the non-cationic helper lipid is a DSPC analog, a DSPC substitute, oleic acid, or an oleic acid analog.
  • a non-cationic helper lipid is a non- phosphatidyl choline (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a 1 ,2-distearoyl-i77- glycero-3- phosphocholine (DSPC) substitute.
  • PC non- phosphatidyl choline
  • DSPC 1 ,2-distearoyl-i77- glycero-3- phosphocholine
  • the non-cationic helper lipids are phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof.
  • phospholipids comprise a phospholipid moiety and one or more fatty' acid moieties.
  • a phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations).
  • a phospholipid or an analog or derivative thereof may include choline.
  • a phospholipid or an analog or derivative thereof may not include choline. Particular phospholipids may facilitate fusion to a membrane.
  • a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2- lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • the lipid component of a lipid nanoparticle of the disclosure may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety' may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.
  • a fatty' acid moiety maybe selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide.
  • Such reactions may be useful in functionalizing a lipid bilayer of a LNP to facilitate membrane permeation or cellular recognition or in conjugating a LNP to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • a targeting or imaging moiety e.g., a dye
  • Phospholipids usefill in the compositions and methods described herein may be selected from the non-limiting group consisting of 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O
  • a LNP includes DSPC.
  • a LNP includes DOPE.
  • a LNP includes DMPE.
  • a LNP includes both DSPC and DOPE.
  • a non-cationic helper lipid for use in a LNP is selected from the group consisting of DSPC, DMPE, and DOPC or combinations thereof.
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • the one or more lipids comprise a non-cationic helper lipid.
  • the non-cationic helper lipid is a phospholipid.
  • the non-cationic helper lipid comprises a diacylglyerol group.
  • the non-cationic helper lipid is a glycerophospholipid, such as a phosphatidylcholine.
  • the non-cationic helper lipid is not a phosphatidylcholine.
  • the phospholipid is distearoylphosphatidylcholine (DSPC).
  • the one or more lipids comprise a PEG-lipid comprising a lipid group covalently bonded to a PEG group.
  • the lipid group is a phospholipid.
  • the lipid group comprises a diacylglycerol, dialkylglycerol, or dialkylamine group.
  • the lipid group is a glycerophospholipid group.
  • the lipid group is dimyristoyl glycerol.
  • the PEG lipids ranges from about 0.1 mol % to about 25 mol %, from about 1 mol % to about 25 mol %, or from about 5 mol % to about 20 mol % of the LNP composition.
  • the one or more lipids comprise a PEG-lipid comprises a lipid group covalently bonded to a PEG group.
  • PEG lipids can affect the length of time the nanoparticles can exist in vivo (e.g., in the blood). PEG lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. PEG lipids used herein may modulate pharmacokinetic properties of the LNPs.
  • the PEG lipid comprises a lipid moiety and a polymer moiety based on PEG (sometimes referred to as polyethylene oxide)) (a PEG moiety).
  • PEG sometimes referred to as polyethylene oxide
  • Additional suitable PEG lipids are disclosed in PCT Publication Nos. WO 2015/095340 (p. 31, line 14 to p. 37, line 6), WO 2006/007712, and WO 2011/076807 ("stealth lipids”), hereby incorporated by reference in their entireties.
  • the lipid moiety may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about €40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester.
  • the alkyl chain length comprises about CIO to C20.
  • the dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups.
  • the chain lengths may be symmetrical or asymmetric.
  • PEG polyethylene glycol or other polyalkylene ether polymer, such as an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide.
  • the PEG moiety is unsubstituted.
  • the PEG moiety may be substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups.
  • the PEG moiety' may comprise a PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., J.
  • the PEG moiety may be a PEG homopolymer.
  • the PEG moiety has a molecular weight of about 130 to about 50,000, such as from about 150 to about 30,000, or even from about 150 to about 20,000.
  • the PEG moiety may have a molecular weight of about 150 to about 15,000, from about 150 to about 10,000, from about 150 to about 6,000, or even from about 150 to about 5,000.
  • the PEG moiety has a molecular weight of about 150 to about 4,000, from about 150 to about 3,000, from about 300 to about 3,000, from about 1,000 to about 3,000, or from about 1,500 to about 2,500.
  • the PEG moiety is a “PEG-2K,” also termed ‘TEG 2000,” which has an average molecular weight of about 2,000 daltons.
  • PEG-2K is represented herein by the following formula I:
  • n 45, meaning that the number averaged degree of polymerization comprises about 45 subunits
  • n may range from about 30 to about 60.
  • n may range from about 35 to about 55.
  • n may range from about 40 to about 50.
  • n may range from about 42 to about 48.
  • n may be 45.
  • R may be selected from H, substituted alkyl, and unsubstituted alkyl.
  • R may be unsubstituted alkyl, such as methyl.
  • the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog # GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (catalog # DSPE-020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG- dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG- cholesterol (l-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- [omega]-methyl-poly(ethylene glycol), PEG-dilauroylglycerol, P
  • the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG.
  • the PEG lipid may be PEG2k-DSPE. In some embodiments, the PEG lipid may be PEG2k-DMA. In yet other embodiments, the PEG lipid may be PEG2k-C-DMA. In certain embodiments, the PEG lipid may be compound S027, disclosed in WO2016/010840 (paragraphs [00240] to [00244]). In some embodiments, the PEG lipid may be PEG2k-DSA. In other embodiments, the PEG lipid may be PEG2k-Cl 1 . In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.
  • PEG lipids include lipids with PEG chains such as hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHE), 1, 2-distearoyl-glycero-3- phosphoethanolamine-N-[methoxy (PEG)-2000] (DSPE-PEG2000), 1, 2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy (PEG)-2000] modified with a maleimidic group in the distal end of the chain 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (PEG)-2000], DSPE-PEG2000-MAL, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] (DMPE-PEG550), 1, 2- dioleoyl-l-3-trimethylammonium propane (DOTAP), and those with PEG chains such as hydrogenated
  • PEG lipids include DMG-PEG (1,2-Dimyristoyl-sn- glycerol, methoxypolyethylene glycol-PEG), DMA-PEG (polyethylene glycol)- dimethacrylate-PEG) and DMPE-PEG550 (l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-550]), PEG), monosialoganglioside Gml, and polyamide oligomers (PAO), such as those described in U.S. Pat. No. 6,320,017.
  • DMG-PEG 1,2-Dimyristoyl-sn- glycerol, methoxypolyethylene glycol-PEG
  • DMA-PEG polyethylene glycol)- dimethacrylate-PEG
  • DMPE-PEG550 l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[
  • the lipid nanoparticles can include DMPE-PEG2000 or DMG-PEG which could be substituted with DMPE-PEG2000 in any of the formulations taught herein.
  • Other suitable PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) (such as those described in U.S. Pat. No.
  • PEG-modified dialkylamines and PEG-modified l,2-diacyloxypropan-3-amines PEG-modified diacylglycerols and dialkylglycerols
  • PEG-DSPE mPEG (mw2000)-diastearoylphosphatidylethanolamine
  • the lipid nanopaiticles optionally comprise neutral lipids.
  • Neutral lipids suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids.
  • Neutral lipids, when present, can be any of a number of lipid species, which exist either in an uncharged or neutral zwitterionic form at physiological pH.
  • Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.
  • the neutral lipid component is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).
  • the neutral lipid comprises saturated fatty acids with carbon chain lengths in the range of CIO to C20, inclusive, In some embodiments, the neutral lipid includes mono- or di-unsaturated fatty acids with carbon chain lengths in the range of CIO to C20, inclusive.
  • Suitable neutral lipids include, but are not limited to, DPPC (Dipalmitoyl phosphatidylcholine), POPC (Palmitoyl-Oleoyl Phosphatidyl Cholin), DOPE (1,2-dioleoyl- sn-glycero-3-phosphoethanolamine), DSPC (disteroylphosphatidyl choline), egg L-alpha- phosphatidylcholine (EPC);l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); and SM (Sphingomyelin).
  • DPPC Dynamic acid phosphatidylcholine
  • POPC Palmitoyl-Oleoyl Phosphatidyl Cholin
  • DOPE 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine
  • DSPC diisteroylphosphatidyl choline
  • EPC egg L
  • the lipid nanoparticles optionally comprise cationic lipids.
  • Cationic lipids suitable for use in a lipid composition of the disclosure include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N- dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), l,2-Dioleoyl-3-Dimethylammonium -propane (DODAP), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),
  • DODAC N,N-dioleyl-N,N-dimethylammonium chloride
  • DDAB N,N-distearyl-N,N- dimethylammonium
  • DOCDAP 1.2-Dioleoylcarbamyl-3-Dimethylammonium-propane
  • DLINDAP l,2-Dilineoyl-3- Dimethylammonium-propane
  • DLTAP dilauryl(C12:0) trimethyl ammonium propane
  • DOGS Dioctadecylamidoglycyl spermine
  • DOSPA Dioleoyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate
  • DMRIE 1.2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide
  • CLinDMA 3- Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12- octadecadienoxy)propane
  • DODMA N,N-dimethyl-2,3-dioleyloxy)propylamine
  • a number of cationic lipids, and methods for making them, are described in, for example, U.S. Pat. Numbers 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; 5,785,992; 5,830,430; 6,056,938; 7,893,302; 7,404,969; 8,034,376; 8,283,333; and 8,642,076; U.S. Pat. Publ. Nos. 2006/0083780 and 2006/0240554; as well as PCT Publ.
  • the lipid nanoparticles optionally comprise anionic lipids.
  • Anionic lipids can also include, but are not limited to, fetty acids (e.g., oleic, linoleic, linolenic acids); cholesteryl hemisuccinate (CHEMS); l,2-di-0-tetradecyl-sn-glycero-3- phospho-(l ’-rac-glycerol) (Diether PG); l,2-dimyristoyl-sn-glycero-3-phospho-(l '-rac- glycerol) (sodium salt); 1 ,2-dimyristoyl-sn-glycero- 3-phospho-L-serine (sodium salt); 1- hexadecanoyl,2-(9Z, 12Z)-octadecadienoyl-sn-glycero-3- phosphate; 1,2-dioleoyl-sn-glycero- 3-[phospho-rac-(l -gly
  • anionic lipids include, but are not limited to fatty acids, such as oleic, linoleic, and linolenic acids; and cholesteryl hemisuccinate. Such lipids can be used alone or in combination, for a variety of purposes, such as to attach ligands to the liposome surface.
  • the lipid nanoparticles described herein further comprise one or more compounds that are capable of enhancing the cellular uptake or cytosolic distribution of the lipid nanoparticle and/or its encapsulated composition (e.g., small molecule drug, protein-based drug, or nucleic acid-based drug etc.).
  • Compounds that can enhance the cellular uptake can include levodopa, naphazoline hydrochloride, acetohexamide, niclosamide, diprophylline, and isoxicam, or a combination thereof.
  • the lipid nanoparticles comprise lipid bilayers encapsulating one or more agents encompassed by the present disclosure, such as small molecule drug, protein-based drug, or nucleic acid-based drug. In some embodiments, the lipid nanoparticles are formulated to facilitate an uptake into cells.
  • the lipid nanoparticles are formulated to facilitate uptake into monocytes, dendritic cells, and/or macrophages.
  • the lipid nanoparticle can, in some embodiments, further comprise additional agents.
  • the lipid nanoparticle further comprises one or more antioxidants.
  • the antioxidant can help stabilize the lipid nanoparticle and prevent, decrease, and/or inhibit degradation of the cationic lipids and/or active agents encapsulated in the lipid nanoparticle.
  • the antioxidant is a hydrophilic antioxidant, a lipophilic antioxidant, a metal chelator, a primary antioxidant, a secondary antioxidant, or salts or mixtures thereof.
  • the antioxidant comprises EDTA, or a salt thereof.
  • the lipid nanoparticle further comprises EDTA in combination with one, two, three, four, five, six, seven, eight, or more additional antioxidants (e.g., primary' antioxidants, secondary antioxidants, or other metal chelators).
  • additional antioxidants e.g., primary' antioxidants, secondary antioxidants, or other metal chelators.
  • antioxidants include, but are not limited to, hydrophilic antioxidants, lipophilic antioxidants, and mixtures thereof.
  • Non-limiting examples of hydrophilic antioxidants include chelating agents (e.g., metal chelators) such as ethylenediaminetetraacetic acid (EDTA), citrate, ethylene glycol tetraacetic acid (EGTA), l,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), diethylene triamine pentaacetic acid (DTPA), 2,3-dimercapto-l-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA), cc-lipoic acid, salicylaldehyde isonicotinoyl hydrazone (SIR), hexyl thioethylamine hydrochloride (HTA), desferrioxamine, salts thereof, and mixtures thereof.
  • metal chelators e.g., metal chelators
  • EDTA ethylenediaminetetraacetic acid
  • Additional hydrophilic antioxidants include ascorbic acid, cysteine, glutathione, dihydrolipoic acid, 2 -mercaptoethane sulfonic acid, 2 -mercaptobenzimidazole sulfonic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, sodium metabisulfite, salts thereof, and mixtures thereof.
  • Non-limiting examples of lipophilic antioxidants include vitamin E isomers such as a-, ⁇ -, y-, and ⁇ -tocopherols and ⁇ -, ⁇ -, y-, and 5-tocotrienols; polyphenols such as 2-tert-butyl-4-methyl phenol, 2-tert-butyl-5-methyl phenol, and 2-tert-butyl-6-methyl phenol; butylated hydroxyanisole (BHA) (e.g., 2-teri-butyl- 4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole); butylhydroxytoluene (BHT); tert- butylhydroquinone (TBHQ); ascorbyl palmitate; rc-propyl gallate; salts thereof; and mixtures thereof.
  • vitamin E isomers such as a-, ⁇ -, y-, and ⁇ -tocopherols and ⁇ -, ⁇ -, y-, and 5-tocotrieno
  • the lipid-based particle is a pH-sensitive nanoparticle.
  • pH-sensitive nanoparticles PPSDS
  • biologically active agents e.g, siRNA molecules (Tang et al., SiRNA Crosslinked Nanoparticles for the Treatment of Inflammation-induced Liver Injury, Advanced Science, 2016, 4(2), el 600228).
  • the lipid nanoparticle (LNP) is formulated to encapsulate an agent, such as a small molecule drug, protein-based drag, or nucleic acid-based drag, using a spontaneous vesicle formation formulation procedure as previously described in Semple et al. (2010) Nat. Biotechnol. 28172-28176.
  • lipid nanoparticles can be engineered to alter the surface properties of particles so the lipid nanoparticles can penetrate the mucosal barrier.
  • Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
  • oral e.g., the buccal and esophageal membranes and tonsil tissue
  • ophthalmic e.g., gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum)
  • nasal, respiratory e.g., nasal, pharyngeal, tracheal and bronchial
  • Lipid nanoparticles engineered to penetrate mucus can comprise, but are not limited to, a polymeric material (i.e., a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer.
  • the polymeric material can include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.
  • the polymeric material can be biodegradable and/or biocompatible.
  • the polymeric material can additionally be irradiated.
  • the polymeric material can be gamma irradiated (e.g., PCT Publ. No.
  • Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone) ?
  • PCL poly(caprolactone)
  • EVA ethylene vinyl acetate polymer
  • PDA poly(lactic acid)
  • PLA poly(L-lactic acid)
  • PGA poly(glycolic acid)
  • PGA poly(lactic acid-co-glycolic acid)
  • PLA poly(L-lactic acid-co-glycolic acid)
  • PDLA poly(L-lactide)
  • PLA poly(D,
  • polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as polyethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(he
  • the lipid nanoparticle can be coated or associated with a co-polymer such as, but not limited to, a block co-polymer, and (polyethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see, e.g., U.S. Pat. Publ. Numbers 2012/0121718 and 2010/0003337; and U.S. Pat. No. 8,263,665).
  • the co-polymer can be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle can be in such a way that no new chemical entities are created.
  • GRAS generally regarded as safe
  • the lipid nanoparticle can comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. (2011) Angew. Chem. Int. Ed. 50:2597-2600).
  • agents encompassed by the present disclosure can be sustained release formulations, such as encapsulated into a nanoparticle or a rapidly eliminated nanoparticle and the nanoparticles or a rapidly eliminated nanoparticle can then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art.
  • the polymer, hydrogel or surgical sealant can be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).
  • Nanoparticles can be encapsulated into any polymer known in the art that can form a gel when injected into a subject.
  • the nanoparticle can be encapsulated into a polymer matrix that can be biodegradable.
  • compositions encompassed by the present disclosure can be formulated as controlled release nanoparticles.
  • the nanoparticle formulation for controlled release and/or targeted delivery can further include at least one controlled release coating.
  • Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).
  • the controlled release and/or targeted delivery formulation can comprise at least one degradable polyester which can contain polycationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • compositions encompassed by the present disclosure can be formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other conjugate-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of therapeutic agents (Aleku et al. (2008) Cancer Res. 68: 9788-9798; Strumberg et al. (2012) Int. J. Clin. Pharmacol. Then (2012) 50:76-78; Santel et al. (2006) Gene Ther.
  • a lipoplex such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other conjugate-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-
  • therapeutic agents and compositions encompassed by the present disclosure can be encapsulated in, linked to and/or associated with synthetic nanocarriers.
  • Synthetic nanocarriers include, but are not limited to, those described in International Pub. Nos. WO 2010/005740, WO 2010/030763, WO 2012/13501, WO 2012/149252, WO 2012/149255, WO 2012/149259, WO 2012/149265, WO 2012/149268, WO 2012/149282, WO 2012/149301, WO 2012/149393, WO 2012/149405, WO 2012/149411, and WO 2012/149454, and U.S. Pat. Publ.
  • the synthetic nanocarrier formulations can be lyophilized, such as by methods described in PCT Publ. No. WO 2011/072218 and U.S. Pat No. 8,211,473, hereby incorporated by reference in their entireties.
  • the synthetic nanocarriers can contain reactive groups to release the conjugates described herein (e.g., PCT Publ. No. WO 2012/0952552 and U.S. Pat. Publ. No.
  • Synthetic nanocarriers can be formulated for targeted release, e.g., to release the therapeutic agents at a specified pH and/or after a desired time interval.
  • the synthetic nanoparticle can be formulated to release the conjugates after 24 hours and/or at a pH of 4.5 (e.g., PCT Publ. Numbers WO 2010/138193 and WO 2010/138194 and U.S. Pai. Publ. Numbers
  • the synthetic nanocarriers can be formulated for controlled and/or sustained release of conjugates described herein.
  • the synthetic nanocarriers for sustained release can be formulated by methods known in the art, described herein and/or as described in PCT Publ. No. WO 2010/138192 and U.S. Pat. Publ. No. 2010/0303850, hereby incorporated by reference in their entireties.
  • LNP size may be measured by various analytical methods known in the art.
  • LNP size may be measured using Asymetric-Flow Field Flow Fractionation - Multi-Angle Light Scattering (AF4-MALS).
  • LNP size may be measured by separating particles in the composition by hydrodynamic radius, followed by measuring the molecular weights, hydrodynamic radii and root mean square radii of the fractionated particles.
  • LNP size and particle concentration may be measured by nanoparticle tracking analysis.
  • LNP samples are diluted appropriately and injected onto a microscope slide. A camera records the scattered light as the particles are slowly infused through field of view.
  • the Nanoparticle Tracking Analysis processes the movie by tracking pixels and calculating a diffusion coefficient. This diffusion coefficient can be translated into the hydrodynamic radius of the particle. Such methods may also count the number of individual particles to give particle concentration.
  • LNP size, morphology, and structural characteristics may be determined by cryo-electron microscopy (“cryo-EM”).
  • the LNPs of the LNP compositions disclosed herein have a size (e.g., Z-average diameter) of about 1 to about 500 nm. In some embodiments, the LNPs have a size of about 10 to about 250 nm. In further embodiments, the LNPs have a size of about 50 to about 200 nm. In some embodiments, the LNPs have a size of about 75 to about 200 nm or about 100 to 200 nm. In some embodiments, the LNPs have a size of about 75 to about 150 nm.
  • the mean lipid particle diameter is greater than 300 nm.
  • the lipid particle has a diameter of about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less, e.g., fiom about 50 to about 150 nm.
  • Smaller particles generally exhibit increased circulatory lifetime in vivo compared to larger particles. Smaller particles have an increased ability to reach tumor sites than larger nanoparticles.
  • the lipid particle has a diameter fiom about 15 to about 50 nm.
  • the cargo delivered via LNP composition may be a biologically active agent.
  • the cargo is or comprises one or more biologically active agent, such as an antibody (e.g., monoclonal, chimeric, humanized, nanobody, and fragments thereof etc.), cholesterol, hormone, peptide, protein, chemotherapeutic and other types of antineoplastic agent, mRNA, low molecular weight drag, vitamin, co-fector, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex forming oligonucleotide, antisense DNA or RNA composition, chimeric DNA:RNA composition, allozyme, aptamer, ribozyme, decoys and analogs thereof, plasmid and other types of vectors, and small nucleic acid molecule, RNAi agent, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double
  • LNPs are formed by mixing an aqueous biologically active agent solution with an organic solvent-based lipid solution.
  • Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol.
  • the organic solvent may be 100% ethanol.
  • a pharmaceutically acceptable buffer e.g., for in vivo administration of LNPs, may be used.
  • a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 6.5.
  • a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 7.0.
  • the composition has a pH ranging from about 7.2 to about 7.7.
  • the composition has a pH ranging from about 7.3 to about 7.7 or ranging from about 7.4 to about 7.6.
  • the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7.
  • the pH of a composition may be measured with a micro pH probe.
  • a cryoprotectant is mixed with the composition comprising LNPs.
  • cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol.
  • the composition comprising LNPs may include up to 10% cryoprotectant, such as, for example, sucrose.
  • the composition comprising LNPs may comprise tris saline sucrose (TSS).
  • TSS tris saline sucrose
  • the composition comprising LNPs may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant.
  • the composition comprising LNPs may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose.
  • the composition comprising LNPs may include a buffer.
  • the buffer may comprise a phosphate buffer (PBS), a Tris buffer, a citrate buffer, and mixtures thereof.
  • the buffer comprises NaCl.
  • the buffer lacks NaCl. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM.
  • the buffer is a Tris buffer.
  • Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM.
  • the buffer comprises NaCl and Tris. Certain exemplary' embodiments of the LNP compositions contain 5% sucrose and 45 mM NaCl in Tris buffer. In other exemplary- embodiments, the composition comprising LNPs may contain sucrose in an amount of about 5% w/v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The salt, buffer, and cryoprotectant amounts may be varied such that the osmolality of the overall composition is maintained.
  • the final osmolality may be maintained at less than 450 mOsm/L.
  • the osmolality is between 350 and 250 mOsm/L.
  • Certain embodiments have a final osmolality of 300 +/- 20 mOsm/L or 310 +/- 40 mOsm/L.
  • microfluidic mixing, T-mixing, or cross-mixing of the aqueous RNA solution and the lipid solution in an organic solvent is used.
  • flow rates, junction size, junction geometry', junction shape, tube diameter, solutions, and/or RNA and lipid concentrations may be varied.
  • LNPs or LNP compositions may be concentrated or purified, e.g., via dialysis, centrifugal filter, tangential flow filtration, or chromatography.
  • the LNPs may be stored as a suspension, an emulsion, or a lyophilized powder, for example.
  • an LNP composition is stored at 2-8° C.
  • the LNP compositions are stored at room temperature.
  • an LNP composition is stored frozen, for example at -20° C or -80° C. In other embodiments, an LNP composition is stored at a temperature ranging from about 0° C to about -80° C. Frozen LNP compositions may be thawed before use, for example on ice, at room temperature, or at 25° C.
  • LNP compositions encompassed by the present disclosure can also be formulated using natural and/or synthetic polymers to reduce or inhibit the attachment of unwanted substances, such as bacteria, to the LNPs, or to enhance drug deliver of the LNPs.
  • Non-limiting examples of polymers which can be used for drug delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, CA) formulations from MIRUS® Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTM (Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers, RONDELTM (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, CA) and pH responsive co-block polymers such as, but not limited to, PHASERXTM (
  • agents and compositions encompassed by the present disclosure can be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof.
  • a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copoly
  • the polymers used in the present disclosure may have undergone processing to reduce or inhibit the attachment of unwanted substances, such as bacteria, to the LNPs.
  • the polymer can be processed by methods known and/or described in the art and/or described in PCT Publ. No. WO 2011/50467, hereby incorporated by reference in its entirety.
  • the LNP compositions disclosed herein comprise apolipoprotein E-mimicking peptides.
  • the ApoE-mimicking peptides can be single domain or dual domain peptides.
  • Disclosed are ApoE-mimicking peptides comprising a receptor binding domain of ApoE and a lipid-associating peptide.
  • the ApoE-mimicking peptide further comprises an amino hexanoic acid, such as an acetylated amino hexanoic acid (Ac- Aha).
  • the Ac- Aha is at the N-terminus of the peptide.
  • the Aha can be inserted between the lipid-associating peptide comprises a class A amphipathic-helical domain.
  • the ApoE-mimicking peptide comprises an Ac-Aha
  • the lipid-associating peptide comprises a class A amphipathic-helical domain.
  • the class A amphipathic-helical domain is DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1), DWLRAFYDKVAEKLREAF (SEQ ID NO: 2), DWLRALYDKVAEKLREAL (SEQ ID NO: 3), DLLRALYDKVAEKLREAW (SEQ ID NO: 4), or FAEKLKEAVKDYFAKLWD (SEQ ID NO: 5).
  • the ApoE-mimicking peptide comprises an Ac-Aha, wherein the lipid-associating peptide comprises a class A amphipathic-helical domain, wherein the receptor binding domain of ApoE can be covalently linked to the lipid- associating peptide.
  • the ApoE-mimicking peptide comprises an Ac-Aha, w'herein said peptide is protected using an amide group at the C-terminus.
  • the ApoE-mimicking peptide comprises an Ac-Aha, wherein the receptor binding domain of ApoE can be LRKLRKRLLR (SEQ ID NO: 6), LRRLRRRLLR (SEQ ID NO: 7), LRKMRKRLMR (SEQ ID NO: 8), or RLTRKRGLK (SEQ ID NO: 9).
  • the receptor binding domain of ApoE can also be, but is not limited to, LRKLRKRFFR (SEQ £D NO: 17), LRKLPKRLLR (SEQ ID NO: 18), LRNVRKRLVR (SEQ ID NO: 19), MRKLRKRVLR (SEQ ID NO: 20), LRRLRRRLLR (SEQ ID NO: 7), LRKLRKRFFR (SEQ ID NO: 17), LRKLRKRLLR (SEQ ID NO: 6), or LRKMRKRLMR (SEQ ID NO: 8).
  • the ApoE-mimicking peptide comprises an Ac-Aha, wherein the ApoE-mimicking peptide can be Ac-Aha-hElSA-NH 2 or Ac-Aha-[R]hEl 8A- NFb.
  • the ApoE-mimicking peptide of Ac-Aha-hElSA-NH 2 is Ac-Aha- LRKLRKRLLRDWLKAFYDKVAEKLKEAF-NH 2 (SEQ ID NO: 21).
  • the ApoE- mimicking peptide of Ac-Aha-[R]hE18A-NH 2 is Ac-Aha- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NHb (SEQ ID NO: 22).
  • the ApoE-mimicking peptide comprises a fatty acid moiety, co-amino fatty acid moiety, or an acetylated co-amino fatty acid moiety.
  • the receptor binding domain of ApoE can be covalently linked to the lipid-associating peptide.
  • the ApoE-mimicking peptide is protected using an amide group at the C-terminus.
  • the ApoE-mimicking peptide can be: butanoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH 2 (SEQ ID NO:
  • gadoleoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH 2 SEQ ID NO:
  • fatty acid moiety is shown at the left side and is linked to the peptide LRRLRRRLLR (SEQ ID NO: 7).
  • EPA indicates a moiety derived from 5,8,11,14, 17- eicosapentaenoic acid
  • DHA indicates a moiety- derived from 4,7,10,13,16,19- docosahexaenoic acid.
  • the ApoE-mimicking peptide comprises a fatty acid moiety derived from a natural oil or fat, e.g., fish oil, wherein the ApoE-mimicking peptide can be:
  • the ApoE-mimicking peptide is a mixture of peptides comprising fatty acid groups derived from the fish oil used to prepare them.
  • the ApoE-mimicking peptide can be any of the disclosed peptides comprising a fatty acid.
  • the ApoE-mimicking peptide can be any of the disclosed peptides comprising an acetylated fatty acid.
  • the ApoE-mimicking peptide comprises an Ac-Aha, wherein the receptor binding domain of apolipoprotein E is scrambled. Examples of scrambled receptor binding domains of ApoE are provided below.
  • the receptor binding domain is covalently linked to said lipid-associating peptide, wherein both the receptor binding domain of apolipoprotein E and the lipid-associating peptide are scrambled. Examples of scrambled receptor binding domains of ApoE and scrambled lipid-associating peptides are provided below.
  • Apolipoprotein E-mimicking peptides have both direct cholesterol lowering effects by providing an alternative ligand for receptors on the liver to clear atherogenic Apolipoprotein B containing lipoproteins (LDL, VLDL, and ⁇ -VLDL), and direct beneficial effects on the artery wall.
  • the Apo E-mimicking peptides can enhance the removal of cholesterol from the artery- wall, working in conjunction with HDL, increasing the formation of lipid poor preP-HDL that accept cholesterol from macrophages.
  • the Apo E-mimicking peptides can stimulate macrophage-mediated clearance of dead and dying cells in the artery wall (eflferocytosis), improve the quality of HDL by increasing PON-1 levels and bringing down plasma lipid hydroperoxide levels, decrease macrophage content in atherosclerotic lesions resulting in more stable lesions, and decrease inflammation in the artery wall.
  • the Apo E-mimicking peptides reduce the size of atherosclerotic lesions more rapidly than apoA-I mimetic peptides and more rapidly than the statins (HMG-CoA reductase inhibitors). Atherosclerotic lesion regression persists in Apo E-mimicking peptides treated animals even when cholesterol levels are the same as in saline treated animals.
  • Apolipoprotein E plays an important role in the metabolism of triglyceride- rich lipoproteins, such as very low density lipoprotein (VLDL) and chylomicrons.
  • Apolipoprotein E mediates the high affinity binding of Apo E-containing lipoproteins to the low density lipoprotein (LDL) receptor (Apo B, E receptor) and the members of its gene family, including LDL receptor related protein (LRP), very low density lipoprotein receptor (VLDLR) and the Apo E2 receptor (Apo E2R).
  • LDL low density lipoprotein
  • LRP LDL receptor related protein
  • VLDLR very low density lipoprotein receptor
  • Apo E2 receptor Apo E2 receptor
  • Apo E is a protein that binds lipid and has two major domains (Mahley, R.W., et al. J. Lipid Res. 1999, 40:622-630).
  • the 22 kDa amino terminal domain has been shown by X- ray crystallographic studies to be a 4-helix bundle (Wilson, C., et al. Science 1991;252: 1817- 1822) and to contain a positively-charged receptor binding domain.
  • VLDL very low-density lipoprotein
  • the VLDL is defective in binding to receptors.
  • the positively charged arginine (Arg)-rich cluster domain of the Apo E and the C-terminal amphipathic helical domain are both required for the enhanced uptake of atherogenic Apo E-containing lipoproteins.
  • Apo E is secreted as a 299 amino acid residue protein with a molecular weight of 34,200. Based on thrombin cleavage of Apo E into two fragments, a two-domain hypothesis was initially suggested to explain the fact that the C-terminal region of Apo E (192-299) is essential for its binding to hypertriglyceridemic VLDL and the N-terminal 22 kDa domain (1- 191), binds to the LDL-R.
  • the LNP compositions disclosed herein can be linked to a fatty' acid moiety, an co- amino fatty acid moiety, or an acetylated co-amino fatty acid moiety.
  • the fatty acid moiety, the co-amino fatty acid moiety, or the acetylated co-amino fatty acid moiety is linked to a disclosed peptide via the N-terminal amino group of the peptide.
  • the linkage between the fatty acid moiety, the co-amino fatty acid moiety, or the acetylated co-amino fatty acid moiety and the N-terminal amino group of the peptide has the a structure represented by the following formulas II-IV, respectively: wherein A is an aliphatic group have 2-32 carbon atoms..
  • the aliphatic group is an alkyl group.
  • the aliphatic group comprises 0-3 double bonds.
  • the aliphatic group is an alkenyl group.
  • the fatty acid moiety linked to the disclosed peptide is derived from a purified fatty acid. In some embodiments, the fatty acid moiety linked to the disclosed peptide is derived from a saturated fatty acid. In a other embodiments, the fatty acid moiety linked to the disclosed peptide is derived from an unsaturated fatty acid, such as a polyunsaturated fatty acid with two or more double bonds.
  • Exemplary fatty acids from which a fatty acid moiety is derived include, without limitation, butyric acid, caproic acid, caprylic acid, capric acid, decanoic acid, lauric acid, myristic acid, palmitic acid, pentadecanoic acid, stearic acid, arachidic acid, behenic acid, erucic acid, lignoceric acid, margaric acid, myristoleic acid, palmitoleic acid, oleic acid, gadoleic acid, ricinoleic acid, vaccenic acid, linoleic acid, linolenic acid, alpha-linolenic acid, gamma-linolenic acid, licanic acid, margaroleic acid, arachidic acid, gadoleic acid, nervonic acid, arachidonic acid, docosapentaenoic (DPA), eicosapentaenoic acid (EPA), doco
  • Exemplary saturated fatty acids include, but are not limited to, propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid, nonacosanoic acid, triacont
  • Exemplary unsaturated fatty acids include, but are not limited to, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA), erucic acid, docosahexaenoic acid (DHA), and docosapentaenoic acid.
  • the tatty acid moiety linked to the disclosed peptide is derived from an unpurified fatty acid or mixture of fatty acids such as natural oil or fat.
  • a natural oil or fat is a heterogeneous mixture of generally hydrophobic compounds comprising one or more fatty acids.
  • the tatty acid source may comprise a natural oil or fat, such as (but not limited to) animal fits, biological oils, or vegetable oils such as soya bean oil, coconut oil, palm oil, palm kernel oil, rapeseed oil, cottonseed oil, linseed oil, sunflower oil, fish oil, algae oil, and the like.
  • the natural oil or fat is one that contains or is enriched for one or more omega-3 fatty acids, for example, marine oil, for example, fish oil, krill oil and algae oil. Any oil containing DHA and/or EP A can be used.
  • the natural oil or fat contains at least 70% or about 70%, by weight, DHA, for example, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, or at least 90% or about 90%, by weight, DHA.
  • the natural oil or fat contains between 5% or about 5% and 15% or about 15% EPA, for example, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15%, by weight, EPA.
  • the natural oil or fat contains not more than 10% or about 10% EPA or less than 10% or about 10%, EPA.
  • the fatty acid moiety is derived from an omega-3 fatty acid.
  • omega-3 polyunsaturated fatty acid(s) or “omega-3 fatty acid” refers to a family of unsaturated fatty carboxylic acids that have in common a carbon-carbon bond in the n-3 position (i.e., the third bond from the methyl end of the molecule). Typically, they contain from about 16 to about 24 carbon atoms and from three to six carbon-carbon double bonds. Omega-3 polyunsaturated fatty acids can be found in nature, and these natural omega-3 polyunsaturated forty acids frequently have all of their carbon-carbon double bonds in the cis-configuration.
  • omega-3 fatty acids include, but are not limited to, 7,10,13- hexadecatrienoic acid (sometimes abbreviated as 16:3 (n-3)); 9,12,15-octadecatetrienoic acid (a-linolenic acid (ALA), 18:3 (n-3)); 6,9,12,15-octadecatetraenoic acid (stearidonic acid (STD), 18:4 (n-3)); 11,14,17-eicosatrienoic acid (eicosatrienoic acid (ETE), 20:3 (n-3)); 8,11,14,17-eicosatetraenoic acid (eicosatetraenoic acid (ETA), 20:4 (n-3)); 5,8,11,14,17- eicosapentaenoic acid (eicosapentaenoic acid (EPA), (20:5 (n-3)); 7,10,13,16,19- docosapentaenoic acid
  • EPA Eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • a fatty acid comprises a chain length between C6 and C24, CIO and C24, CIO and C28, or CIO and C32, including synthetic fatty acids with odd carbon numbers.
  • a fatty acid comprises a chain length selected from: CIO, C12, C14, C16, C18, C20, C20, C22 and C24, preferably fiom C14, C16 and C18.
  • the fetty acid has a chain length selected from C13, C15 and C17.
  • the fatty acid has between 4 and 28 carbons.
  • the fatty acid aliphatic chain comprises 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 carbon atoms.
  • the fatty' acid is a naturally-occurring fatty acid.
  • the fetty acid is a short chain fatty acid (e.g., less than six carbons), a medium chain fatty' acid (e.g., 6-12 carbons), long chain fatty acids (e.g., longer than 12 carbons), or a ven- long chain fatty acid (e.g., longer than 22 carbons).
  • the fatty acid is an unsaturated fatty acid in the cis configuration. In other embodiments, the fatty acid is an unsaturated fatty' acid in the trans configuration.
  • the ApoE-mimicking peptide comprises a co-amino fatty acid moiety.
  • Exemplary- co-amino-fatty acid moieties are derived from co-amino-fatty acids including, without limitation, 4-amino-butyric acid, 6-amino-caproic acid, 8-amino-caprylic acid, 10-amino-capric acid (10-amino-decanoic acid), 12-amino-lauric acid (12-amino- dodecanoic acid), 14-amino-myristic acid (14-amino-tetradecanoic acid), 14-amino- myristoleic acid, 16-amino-palmitic acid (16-amino-hexadecanoic acid), 18-amino-stearic acid, 18-amino-oleic acid, 16-amino-palmitoleic acid, 18-amino-linoleic acid, 18-amino- linolenic acid and 20-amino-arachidonic acid.
  • the co-amino-fatty acids including, without
  • the co-amino fatty acid moiety is 4-amino-butanoyl, 6-amino- caproyl, 8-amino-octanoyl, 10-amino-decanoyl, 12-amino-lauroyl, 14-amino-myristoyl, 14- amino-myristoleoyl, 16-amino-palmiteoyl, 18-amino-stearoyl, 18-amino-oleoyl, 16-amino- palmitoleoyl, 18-amino-linoleoyl, 18-amino-linolenoyl, or 20-amino-arachidonoyl.
  • co-amino fatty acid moiety is 6-amino-caproyl (or alternatively referred to as 6-amino hexanoyl).
  • the co-amino-fatty acid moiety' is derived from a co-amino- fatty acid having the structure: wherein A is an aliphatic group have 2-32 carbon atoms.
  • the aliphatic group is an alkyl group.
  • the aliphatic group comprises 0-3 double bonds.
  • the aliphatic group is an alkenyl group.
  • A is -(CH 2 ) 5- .
  • the co-amino-fatty acid moiety is linked to the peptide via the N-terminal amino group of the peptide, and following linking to the peptide, the co-amino- fatty acid moiety has the structure: wherein A is an aliphatic group have 2-32 carbon atoms..
  • the aliphatic group is an alkyl group.
  • the aliphatic group comprises 0-3 double bonds.
  • the aliphatic group is an alkenyl group.
  • A is an aliphatic group have 2-32 carbon atoms.
  • the aliphatic group is an alkyl group.
  • the aliphatic group comprises 0-3 double bonds.
  • the aliphatic group is an alkenyl group.
  • the ApoE-mimicking peptide comprises an acetylated co- amino fatty acid moiety.
  • the disclosed peptides can be linked to any of the disclosed co-amino-fatty acids, and then further comprise an acetyl moiety' on the co- amino group.
  • the co-amino-fatty acid moiety is linked to the peptide via the N-terminal amino group of the peptide, and following linking to the peptide, the co-amino group is acetylated, and the co-amino-fatty acid moiety has the structure: wherein A is an aliphatic group have 2-32 carbon atoms..
  • the aliphatic group is an alkyl group.
  • the aliphatic group comprises 0-3 double bonds.
  • the aliphatic group is an alkenyl group.
  • the co-amino-fatty acid moiety is linked to the peptide via the N-terminal amino group of the peptide, and following linking to the peptide, the co-amino group is acetylated, and the co-amino-fatty acid moiety has the structure: wherein A is an aliphatic group have 2-32 carbon atoms..
  • the aliphatic group is an alkyl group.
  • the aliphatic group comprises
  • fatty acids from which the fatty acid moiety is derived are commercially available and can be prepared by different chemical methods (Recent Developments in the Synthesis of Fatty Acid Derivatives, Editors: Knothe G and Derksen JTB, AOCS Press 1999, ISBN 1-893997-00-6.) Receptor binding domain peptides
  • the receptor binding domain peptide for the Apo E mimetics can be a human receptor binding domain peptide of Apo E.
  • receptor binding domain peptide of the disclosed Apo E mimetics can comprise the amino acid sequence of LRKLRKRLLR (SEQ ID NO: 6), LRRLRRRLLR (SEQ ID NO: 7), or LRKLRKRFFR (SEQ ID NO: 17).
  • the receptor binding domain peptide of such Apo E mimetics can also be from a species selected from the group consisting of mouse, rabbit, monkey, rat, bovine, pig and dog.
  • the receptor binding domain peptide for the Apo E mimetics can also be the LDL receptor (LDLR) binding domain of apolipoprotein B (ApoB).
  • the LDL receptor (LDLR) binding domain of ApoB can have the sequence RLTRKRGLK (SEQ ID NO: 9).
  • ApoB- 100 is a 550,000 Da glycoprotein with nine amino acids (3359-3367) serving as the binding domain for the LDL receptor (Segrest et al., J. Lipid. Res. 42, pp. 1346-1367 (2001)).
  • LDL Upon binding to LDLR in clathrin coated pits, LDL is internalized via endocytosis and moves into the endosome where a drop in pH causes the receptor to dissociate from the LDL. The receptor is recycled back to the surface of the cell while the LDL is moved into the lysosome where the particle is degraded (Goldstein et al., Ann. Rev. Cell Biol. 1, pp. 1-39 (1985)).
  • the LDL receptor (LDLR) binding domain of ApoB when used with the disclosed peptides can also be altered and/or modified as described throughout this application for Apo E.
  • LDL receptor (LDLR) binding domain of ApoB can be used with the disclosed lipid-associating peptides, wherein the LDL receptor (LDLR) binding domain of ApoB is covalently linked to said lipid-associating peptide.
  • the LDL receptor (LDLR) binding domain of ApoB can be scrambled, reverse-oriented, can be part of a domain switched peptide as described below.
  • Lipid-associating peptides can be used alone or in combination with the Apo E- mimicking peptides.
  • the lipid associating peptide for these Apo E mimetics can be, but are not limited to, class A amphipathic helical peptides, class A amphipathic helical peptide mimetics of apoA-I having aromatic or aliphatic residues in the non-polar face, small peptides including pentapeptides, tetrapeptides, tripeptides, dipeptides and pairs of amino acids, Apo-J (G* peptides), and peptide mimetics, e.g., as described below.
  • the lipid-associating peptides for use in the disclosed methods include class A amphipathic helical peptides, e.g., as described in U.S. Pat. No. ⁇ ,664,230, and PCT Publications WO 2002/15923 and WO 2004/034977, hereby incorporated by reference in their entireties It was discovered that peptides comprising a class A amphipathic helix (“class A peptides”), are capable of mitigating one or more symptoms of atherosclerosis as well as treating other disorders.
  • class A peptides class A amphipathic helix
  • Class A peptides are characterized by formation of an a-helix that produces a segregation of polar and non-polar residues thereby forming a polar and a nonpolar face with the positively charged residues residing at the polar-nonpolar interface and the negatively charged residues residing at the center of the polar face (see, e.g., Anantharamaiah (1986) Meth. Enzymol, 128: 626-668). It is noted that the fourth exon of apo A-I, when folded into 3.667 residues/tum produces a class A amphipathic helical structure.
  • One class A peptide designated 18A (see, e.g., Anantharamaiah (1986) Meth. Enzymol, 128: 626-668) was modified as described herein to produce peptides orally administrable and highly effective at inhibiting or preventing one or more symptoms of atherosclerosis and/or other indications described herein. Without being bound by a particular theory, it is believed that the disclosed peptides can act in vivo by picking up seeding molecule(s) that mitigate oxidation of LDL.
  • a number of these class A peptides were made including, the peptide designated 4F, D4F, 5F, and D5F, and the like.
  • Various class A peptides inhibited lesion development in atherosclerosis-susceptible mice and rabbits.
  • the peptides show varying, but significant degrees of efficacy in mitigating one or more symptoms of the various pathologies described herein.
  • a number of such peptides are illustrated in Table 2. * Linkers are underlined; NMA is N-Methyl Anthranilyl
  • the peptides include variations of 4F (D-W-F-K-A-F-Y -D- K-V-A-E-K-F-K-E-A-F in Table 2 (SEQ ID NO: 47)), also known as L-4F, where all residues are L form amino acids) or D-4F where one or more residues are D form amino acids).
  • 4F D-W-F-K-A-F-Y -D- K-V-A-E-K-F-K-E-A-F in Table 2 (SEQ ID NO: 47)
  • L-4F L-4F, where all residues are L form amino acids
  • D-4F where one or more residues are D form amino acids
  • the C-terminus, and/or N-terminus, and/or internal residues can be blocked with one or more blocking groups as described herein.
  • peptides of Table 2 are illustrated with an acetyl group or an N- methylanthranilyl group protecting the amino terminus and an amide group protecting the carboxyl terminus, any of these protecting groups may be eliminated and/or substituted with another protecting group as described herein.
  • the peptides can comprise one or more D-form amino acids as described herein. In certain embodiments, every amino acid (e.g., every chiral amino acid) of the peptides of Table 2 is a D-form amino acid.
  • Table 2 is not folly inclusive.
  • other suitable class A amphipathic helical peptides can routinely be produced (e.g., by conservative or semi-conservative substitutions (e.g., D replaced by E), extensions, deletions, and the like).
  • truncations may be made of any one or more of peptides shown herein (e.g., peptides identified as 2F, 3F, 3F 14 , 4F, 5F, 6F, or 7F— in Table 2).
  • A-F-Y-D-K-V-A-E-K-L-K-E-A-F illustrates a peptide comprising 14 amino acids from the C-terminus of 18A comprising one or more D amino acids, while others illustrate other truncations.
  • Longer peptides are also suitable. Such longer peptides may entirely form a class A amphipathic helix, or the class A amphipathic helix (helices) can form one or more domains of the peptide.
  • this invention contemplates multimeric versions of the peptides (e.g., concatamers).
  • the peptides illustrated herein can be coupled together (directly or through a linker (e.g., a carbon linker, or one or more amino acids) with one or more intervening amino acids).
  • Illustrative polymeric peptides include 18A-Pro-18A and the peptides in the following table (Table 2B), in certain embodiments comprising one or more D amino acids, more preferably with every amino acid a D amino acid as described herein and/or having one or both termini protected.
  • Table 2B Table 2B
  • the LNP compositions disclosed herein comprise modified class A amphipathic helix peptides.
  • Certain preferred peptides incorporate one or more aromatic residues at the center of the nonpolar face, e.g. , , (as present in 4F), or with one or more aliphatic residues at the center of the nonpolar fece, e.g. see, e.g., Table 3.
  • the central aromatic residues on the nonpolar fece of the peptide due to the presence of it electrons at the center of the nonpolar fece can allow water molecules to penetrate near the hydrophobic lipid alkyl chains of the peptide-lipid complex, which in turn would enable the entry of reactive oxygen species (such as lipid hydroperoxides) shielding them from the cell surface.
  • the peptides with aliphatic residues at the center of the nonpolar face e.g. can act similarly but not quite as effectively as
  • the peptides can convert pro-inflammatory HDL to anti- inflammatory HDL or make anti-inflammatory HDL more anti-inflammatory, and/or decrease LDL-induced monocyte chemotactic activity generated by artery wall cells equal to or greater than D4F or other peptides shown in Table 2.
  • c. Other class A and some Class Y Amphipathic Helical Peptides.
  • Class A amphipathic helical peptides that have an amino acid composition identical to one or more of the class A amphipathic helical peptides described above. Thus, for example, in certain embodiments this invention contemplates peptides having an amino acid composition identical to 4F.
  • this invention includes active agents that comprise a peptide that consists of 18 amino acids, where the 18 amino acids consist of 3 alanines (A), 2 aspartates (D), 2 glutamates (E), 4 phenylalanines (F), 4 lysines (K), 1 valine (V), 1 tryptophan (W), and 1 tyrosine (Y); and where the peptide forms a class A amphipathic helix; and protects a phospholipid against oxidation by an oxidizing agent.
  • the peptides comprise least one "D” amino acid residue; and in certain embodiments, the peptides comprise all "D” form amino acid residues. A variety of such peptides are illustrated in Table 4.
  • Lipid- associating peptides can comprise a peptide that is 18 amino acids in length and forms a class A amphipathic helix.
  • the peptide comprises the following amino acid composition: 2 aspartates, 2 glutamates, 4 lysines, 1 tryptophan, 1 tyrosine, no more than one leucine, no more than 1 valine, no less than 1 and no more than 3 alanines, and with 3 to 6 amino acids from the group: phenylalanine, alpha-naphthalanine, beta-naphthalanine, histidine.
  • the peptide comprises either 9 or 10 amino acids on the polar face in a helical wheel representation of the class A amphipathic helix.
  • the peptide comprises 4 amino acids with positive charge at neutral pH.
  • two of the positively charged residues reside at the interlace between the polar and non-polar faces. In some embodiments, two of the four positively charged residues reside on the polar face that are contiguous. In some embodiments, two of the amino acid residues on the non-polar face are from the group: phenylalanine, alpha-naphthalanine, beta- naphthalanine, histidine. In some embodiments, two of the amino acid residues on the non- polar face are are also contiguous, and if there are 4 or more amino acids from this group on the non-polar face, there are also at least 2 residues from this group that are not contiguous.
  • the acidic amino acids are glutamic acid rather than having two aspartic acids and two glutamic acids.
  • the lipid associating peptide can be 18A, wherein each of the acidic amino acids of 18A are Glu residues.
  • Class Y amphipathic helical peptides are known to those of skill in the art (see, e.g., Segrest et al. (1992) J. Lipid Res. 33: 141-166; Oram and Heinecke (2005) Physiol Rev. 85: 1343-1372, and the like).
  • peptides include, but are not limited to, an 18 amino acid peptide that forms a class A amphipathic helix or a class Y amphipathic helix described by formula V: D X X KY X X D K XY D K X K D Y X (V) where the D's are independently Asp or Glu; the Ks are independently Lys or Arg; the Xs are independently Leu, nor Leu, Vai, lie, Trp, Phe, Tyr , or a-Nal and all X residues are on the non-polar face (e.g., when viewed in a helical wheel diagram) except for one that can be on the polar face between two K residues; the Y's are independently Ala, His, Ser, Gin, Asn, or Thr non-polar face (e.g., when viewed in a helical wheel diagram) and the Y's are independentiy one Ala on the polar face, one His, one Ser, one Gin
  • peptides of this kind which include peptides with histidine, and/or alpha- and/or beta-napthalanine are shown in Table 5. Reverse (retro-), inverse, retro-inverso-, and circularly permuted forms of these peptides are also contemplated.
  • alpha-Nph analogs can be designed. Furthermore, His can be incorporated to Nph analogs. D>E analogs, E>D analogs and D-E switch analogs are additional possibilities.. [00212] As described above for 4Nph, a minimum of 7 additional analogs for each of the analogs given below.
  • any of the peptides described herein can comprise non-natural amino acids in addition to or instead of the corresponding the natural amino acids identified herein.
  • Such modifications include, but are not limited to acetylation, amidation, fbrmylation, methylation, sulfation, and the like.
  • Illustrative non-natural amino acids include, but are not limited to Ornithine, norleucine, norvaline, N-methylvaline, 6-N-methyllysine, N- methylisoleucine, N-methylglycine, sarcosine, inosine, allo-isoleucine, isodesmolysine, 4- hydroxyproline, 3-hydroxyproline, allo-hydroxylysine, hydoxylisine, N-ethylasparagine, N- ethylglycine, 2,3-diaminopropionic acid, 2,2'-diaminopropionic acid, desmosine, 2,4- diaminobutyric acid, 2-aminopimelic acid, 3-aminoisobutyric acid, 2 -aminoisobutyric acid, 2- aminoheptanoic acid, 6-aminocaproic acid, 4-aminobutyric acid, 2-aminobutyric acid, beta- alanine,
  • this invention contemplates particularly the use of modified lysines.
  • modifications include, but are not limited to, biotin modification of epsilon lysines and/or methylation of the epsilon lysines.
  • Illustrative peptide comprising epsilon methylated lysines include, but are not limited to: Ac-D-W-F-K(eCH 3 )2-A-F-Y-D-K(eCH 3 )2-
  • Other modified amino acids include but are not limited to ornithine analogs and homoaminoalanine analogs (instead of for Haa and for Om] and the like. It is noted that these modifications are illustrative and not intended to be limiting.
  • Illustrative 4F analogues that possess modified amino acids are shown in Table 6.
  • the single-domain Apo E mimetics can comprise a receptor binding domain of Apo E or a lipid-associating peptide.
  • Dual domain peptides are also disclosed. Dual domain peptides can be Apo E- mimicking peptides, comprising of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide.
  • Apo E-mimicking peptides comprising of a combination of the disclosed receptor binding domains of apolipoprotein B and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide.
  • Non-limiting examples of the disclosed Apo E-mimicking peptides are provided in Table 7.
  • the disclosed Apo E-mimicking peptides can also be N-terminally protected using acetyl and amino groups.
  • Table 7 provides non-limiting representative examples of the disclosed Apo E-mimicking peptides comprising a dual domain.
  • Apo E mimetics comprising of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a domain switched orientation.
  • Apo E mimetics comprising of a combination of the disclosed receptor binding domains of apotipoprote in B and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a domain switched orientation.
  • These peptides can be referred to as “domain switched” “switched domain”, or “switched” peptides.
  • Apo E mimetics consisting of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a domain switched orientation to those described above and in Table 7.
  • the lipid-associating peptide is covalently linked to the receptor binding domain of apolipoprotein E such that the lipid-associating peptide is at the N-terminus of the apolipoprotein E-mimicking peptide.
  • Table 8 provides non-limiting examples of the disclosed Apo E mimetics comprising a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a domain switched orientation.
  • the domain switched Apo E mimetics can also be N- terminally protected using acetyl and amino groups.
  • the LNP compositions disclosed herein comprise Apo E-mimicking peptides, comprising of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a reversed orientation.
  • Apo E-mimicking peptides comprising of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein either the sequence of the receptor binding domain or the sequence of the lipid- associating peptide or both sequences are in the reversed orientation.
  • Apo E-mimicking peptides comprising of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein either the sequence of the receptor binding domain or the sequence of the lipid- associating peptide or both sequences are in the
  • E-mimicking peptides comprising of a combination of the disclosed receptor binding domains of apolipoprotein B and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a reversed orientation.
  • Table 9 provides non-limiting examples of the disclosed Apo E-mimicking peptides comprising a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a reversed orientation.
  • the LNP compositions disclosed herein comprise Apo E-mimicking peptides, comprise of: a receptor binding domain of apolipoprotein E and a lipid-associating peptide, wherein said receptor binding domain is covalently linked to said lipid-associating peptide, wherein the receptor binding domain of apolipoprotein E is scrambled.
  • a apolipoprotein E-mimicking peptide comprising of: a receptor binding domain of apolipoprotein E comprising the amino acid sequence of D-W-L-K-A-F-V-Y-D-K-V-F- K-L-K-E-F-F (SEQ ID NO: 94); and a lipid-associating peptide, w'herein said receptor binding domain is covalently linked to said lipid-associating peptide.
  • Apo E-mimicking peptides comprising of: a receptor binding domain of apolipoprotein B and a lipid-associating peptide, wherein said receptor binding domain is covalently linked to said lipid-associating peptide, wherein the receptor binding domain of apolipoprotein B is scrambled.
  • the Apo E-mimicking peptides comprise of: a receptor binding domain of apolipoprotein E and a lipid-associating peptide, wherein said receptor binding domain is covalently linked to said lipid-associating peptide, wherein the lipid- associating peptide is scrambled.
  • a Apo E-mimicking peptides comprising: a lipid binding domain of apolipoprotein E comprising the amino acid sequence of E-W-L-K-A-F-V-Y-E-K-V-F-K-L-K-E-F-F (SEQ ID NO: 95) and a receptor binding domain peptide, wherein said lipid binding domain is covalently linked to said receptor binding domain peptide.
  • the Apo E mimetics comprise of: a receptor binding domain of apolipoprotein E and a lipid-associating peptide of apolipoprotein E, wherein receptor binding domain is covalently linked to said lipid-associating peptide, wherein both the receptor binding domain and the lipid-associating peptide are scrambled.
  • Table 10 provides non-limiting examples of the disclosed scrambled Apo E mimetics comprising a receptor binding domain of apolipoprotein E and a lipid-associating peptide, wherein said receptor binding domain is covalently linked to said lipid-associating peptide, wherein the receptor binding domain of apolipoprotein E is scrambled.
  • Table 10 Scrambled Domain Peptides.
  • the disclosed scrambled Apo E mimetics can also be N-terminally and C-terminally protected using acetyl and amide groups.
  • the disclosed scrambled Apo E mimetics can also be reverse-oriented as described above.
  • the ApoE-mimicking peptides may comprise a linker to connect the receptor binding domain of ApoE and the lipid-associating peptide together.
  • Any suitable linker can be used in accordance with the present disclosure.
  • the peptide linkages can be selected from the group consisting of etc. by methods known in the art and further described in the following references: Spatola (1983) p. 267 in Chemistry' and Biochemistry' of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York; Spatola (1983) Vega Data 1(3) Peptide Backbone Modifications, (general review); Morley (1980) Trends Pharm Sci pp.
  • linker is a cleavable linker.
  • cleavable linkers include protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH sensitive linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, x-ray cleavable tinkers, etc.
  • protease cleavable peptide linkers include protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH sensitive linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers
  • the receptor binding domain or the lipid-assodating peptide can be modified or altered as described above.
  • the receptor binding domain or the lipid-associating peptide can be mutated, scrambled, and/or reverse-oriented. Any other modifications or alterations disclosed herein for the dual-domain polypeptides can also be used for the single- domain peptides.
  • peptides disclosed herein are also contemplated.
  • scrambled peptides can also be reverse-oriented, or can be in a switched orientation.
  • reverse-oriented peptides can be in a switched orientation. All other combinations of the disclosed peptides are also contemplated.
  • Non- limiting examples of the peptides have been described herein (see Tables 1-5, for example).
  • the term “analog” is used interchangeably with “variant” and “derivative.”
  • variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. Such, amino acid sequence modifications typically fall into one or more of three classes: substantial; insertional; or deletional variants.
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily are smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once.
  • substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final derivative or analog.
  • Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with Tables 11 and 12 and are referred to as conservative substitutions.
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 11, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in the protein properties are those in which: (a) the hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; tryptophan, tyrosinyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or hystidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine
  • the Apo E mimetics and other proteins or peptides herein disclosed which have at least, 70% or at least 75% or at least 80% or at least 85% or at least 90% or at least 95% homology to the Apo E mimetics specifically recited herein. Those of skill in the art readily understand how to determine the homology of two proteins.
  • polypeptides can be modified by either natural processes, such as post- translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications.
  • Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation.
  • Variants can also include peptidomimetics.
  • Peptidomimetics typically are short sequences of amino acids that in biological properties mimic one or more fimction(s) of a particular protein. Peptide analogs enhance some property of the original peptide, such as increases stability, increased efficacy, enhanced delivery-, increased half-life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position.
  • the present disclosure encompasses peptidomimetics having a bond, a peptide backbone, or an amino acid component replaced with a suitable mimic.
  • suitable amino acid mimics include 0 alanine, L -amino butyric acid, L amino butyric acid, L- amino isobutyric acid, L amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N- Boc-N- CBZ-L-lysine, N- -Boc-N-a-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L- norvaline, N-a-Boc-N- -L-omithine, N- -Boc-N-a-CBZ-L-omithine, Boc-p-nitro-L- phenylalanine, Boc-hydroxyproline, and
  • nucleic acids that can encode the polypeptide sequences described herein are also disclosed. This would include all degenerate sequences related to a specific polypeptide sequence, i.e. all nucleic acids having a sequence that encodes one particular polypeptide sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in feet disclosed and described herein through the disclosed polypeptide sequences.
  • the LNP compositions provided herein can comprise an acetyl group followed by a protecting group.
  • the protecting group can be, but is not limited to, a fatty acid.
  • the fatty acids can be saturated, unsaturated or essential fetty acids.
  • Fatty acids can include but are not limited to DHA, EPA, linoleic acid, or any other saturated amino acid such as myristic acid.
  • the LNP compositions provided herein can bear one, two, three, four, or more protecting groups.
  • the protecting groups can be coupled to the C- and/or N-terminus of the peptide(s) and/or to one or more internal residues comprising the peptide(s) (e.g., one or more R-groups on the constituent amino acids can be blocked).
  • any of the peptides described herein can bear, e.g., an acetyl group protecting the amino terminus and/or an amide group protecting the carboxyl terminus.
  • a “dual protected peptide” is Ac- LRKLRKRLLRDWLKAFYDKVAEKLKEAF-NH 2 (SEQ ID NO: 10) with blocking groups), either or both of these protecting groups can be eliminated and/or substituted with another protecting group as described herein.
  • blockage, particularly of the amino and/or carboxyl termini of the subject peptides of this invention can improve oral deliver ⁇ ' and can also increase serum half-life.
  • a wide number of protecting groups are suitable for this purpose.
  • Such groups include, but are not limited to acetyl, amide, and alkyl groups with acetyl and alkyl groups being particularly preferred for N-terminal protection and amide groups being preferred for carboxyl terminal protection.
  • the protecting groups can include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl, and others.
  • Carboxyl protecting groups include amides, esters, and ether-forming protecting groups can also be used.
  • an acetyl group can be used to protect the amino terminus and an amide group can be used to protect the carboxyl terminus.
  • Additional blocking groups include alkyl groups of various lengths, e.g., groups having the formula: where n ranges from about 1 to about 20, preferably from about 1 to about 16 or 18, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
  • the protecting groups include, but are not limited to alkyl chains as in fatty 7 acids, propeonyl, formyl, and others.
  • carboxyl protecting groups can include amides, esters, and ether-forming protecting groups. These blocking groups can enhance the helix-forming tendencies of the peptides.
  • Blocking groups can include alkyl groups of various lengths, e.g., groups having the formula: where n ranges from about 3 to about 20, preferably from about 3 to about 16, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
  • Other protecting groups include, but are not limited to Fmoc, t-butoxycarbonyl (t- BOC), 9-fhioreneacetyl group, 1 -fluorenecarboxylic group, 9-florenecaiboxylic group, 9- fluorenone-1 -carboxylic group, benzyloxycarbonyl, xanthyl (Xan), trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), tosyl (Tos), 2,2,5 ,7,8- pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (M
  • Protecting/blocking groups are well known to those of skill as are methods of coupling such groups to the appropriate residue(s) comprising the peptides of this invention (see, e.g., Greene etal., (1991) Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc. Somerset, N.J.). For example, acetylation can be accomplished during the synthesis when the peptide is on the resin using acetic anhydride. Amide protection can be achieved by the selection of a proper resin for the synthesis.
  • compositions disclosed herein can also comprise one or more D-form (dextro rather than levo) amino acids as described herein.
  • D-form amino acids for example, at least two enantiomeric amino acids, at least 4 enantiomeric amino acids or at least 8 or 10 enantiomeric amino acids can be in the “D” form amino acids.
  • every' other, or even every amino acid (e.g., every enantiomeric amino acid) of the peptides described herein is a D-form amino acid.
  • At least 50% of the enantiomeric amino acids can be “D” form, at least 80% of the enantiomeric amino acids are “D” form, at least 90%, or even all of the enantiomeric amino acids can be in the “D” form amino acids.
  • FMOC-Aha can be added to the growing chain as the last amino acid using the normal amino acid chain extension procedure (use of HOBt+DCC or HBTU as condensing agents). After the removal of the FMOC group using 20% piperidine in DMF, the NH 2 can be acetylated using either excess of acetic anhydride under basic conditions or by condensing acetic acid using amino acid condensing agents used for peptide chain elongation.
  • compositions comprising a lipid nanoparticle (LNP) comprising one or more lipids and an apolipoprotein E (ApoE)- mimicking peptide, wherein the ApoE-mimicking peptide comprises a receptor-binding domain of ApoE and a lipid-associating domain.
  • LNP lipid nanoparticle
  • ApoE apolipoprotein E
  • compositions and methods provided herein can be utilized to treat a subject in need thereof.
  • the subject is a mammal such as a human, or a non-human mammal.
  • the composition is preferably administered as a pharmaceutical composition and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • the aqueous solution is pyrogen-free, or substantially pyrogen-free.
  • the excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.
  • the pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like.
  • the composition can also be present in a transdermal delivery- system, e.g., a skin patch.
  • the composition can also be present in a solution suitable for topical administration, such as an eye drop.
  • the pharmaceutical compositions provided herein comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a composition.
  • physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent depends, for example, on the route of administration of the composition.
  • the preparation or pharmaceutical composition can be a self-emulsifying drug deliverysystem or a self-microemulsifying drug delivery system.
  • the pharmaceutical composition also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a therapeutic compound.
  • Liposomes for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • the pharmaceutical compositions provided herein can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop).
  • routes of administration including, for example, orally (for example, drenches as in
  • compositions may also be formulated for inhalation.
  • a composition may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973; 5,763,493; 5,731,000; 5,541,231; 5,427,798; 5,358,970; and 4,172,896, as well as in patents cited therein.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association an active composition with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a composition with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water- in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a composition as an active ingredient.
  • Compositions may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered composition moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredients) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art,
  • oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfinning, and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfinning, and preservative agents.
  • Suspensions in addition to the active compositions, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active composition with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active composition.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active composition.
  • compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.
  • Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active composition may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an active composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary' propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
  • compositions suitable for parenteral administration comprise one or more active compositions in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and
  • Injectable depot forms are made by forming microencapsulated matrices of the subject compositionsin biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drag to polymer, and the nature of the particular polymer employed, the rate of drag release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drag in liposomes or microemulsions that are compatible with body tissue.
  • biodegradable polymers such as polylactide-polyglycolide.
  • Depot injectable formulations are also prepared by entrapping the drag in liposomes or microemulsions that are compatible with body tissue.
  • active compositions can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Methods of introduction may also be provided by rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery' of drags, including proteinacious biopharmaceuticals.
  • a variety of biocompatible polymers including hydrogels, including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compositional a particular target site.
  • compounds can be used alone or conjointly administered with another type of therapeutic agent (e.g., an antiviral agent).
  • another type of therapeutic agent e.g., an antiviral agent.
  • conjoint administration of therapeutic compositions with one or more additional therapeutic agent(s) provides improved efficacy relative to each individual administration of the composition or the one or more additional therapeutic agent(s).
  • the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the therapeutic composition and the one or more additional therapeutic agent(s).
  • contemplated salts include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts.
  • contemplated salts include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.
  • contemplated salts include, but are not limited to, Na, Ca, K, Mg, Zn, copper, cobalt, cadmium, manganese, or other metal salts.
  • compositions described herein may comprise wetting agents, emulsifiers and/or lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coating agents such as sweetening, flavoring and perfuming agents, preservatives and antioxidants.
  • antioxidants examples include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfete, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfete, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (
  • a therapeutically effective dose of a composition comprising a lipid nanoparticle (LNP) comprising one or more lipids and an apolipoprotein E (ApoE)-mimicking peptide is about 0.01 mg/kg to about 500 mg/kg, about 0.05 mg/kg to about 250 mg/kg, about 0.1 mg/kg to about 100 mg/kg, about 0.5 mg/kg to about 50 mg/kg, or about 1 mg/kg to about 25 mg/kg.
  • a therapeutically effective dose of a composition comprising a lipid nanoparticle (LNP) comprising one or more lipids and an apolipoprotein E (ApoE)-mimicking peptide is about 0.01 mg/kg to about 500 mg/kg. In some embodiments, the therapeutically effective dose of a composition comprising a lipid nanoparticle (LNP) is preferably about 0.01 mg/kg to 100 mg/kg.
  • the lipid nanoparticle can be administered, for example, once daily, twice daily, once every two weeks, or once monthly. The duration of administration of the lipid nanoparticle can be dependent on the severity of a disease or the stage of disease a subject is afflicted with. For example, the lipid nanoparticle can be administered for at least 1 or 2 weeks. Longer durations of administration are also contemplated.
  • the effective daily dose of the active composition can be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments, the active compositionis administered two or three times daily. In some embodiments, the active compositionis administered once daily.
  • compositions and methods provided herein are useful for the treatment or prevention of a disease, disorder, or condition.
  • compositions and methods described herein relate to the treatment or prevention of a disease or disorder associated a pathological immune response, such as an autoimmune disease, an allergic reaction and/or an inflammatory disease.
  • the disease or disorder is an inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis).
  • the disease or disorder is psoriasis.
  • the disease or disorder is atopic dermatitis.
  • compositions and methods described herein can be used to treat any subject in need thereof.
  • the pharmaceutical compositions described herein can be used, for example, as a pharmaceutical composition for preventing or treating an autoimmune disease, such as chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, muckle-wells syndrome, rheumatoid arthritis, multiple sclerosis, or Hashimoto's disease; an allergic disease, such as a food allergy, pollenosis, or asthma; an infectious disease, such as an infection with Clostridium difficile; an inflammatory' disease such as a TNF-mediated inflammatory disease (e.g., an inflammatory disease of the gastrointestinal tract, such as pouchitis, a cardiovascular inflammatory condition, such as atherosclerosis, or an inflammatory lung disease, such as chronic obstructive pulmonary disease, fibrotic disease, or cystic fibrosis); a pharmaceutical composition for suppressing rejection in organ transplantation or other situations in which tissue rejection might occur; a supplement, food, or beverage for improving immune functions; or a reagent for
  • compositions and methods provided herein are usefill for the treatment of inflammation.
  • the pharmaceutical compositions described herein can be used for preventing or treating inflammation of any tissue and organs of the body, including musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, ocular inflammation, inflammation of the reproductive system, and other inflammation, as discussed below.
  • Immune disorders of the musculoskeletal system include, but are not limited, to those conditions affecting skeletal joints, including joints of the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle, and foot, and conditions affecting tissues connecting muscles to bones such as tendons.
  • immune disorders which may be treated with the compositions and methods described herein include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, osteitis pubis, and osteitis fibrosa cystic).
  • arthritis including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis
  • tendonitis synovitis, ten
  • Ocular immune disorders refers to an immune disorder that affects any structure of the eye, including the eye lids.
  • ocular immune disorders which may be treated with the compositions and methods described herein include, but are not limited to, blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis.
  • Examples of nervous system immune disorders which may be treated with the compositions and methods described herein include, but are not limited to, encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis and schizophrenia.
  • Examples of inflammation of the vasculature or lymphatic system which may be treated with the compositions and methods described herein include, but are not limited to, arthrosclerosis, arthritis, phlebitis, vasculitis, and lymphangitis.
  • Examples of digestive system immune disorders which may be treated with the compositions and methods described herein include, but are not limited to, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, and proctitis.
  • Inflammatory bowel diseases include, for example, certain art- recognized forms of a group of related conditions.
  • Crohn's disease regional bowel disease, e.g., inactive and active forms
  • ulcerative colitis e.g., inactive and active forms
  • the inflammatory' bowel disease encompasses irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis.
  • Other less common forms of IBD include indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet’s disease, sarcoidosis, scleroderma, IBD- associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis.
  • reproductive system immune disorders which may be treated with the compositions and methods described herein include, but are not limited to, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis, and vulvodynia.
  • compositions and methods described herein may be used to treat autoimmune conditions having an inflammatory' component.
  • autoimmune conditions include, but are not limited to, acute disseminated alopecia universalise, Behcet's disease, Chagas' disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, diabetes mellitus type 1, giant cell arteritis, goodpasture's syndrome.
  • Grave's disease Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonlein purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, Muckle-Wells syndrome, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmune haemolytic anemia, interstitial cystitis, Lyme disease, morphea, psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.
  • compositions and methods described herein may be used to treat T-cell mediated hypersensitivity diseases having an inflammatory component.
  • Such conditions include, but are not limited to, contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dustmite allergy) and gluten-sensitive enteropathy (Celiac disease).
  • compositions and methods described herein include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, ulceris, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel, skin allografts, skin homografts, and heart valve xengrafts, sewrum sickness, and
  • Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Type 1 diabetes, asthma, inflammatory' bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease, and inflammation accompanying infectious conditions (e.g., sepsis).
  • compositions and methods described herein relate to the treatment or prevention of a metabolic disease or disorder a, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), Nonalcoholic Steatohepatitis (NASH) or a related disease.
  • a metabolic disease or disorder a such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • the compositions and methods described herein relate to the treatment of Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH).
  • NAFLD Nonalcoholic Fatty Liver Disease
  • NASH Nonalcoholic Steatohepatitis
  • the compositions and methods described herein can be used to treat any subject in need thereof.
  • compositions and methods described herein relate to the treatment of hypertension, coronal ⁇ ' artery disease, stroke, cardiovascular disease, peripheral artery- disease, cerebral vascular disease, diabetes-derived cardiovascular diseases, or congestive heart failure.
  • compositions described herein relate to the treatment of heart diseases, vascular diseases and/or cardiovascular diseases or disease of the cardiovascular system, e.g., acute and chronic heart failure, arterial hypertension, coronary heart disease, stable and instable angina pectoris, myocardial ischemia, myocardial infarction, coronary microvascular dysfunction, microvascular obstruction, no-reflow-phenomenon, shock, atherosclerosis, coronary artery disease, peripheral artery disease, peripheral arterial disease, intermittent claudication, severe intermittent claudication, limb ischemia, critical limb ischemia, hypertrophy of the heart, cardiomyopathies of any etiology (such as, e.g., dilatative cardiomyopathy, restrictive cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy), fibrosis of the heart, atrial and ventricular arrhythmias, transitory and/or ischemic attacks, apoplexy, ischemic and/or hemorr
  • LDL LDL
  • PAI-1 plasminogen-activator inhibitor 1
  • peripheral vascular and cardiac vascular diseases peripheral circulatory disorders, primary' and secondary Raynaud syndrome, disturbances of the microcirculation, arterial pulmonary' hypertension, spasms of coronary and peripheral arteries, thromboses, thromboembolic diseases, edema- formation, such as pulmonary edema, brain-edema, renal edema, myocardial edema, myocardial edema associated with heart failure, restenosis after i.e.
  • thrombolytic therapies percutaneous-transluminal angioplasties (PTA), transluminal coronary angioplasties (PTCA), heart transplantations, bypass-surgeries as well as micro- and macrovascular injuries (e.g., vasculitis), reperfusion-damage, arterial and venous thromboses, microalbuminuria, cardiac insufficiency, endothelial dysfunction.
  • PTA percutaneous-transluminal angioplasties
  • PTCA transluminal coronary angioplasties
  • heart transplantations bypass-surgeries as well as micro- and macrovascular injuries (e.g., vasculitis), reperfusion-damage, arterial and venous thromboses, microalbuminuria, cardiac insufficiency, endothelial dysfunction.
  • heart failure includes more specific or related kinds of diseases such as acute decompensated heart failure, right heart feilure, left heart failure, global insufficiency, ischemic cardiomyopathy, dilataiive cardiomyopathy, congenital heart defects), valve diseases, heart feilure related to valve diseases, mitral valve stenosis, mitral valve insufficiency', aortic valve stenosis, aortic valve insufficiency, tricuspid valve stenosis, tricuspid valve insufficiency, pulmonary valve stenosis, pulmonary valve insufficiency, combined valvular defects, inflammation of the heart muscle (myocarditis), chronic myocarditis, acute myocarditis, viral myocarditis, bacterial myocarditis, diabetic heart feilure, alcohol-toxic cardiomyopathy, cardiac storage diseases, heart feilure with preserved ejection fraction (HFpEF), diasto
  • Atrial arrhythmias and ventricular arrhythmias also include more specific and related disease-entitites, such as: Atrial fibrillation, paroxysmal atrial fibrillation, intermittent atrial fibrillation, persistent atrial fibrillation, permanent atrial fibrillation, atrial flutter, sinus arrhythmia, sinus tachycardia, passive heterotopy, active heterotopy, replacement systoles, extrasystoles, disturbances in the conduction of impulses, sick-sinus syndrome, hypersensitive carotis-sinus, tachycardias, AV- node re-entry tachycardias, atrioventricular re-entry tachycardia, WPW-syndrome (Wolff - Parkinson-White syndrome), Mahaim-tachycardia, hidden accessory' pathways/tracts, permanent junctional re-entry tachycardia, focal atrial tachycardia
  • coronary heart disease also include more specific or related diseases entities, such as: Ischemic heart disease, stable angina pectoris, acute coronary syndrome, instable angina pectoris, NSTEMI (non-ST-segement-elevation myocardial infarction), STEM1 (ST-segement-elevation myocardial infarction), ischemic damage of the heart, arrhythmias, and myocardial infarction.
  • diseases entities such as: Ischemic heart disease, stable angina pectoris, acute coronary syndrome, instable angina pectoris, NSTEMI (non-ST-segement-elevation myocardial infarction), STEM1 (ST-segement-elevation myocardial infarction), ischemic damage of the heart, arrhythmias, and myocardial infarction.
  • compositions and methods described herein relate to the treatment of cancer.
  • any cancer can be treated using the methods described herein.
  • cancers that may treated by compositions and methods described herein include, but are not limited to, cancer cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be one of the following histological types, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coh; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
  • the cancer comprises breast cancer (e.g., triple negative breast cancer).
  • the cancer comprises colorectal cancer (e g., microsatellite stable (MSS) colorectal cancer).
  • MSS microsatellite stable
  • the cancer comprises renal cell carcinoma.
  • the cancer comprises lung cancer (e.g., non-small cell lung cancer).
  • the cancer comprises bladder cancer.
  • the cancer comprises gastroesophageal cancer.
  • the compositions and methods provided herein relate to the treatment of a leukemia.
  • leukemia includes broadly progressive, malignant diseases of the hematopoietic organs/systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • Non-limiting examples of leukemia diseases include acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia.
  • compositions and methods provided herein relate to the treatment of a carcinoma.
  • carcinoma refers to a malignant growth made up of epithelial cells tending to infiltrate the surrounding tissues, and/or resist physiological and non-physiological cell death signals and gives rise to metastases.
  • Non-limiting exemplary types of carcinomas include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma,
  • compositions and methods provided herein relate to the treatment of a sarcoma.
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance.
  • Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing ' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic s
  • Additional exemplary neoplasias that can be treated using the compositions and methods described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary' tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, plasmacytoma, colorectal cancer, rectal cancer, and adrenal cortical cancer.
  • the cancer treated is a melanoma.
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
  • tumors that can be treated using compositions and methods described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above.
  • tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypemephroid adenocarcinoma, bile duct carcinoma, chor
  • Cancers treated in certain embodiments also include precancerous lesions, e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis and cervical dysplasia.
  • precancerous lesions e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen
  • Tumors treated in some embodiments include non-cancerous or benign tumors, e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to cholangioma, colonic polyp, adenoma, papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renal tubular adenoma, squamous cell papilloma, gastric polyp, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.
  • non-cancerous or benign tumors e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to cholangioma, colon
  • the compositions and methods described herein relate to the treatment of liver diseases.
  • diseases include, but are not limited to, Alagille Syndrome, Alcohol-Related Liver Disease, Alpha- 1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign Liver Tumors, Biliary Atresia, Cirrhosis, Galactosemia, Gilbert Syndrome, Hemochromatosis, Hepatitis A, Hepatitis B, Hepatitis C, Hepatic Encephalopathy, Intrahepatic Cholestasis of Pregnancy (ICP), Lysosomal Acid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice, Primary Biliary Cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Reye Syndrome, Type I Glycogen Storage Disease, and Wilson Disease.
  • ICP Pregnancy
  • LAL-D Lysosomal Acid Lipase Deficiency
  • compositions and methods and/or solid dosage forms described herein may be used to treat neurodegenerative and neurological diseases.
  • the neurodegenerative and/or neurological disease is Parkinson’s disease, Alzheimer’s disease, prion disease, Huntington’s disease, macular degeneration, motor neuron diseases (MND), spinocerebellar ataxia, spinal muscular atrophy, dystonia, idiopathicintracranial hypertension, epilepsy, nervous system disease, central nervous system disease, movement disorders, multiple sclerosis, encephalopathy, peripheral neuropathy or post-operative cognitive dysfunction. Delivery and internalization of LNPs by cells
  • compositions and methods provided herein are usefol for the delivery and internalization of LNPs by cells.
  • compositions and methods described herein are useful for delivering cargo (e.g., a biologically active agent) to liver cells (e.g., a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a liver sinusoidal cell) or to a cancer cell or a tumor cell (e.g., a primary tumor or metastatic cancer cells).
  • cargo e.g., a biologically active agent
  • liver cells e.g., a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a liver sinusoidal cell
  • cancer cell or a tumor cell e.g., a primary tumor or metastatic cancer cells
  • the compositions and methods are usefol for delivering cargo (e.g., a biologically active agent) to the skin, adipose, muscle, or lymph nodes (e.g., by subcutaneous dosing).
  • compositions and methods are usefol for delivering cargo (e.g., a biologically active agent) to a brain cell, splenic cell (e.g., a splenocytes), ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell, a skeletal muscle cell, a non-immune cell, T-cell, a stem cell(e.g., a hematopoietic cell), a lung cell, or a kidney cell.
  • cargo e.g., a biologically active agent
  • a brain cell e.g., a splenocytes
  • ovarian cell e.g., a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell, a skeletal muscle cell, a non-immune cell, T-cell, a stem cell(e.g., a hematopoietic cell), a lung cell, or a kidney cell.
  • the liver or liver cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, portal vein injection, catheterization, stenting) to facilitate delivery of cargo (e.g., a biologically active agent).
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., direct injection, portal vein injection, catheterization, stenting
  • cargo e.g., a biologically active agent
  • kidney or kidney cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stenting) to facilitate delivery of cargo (e.g., a biologically active agent).
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., direct injection, catheterization, stenting
  • cargo e.g., a biologically active agent
  • the tumor or tumor cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stenting), to facilitate delivery of cargo (e.g., a biologically active agent).
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., direct injection, catheterization, stenting
  • cargo e.g., a biologically active agent
  • the CNS or CNS cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stenting, osmotic pump administration (e.g., intrathecal or ventricular)), to facilitate delivery of cargo (e.g., a biologically active agent).
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., direct injection, catheterization, stenting, osmotic pump administration (e.g., intrathecal or ventricular)
  • cargo e.g., a biologically active agent
  • the PNS or PNS cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection), to facilitate delivery of cargo (e.g., a biologically active agent).
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., direct injection
  • cargo e.g., a biologically active agent
  • the lung or lung cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., pulmonary administration directly to lung tissues and cells), to facilitate delivery' of cargo (e.g., a biologically active agent).
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., pulmonary administration directly to lung tissues and cells
  • cargo e.g., a biologically active agent
  • the vasculature or vascular cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., clamping, catheterization, stenting), to facilitate delivery of cargo (e.g., a biologically active agent).
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., clamping, catheterization, stenting
  • cargo e.g., a biologically active agent
  • the skin or skin cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct dermal application, iontophoresis), to facilitate delivery of cargo (e.g., a biologically active agent).
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., direct dermal application, iontophoresis
  • cargo e.g., a biologically active agent
  • the eye or ocular cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eyedrops, implants), to facilitate delivery' of cargo (e.g., a biologically active agent).
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eyedrops, implants
  • cargo e.g., a biologically active agent
  • the ear or cells of the ear are contacted with the composition described herein as is generally known in the art, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection), to facilitate delivery-.
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous administration
  • local administration e.g., direct injection
  • cells of the immune system are contacted with the composition described herein intramuscularly, after which immune cells can infiltrate the delivery site and process delivered RNA and/or process encoded antigen produced by non-immune cells, such as muscle cells.
  • immune cells can include macrophages (e.g., bone marrow derived macrophages), dendritic cells (e.g., bone marrow derived plasmacytoid dendritic cells and/or bone marrow derived myeloid dendritic cells), monocytes (e.g., human peripheral blood monocytes), etc. (for example, WO2012/006372, incorporated herein by reference in its entirety.

Abstract

Provided herein are lipid nanoparticle (LX P) compositions and methods related, to the delivery' of biologically active agents and treatment of disease.

Description

METHOD OF USE FOR APOE PEPTIDES
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application serial number 63/222,007, filed July 15, 2021, the entire contents of which are incorporated herein by reference.
BACKGROUND
[0001] The delivery of biologically active agents to cells is often made difficult by the relative instability and low cell permeability of such agents. Biologically active agents that are particularly difficult to deliver into cells include small molecular drugs, protein-based drug, nucleic acid-based drugs, and derivatives thereof. Lipid nanoparticles (LNPs) formulated with ionizable lipids can serve as cargo vehicles for delivery- of biologically active agents into cells. Although a variety of LNP compositions have been recently developed, effective delivery- vehicles for reaching desired cell populations while maintaining safety, and efficacy- are still lacking. Therefore, LNP compositions for efficient delivery of biologically active agent for the treatment of disease are needed.
SUMMARY
[0002] The present disclosure provides, in some embodiments, lipid nanoparticles (LNPs) comprising an apolipoprotein E (ApoE)-mimicking peptide and one or more lipids. The LNPs benefit from enhanced association with target cells and delivery of an agent into the target cells, compared to LNPs without the ApoE-mimicking peptide. Enhanced delivery of agents to target cells (e.g., liver cells or splenic cells) can be usefill for the treatment of a variety of diseases.
[0003] In certain aspects, a lipid nanoparticle (LNP) comprises one or more lipids and an apolipoprotein E (ApoE)-mimicking peptide, wherein the ApoE-mimicking peptide comprises a receptor-binding domain of ApoE and a lipid-associating domain.
[0004] In some embodiments, the LNP is an ionizable LNP.
[0005] In other embodiments, the one or more lipids comprise an ionizable lipid.
[0006] In some embodiments, ionizable lipid comprises an amino group. In some such embodiments, the amino group is an amine head group.
[0007] In some embodiments, the ionizable lipid comprises two or more aliphatic tail groups, e.g., two to four aliphatic tail groups. In certain embodiments, the aliphatic tail groups are hydrophobic tail groups, e.g., each independently alkyl or alkenyl groups. [0008] In some embodiments, the ionizable lipid is a cationic lipid. In other embodiments, the ionizable lipid is a neutral lipid. In some embodiments, the ionizable lipid is a zwitterionic lipid.
[0009] In preferred embodiments, the ionizable lipid is protonated at physiological pH. [0010] In some embodiments, the one or more lipids comprise a structural lipid, optionally a steroid, such as a sterol, e.g., cholesterol.
[0011] In certain embodiments, the one or more lipids comprise a non-cationic helper lipid. In some embodiments, the non-cationic helper lipid is a phospholipid. In some embodiments, the non-cationic helper lipid comprises a diacylglyerol group. In certain embodiments, the non-cationic helper lipid is a glycerophospholipid, such as a phosphatidylcholine. Alternatively, the non-cationic helper lipid is not a phosphatidylcholine.
[0012] In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC). [0013] In some embodiments, the one or more lipids comprise a PEG-lipid comprising a lipid group covalently bonded to a PEG group. In some such embodiments, the lipid group is a phospholipid. In certain embodiments, the lipid group comprises a diacylglycerol, dialkylglycerol, or dialkylamine group. In certain preferred embodiments, the lipid group is a glycerophospholipid group. In some embodiments, the lipid group is dimyristoyl glycerol. [0014] In some embodiments, the lipid-associating domain comprises a class A amphipathic-helical domain, e.g., DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1), DWLRAFYDKVAEKLREAF (SEQ ID NO: 2), DWLRALYDKVAEKLREAL (SEQ ID NO: 3), DLLRALYDKVAEKLREAW (SEQ ID NO: 4), or FAEKLKEAVKDYFAKLWD (SEQ ID NO: 5), most preferably DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1).
[0015] In some embodiments, the receptor-binding domain of ApoE is capable of binding to an LDL receptor.
[0016] In certain embodiments, the receptor-binding domain of ApoE is LRKLRKRLLR (SEQ ID NO: 6), LRRLRRRLLR (SEQ ID NO: 7), LRKMRKRLMR (SEQ ID NO: 8), or RLTRKRGLK (SEQ ID NO: 9). In preferred embodiments, the receptor-binding domain of ApoE is LRKLRKRLLR (SEQ ID NO: 6) or LRRLRRRLLR (SEQ ID NO: 7), most preferably LRRLRRRLLR (SEQ ID NO: 7).
[0017] In certain embodiments, the receptor-binding domain of ApoE is covalently bonded to the lipid-associating domain. In some embodiments, the C-terminus of the receptor- binding domain of ApoE is covalently bonded to the N-terminus of the lipid-associating domain. In other embodiments, an amino group is bonded to the C-terminus of the lipid- associating domain. In certain embodiments, an acyl group is bonded to the N -terminus of the receptor-binding domain of ApoE.
[0018] In some embodiments, the ApoE-mimicking peptide comprises a fatty acid moiety e.g., butanoyl, caproyl, octanoyl, decanoyl, lauroyl, myristoyl, myristoleoyl, palmitoyl, stearoyl, oleoyl, pahnitoleoyl, linoleoyl, linolenoyl, or arachidonoyl. In preferred embodiments, the fatty acid moiety is octanoyl, myristoyl, palmitoyl, or oleoyl.
[0019] In certain embodiments, the ApoE-mimicking peptide comprises a fatty acid moiety, and the fatty acid moiety is bonded to the N -terminus of the receptor-binding domain of ApoE. In some embodiments, the fatty' acid moiety comprises a chain of 4 to 20 carbon atoms.
[0020] In certain embodiments, the fatty acid moiety is a saturated fatty' acid moiety.
[0021] In some embodiments, the fatty acid moiety comprises one, two, or three alkene groups.
[0022] In certain embodiments, the fatty acid moiety comprises an co-amino group. In certain such embodiments, the co-amino group comprises an acyl group.
[0023] In preferred embodiments, the fetty acid moiety is 4-amino-butanoyl, 6-amino- caproyl, 8-amino-octanoyl, 10-amino-decanoyl, 12-amino-lauroyl, 14-amino-myristoyl, 14- amino-myristoleoyl, 16-amino-palmitoyl, 18 -amino -stearoyl, 18-amino-oleoyl, 16-amino- pahnitoleoyl, 18-amino-linoleoyl, 18-amino-linolenoyl, or 20-amino-arachidonoyl.
[0024] In certain embodiments, the fatty acid moiety is Ac-Aha. For example, the Apo-E mimicking peptide is Ac-Aha-hEl8A-NH2 or Ac-Aha-[R]hE18A-NH2.
[0025] In some embodiments, the ApoE-mimicking peptide is Ac- LRKLRKRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 10); octanoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 11); myristoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 12); palmitoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 13); or oleoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 14).
[0026] In certain embodiments, the LNP further comprises a payload, such as a drug, a peptide, a polypeptide, a protein, or a nucleic acid (e.g., RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR/Cas9, ssDNA, and DNA). In some embodiments, the payload comprises an mRNA encoding a secreted protein, membrane-bound protein, intracellular protein, antibody molecule, or enzyme.
[0027] In certain embodiments, the LNP is formulated for systemic delivery. [0028] In some embodiments, the LNP is formulated for parenteral, e.g., intravenous, intramuscular, subcutaneous, intrathecal, or intradermal; or enteral, e.g., oral, rectal, or sublingual, delivery.
[0029] In certain embodiments, the LNP is not in an organism.
[0030] Pharmaceutical compositions are described that comprise an LNP as described herein and a pharmaceutically acceptable carrier.
[0031] Further, the compositions and methods described herein can be used to treat disease by administering an LNP as described herein to a subject. In some embodiments, the disease is coronary artery disease, rheumatoid arthritis, diabetes, neurodegenerative disease, Alzheimer’s disease, peripheral artery disease, cerebral vascular disease, diabetes-derived cardiovascular diseases, macular degeneration, congestive heart failure, systemic lupus, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, cardiovascular, renovascular diseases, metabolic diseases, immune disorders, fibrotic disease, inflammation, and/or infectious disease. In certain embodiments, the LNP is systemically delivered to the subject. In some embodiments, the LNP is parenterally, e.g., intravenously, intramuscularly, subcutaneously, intrathecally, or intradermally; or enterally, e.g., orally, rectally, or sublingually, delivered to the subject.
[0032] A therapeutically effective dose of a LNP composition described herein is about 0.01 mg/kg to about 500 mg/kg, about 0.05 mg/kg to about 250 mg/kg, about 0.1 mg/kg to about 100 mg/kg, about 0.5 mg/kg to about 50 mg/kg, or about 1 mg/kg to about 25 mg/kg. In preferable embodiments, the therapeutically effective dose of a LNP composition described herein is about 0.01 mg/kg to about 100 mg/kg.
[0033] In some embodiments, the LNP is internalized by a cell. In certain embodiments, the LNP increases internalization by a cell compared to the same LNP without the ApoE- mimicking peptide. In some embodiments, the cell is a liver cell, e.g., a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a liver sinusoidal cell. In other embodiments, the cell is a brain cell. In some embodiments, the cell is a splenic cell, e.g., a splenocyte. In other embodiments, the cell is an ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell, or a skeletal muscle cell. In some embodiments, the cell is a non-immune cell. In other embodiments, the cell is a cancer cell. In yet other embodiments, the cell is a T-cell. In some embodiments, the cell is a stem cell, such as a hematopoietic cell. In other embodiments, the cell is a lung cell . In yet other embodiments, the cell is a kidney cell. [0034] In some embodiments, the total plasma concentration of cholesterol in the subject is lowered. In certain embodiments, the plasma LDL concentration, the plasma VLDL concentration, or both in the subject are lowered.
DETAILED DESCRIPTION
[0035] The disclosed compositions and methods may be understood more readily by reference to the following detailed description of particular embodiments.
[0036] It is to be understood that the disclosed compositions and methods are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
[0037] The present disclosure relates to lipid nanoparticles and methods for treating disease with lipid nanoparticles, e.g., by binding apolipoprotein E (ApoE) and inhibiting the downstream effects of ApoE. In certain aspects, lipid nanoparticles (LNPs) are provided comprising one or more lipids and an apolipoprotein E (ApoE)-mimicking peptide, wherein the ApoE-mimicking peptide comprises a receptor-binding domain of ApoE and a lipid- associating domain. In certain aspects, pharmaceutical compositions are provided herein comprising an LNP disclosed herein. In certain aspects, methods of treating a disease are provided herein, comprising administering an LNP disclosed herein or a pharmaceutical composition disclosed herein.
Definitions
[0038] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.
[0039] The articles “a” and “an” are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
[0040] Numeric ranges are inclusive of the numbers defining the range. Measured and measureable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. As used in this application, the terms “about” and “approximately” have their art-understood meanings; use of one vs the other does not necessarily imply different scope. Unless otherwise indicated, numerals used in this application, with or without a modifying term such as “about” or “approximately”, should be understood to encompass normal divergence and/or fluctuations as would be appreciated by one of ordinary' skill in the relevant art. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0041] “Administering” or “administration of’ a substance, a composition or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a composition or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A composition or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the composition or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
[0042] Appropriate methods of administering a substance, a composition or an agent to a subject will also depend, for example, on the age and/or the physical condition of the subject and the chemical and biological properties of the composition or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a composition or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered composition or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.
[0043] The term "alkyl" as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s- butyl, r-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched (i.e., linear). The alkyl group can also be substituted or unsubstituted (preferably unsubstituted). For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, carboxylate, or thiol, as described herein. A "lower alkyl" group is an alkyl group containing from one to six (e.g. , from one to four) carbon atoms.
[0044] The term “alkenyl”, as used herein, refers to an aliphatic group containing at least one carbon-carbon double bond and is intended to include both "unsubstituted alkenyls" and "substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed below, except where stability is prohibitive. For example, an alkenyl group may be substituted by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated. Exemplary alkenyl groups include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CHCH2). [0045] An “alkylene” group refers to a divalent alkyl radical, which may be branched or unbranched (i.e., linear). Any of the above mentioned monovalent alkyl groups may be converted to an alkylene by abstraction of a second hydrogen atom from the alkyl. Representative alkylenes include
Figure imgf000008_0001
alkylene and C2-3 alkylene. Typical alkylene groups include, but are not limited to
Figure imgf000008_0002
CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and the like. The alkylene group can also be substituted or unsubstituted. For example, the alkylene group can be substituted with one or more groups including, but not limited to, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonate, sulfonamide, urea, amide, carbamate, ester, carboxylate, or thiol, as described herein.
[0046] The term “alkenylene” includes divalent, straight or branched, unsaturated, acyclic hydrocarbyl groups having at least one carbon-carbon double bond and, in some embodiments, no carbon-carbon triple bonds. Any of the above-mentioned monovalent alkenyl groups may be converted to an alkenylene by abstraction of a second hydrogen atom from the alkenyl. Representative alkenylenes include
Figure imgf000008_0003
[0047] The term “Cx-y” when used in conjunction with a chemical moiety, such as alkyl or alkylene, is meant to include groups that contain from x to y carbons in the chain. For example, the term “Cx-y alkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain and branched-chain alkyd and alkylene groups that contain from x to y carbons in the chain. [0048] As defined herein, “Cn,” where “n” is an integer, describes a hydrocarbon molecule or fragment (e.g., an alkyl group) wherein “n” denotes the number of carbon atoms in the fragment or molecule.
[0049] The term “antibody” as used to herein includes whole antibodies and any antigen binding fragments (i.e., “antigen-binding portions”) or single chains thereof In some embodiments, an “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring antibodies, the heavy chain constant region is comprised of three domains, CHI, CH2 and CHS. In certain naturally occurring antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy - terminus in the following order: FR1, CDR1, FR2, CDR2, FRS, CDRS, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
[0050] As used herein, the term “ApoE mimetic” is interchangeable with apolipoprotein-E mimicking peptide. ApoE mimetics are peptides that are related to, characteristic of, or mimic ApoE. ApoE mimetics include ApoE peptides (i.e., peptides derived from full length ApoE).
[0051] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; embodiments in the specification that recite “about” various components are also contemplated as “at” the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims). [0052] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compositions such that the second composition is administered while the previously administered therapeutic composition is still effective in the body (e.g., the two compositions are simultaneously effective in the patient, which may include synergistic effects of the two compositions). For example, the different therapeutic compositions can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compositions can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compositions. [0053] As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and a nanoparticle are made to share a physical connection. Methods of contacting cells with external entities both in vivo and ex vivo are well known in the biological arts. For example, contacting a nanoparticle composition and a mammalian cell disposed within a mammal may be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve varied amounts of nanoparticle compositions. Moreover, more than one mammalian cell may be contacted by a nanoparticle composition.
[0054] As used herein, the term “delivering” means providing an entity to a destination. For example, delivering a therapeutic and/or prophylactic to a subject may involve administering a nanoparticle composition including the therapeutic and/or prophylactic to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.
[0055] As used herein “domain switched”, “switched domain”, or “switched” peptide refers to the lipid-associating peptide is covalently linked to the receptor binding domain of apolipoprotein E such that the lipid-associating peptide is at the N-terminus of the synthetic apolipoprotein E-mimicking peptide. For example, the peptide 18A-hE is exemplary of a domain switched peptide. [0056] As used herein the term “fatty acid” refers to a mono-carboxylic acid having an aliphatic chain (“tail”), wherein said aliphatic chain may be either saturated, mono- unsaturated (having one unsaturated bond anywhere on the aliphatic chain) or poly unsaturated (having at least two unsaturated bonds anywhere on the aliphatic chain). An unsaturated bond on the aliphatic chain may be a double (in the cis and/or trans configuration) or a triple bond. The length of the aliphatic chain (being either saturated, monounsaturated or polyunsaturated) of a fatty acid may vary from 8 to 32 carbon atoms. Fatty acids may be derived from a natural source (either an animal or plant source), synthetic source or semi-synthetic source.
[0057] As used herein, “high-density lipoprotein” (HDL) refers to a class of lipoproteins, varying somewhat in their size (8-11 run in diameter), that can transport cholesterol. HDL cholesterol is cholesterol that is associated with HDLs. About one-fourth to one-third of blood cholesterol is carried by high-density lipoprotein (HDL). HOL cholesterol is known as “good” cholesterol, because high levels of HDL seem to protect against heart attack. Low levels of HDL (less than 40 mg/dL in men and less than 50 mg/dL in women) also increase the risk of heart disease.
[0058] As used herein, the term “LDL receptor” refers to a receptor that mediates the endocytosis of cholesterol-rich low-density lipoprotein (LDL).
[0059] “Lipid” refers to structurally diverse group of organic compounds that are fatty acid derivatives or sterols or could be lipid like materials as in lipidoids (example C 12-200) or polymer conjugated lipids and are characterized by being insoluble in water but soluble in many organic solvents.
[0060] As used herein, “lipoprotein” or “lipoproteins” refers to a biochemical assembly that contains both proteins and lipids. The lipids or their derivatives may be covalently or non- covalently bound to the proteins. Many enzymes, transporters, structural proteins, antigens, adhesins, and toxins are lipoproteins. Examples include the high density and low density lipoproteins of the blood, the transmembrane proteins of the mitochondrion and the chloroplast, and bacterial lipoproteins.
[0061] As used herein, by “liposome” is a structure including a lipid containing membrane enclosing an aqueous interior. Liposomes may have one or more lipid membranes.
Liposomes include single-layered liposomes (also known in the art as unilamellar liposomes) and multi-layered liposomes (also known in the art as multilamellar liposomes). [0062] As used herein, “low-density lipoprotein” or “LDL” is a lipoprotein that varies in size (approx. 22 nm) and can contain a changing number of triglycerides and cholesteryl esters they actually have a mass and size distribution. Each native LDL particle contains a single apolipoproteinB-100 molecule (Apo B-100, a protein with 4536 amino acid amino acid residues) and a phospholipid coat that circles the triglycerides and cholesteryl esters, keeping them soluble in the aqueous environment. LDL is commonly referred to as bad cholesterol. LDL cholesterol is cholesterol that is associated with LDLs. When too much LDL cholesterol circulates in the blood, it can slowly build up in the inner walls of the arteries that feed the heart and brain. Together with other substances, it can form plaque, a thick, hard deposit that can narrow the arteries and make them less flexible. This condition is known as atherosclerosis. If a clot forms and blocks a narrowed artery, then heart attack or stroke can result.
[0063] As used herein, an “mRNA” refers to a messenger ribonucleic acid. An mRNA may be naturally or non-naturally occurring. For example, an mRNA may include modified and/or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An mRNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and/or a polyadenylation signal. An mRNA may have a nucleotide sequence encoding a polypeptide. Translation of an mRNA, for example, in vivo translation of an mRNA inside a mammalian cell, may produce a polypeptide.
[0064] As used herein, “nanoparticle” refers to a particle having any one structural feature on a scale of less than about lOOOnm that exhibits novel properties as compared to a bulk sample of the same material. Routinely, nanoparticles have any one structural feature on a scale of less than about 500 nm, less than about 200 nm, or about 100 nm. Also routinely, nanoparticles have any one structural feature on a scale of from about 50 nm to about 500 nm, from about 50 nm to about 200 nm or from about 70 to about 120 nm. In exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 1 - lOOOnm. In other exemplary' embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10- 500 nm. In other exemplary- embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 50- 200 nm. A spherical nanoparticle would have a diameter, for example, of between about 50-100 or 70- 120 nanometers. A nanoparticle most often behaves as a unit in terms of its transport and properties. It is noted that novel properties that differentiate nanoparticles from the corresponding bulk material typically develop at a size scale of under 1000 nm, or at a size of about lOOnm, but nanoparticles can be of a larger size, for example, for particles that are oblong, tubular, and the like. Although the size of most molecules would fit into the above outline, individual molecules are usually not referred to as nanoparticles.
[0065] As used herein, the term “nucleic acid” is used in its broadest sense and encompasses any compound and/or substance that includes a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having ab-D-ribo configuration, a- LNA having an a-L-ribo configuration (a diastereomer of LNA), 2*-amino-LNA having a 2'- amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization) or hybrids thereof. A polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation with a labeling component.
[0066] The phrase "pharmaceutically acceptable" is employed herein to refer 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. [0067] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
[0068] Compositions may also include salts of one or more compounds. Salts may be pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety to its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfide, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, tiimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.
[0069] As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains.
[0070] The term "polypeptide" refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, natural amino acids, etc.), polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-naturally occurring.
[0071] As used herein, a therapeutic composition that “prevents” a disorder or condition refers to a composition that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
[0072] As used herein, “reverse oriented”, “reversed orientation”, “reverse analog” or “reverse sequence” refers to a peptide, or a portion of the peptide, has a reverse amino acid sequence as compared to a non-reverse oriented peptide (i.e., the original sequence is read (or written) from right to left). For example, if one peptide has the amino acid sequence ABCDE, its reverse analog or a peptide having its reverse sequence is as follows: EDCBA. In a dual domain peptide for example, Ac-hE-18A-NH2, either the hE sequence is read from right to left or the 18A sequence is read from right to left. For a reverse analog of, LRKLRKRLLR- DWLKAFYDKVAEKLKEAF (SEQ ID NO: 10) can be RLLRKRLKRL- DWLKAFYDKVAEKLKEAF (SEQ ID NO: 15) or LRKLRKRLLR- FAEKLKEAVKDYFAKLWD (SEQ ID NO: 16).
[0073] As used herein, an “RNA” refers to a ribonucleic acid that may be naturally or non- naturally occurring. For example, an RNA may include modified and/or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and/or a polyadenylation signal . An RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, an RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, for example, in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide. RNAs may be selected from the non- liming group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, long non-coding RNA (IncRNA) and mixtures thereof.
[0074] As used herein, “scrambled” “scrambled version”, or “scrambled peptide” is refers to the composition of the amino acid sequence is the same as the unscrambled peptide, however the sequence of the amino acids is altered thus rendering the peptide unable to form either an a-amphipathic helix or does not possess lipid associating (or HSPG associating) properties. However, in some cases, as described in this invention, the scrambled peptide remains able to form a different helical structure, such as a n-helix. For example, if one peptide has the amino acid sequence ABCDE, the scrambled version of the peptide could have the amino acid sequence DEABC. Scrambled peptides are often denoted as having a “Sc” prior to the portion of the peptide that is scrambled. For example, Sc-hE-18A denoted that the hE portion of the peptide is scrambled.
[0075] As used herein, “transfection” refers to the introduction of a species (e.g., an RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.
[0076] The term “treating” includes prophylactic and/or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to a subject of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
[0077] As used herein, “very Low Density Lipoproteins” (VLDL) is a lipoprotein subclass. VLDL is assembled in the liver from cholesterol and apolipoproteins. The lipoprotein is converted in the bloodstream to low density lipoprotein (LDL). VLDL particles have a diameter of 30-80 tun. VLDL transports endogenous products where chylomicrons transport exogenous (dietary) products.
[0078] “Zwitterionic lipid” refers to a lipid molecule having both a positive and a negative charge. Lipid Nanopartide (LNP) Compositions
[0079] The present disclosure provides lipid-based compositions, specifically lipid nanoparticles (LNPs), that comprise an apolipoprotein E (ApoE)-mimicking peptide and one or more lipids. In various aspects, the present disclosure provides LNPs comprising an agent (e.g., a protein or a nucleic acid molecule) for delivery- to a target cell or population of target cells, methods for enhancing delivery- of an agent (e.g., a protein or a nucleic acid molecule) to a target cell or population of target cells, methods of delivering such LNPs to subjects that would benefit from modulation of target cell activity, and methods of treating diseases or disorders in such subjects.
Lipid Components
[0080] Accordingly, provided herein are LNP compositions comprising an apolipoprotein E (ApoE)-mimicking peptide and one or more lipids.
[0081] The disclosure provided herein describes lipid particles, which are assemblies comprising lipid components and cargo. Lipid particles may be, for example, liposomes, lipid micelles, solid lipid particles, lipoplexes, lipid nanoparticles (LNPs), or lipid-stabilized polymeric particles, comprised of one or a mixture of different biocompatible lipids. The structural organization of these lipid particles may lead to an aqueous interior with a minimum bilayer as in liposomes or the lipid particles may have a solid interior as in solid nucleic acid lipid nanoparticles.
[0082] In preferred embodiments, the lipid particles provided herein are lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise ionizable lipids, structural lipids, non- cationic helper lipids, and/or PEG lipids. In some embodiments, the LNPs comprise other lipid components that are described below.
Ionizable Lipids
[0083] In certain embodiments, the one or more lipids comprise an ionizable lipid. Ionizable lipids of the disclosure may comprise a central amine moiety and at least one biodegradable group. The ionizable lipids described herein may be advantageously used in lipid nanoparticles of the disclosure for the delivery- of cargo to mammalian cells or organs. [0084] In some embodiments, the ionizable lipid ranges from about 20 mol % to about 80 mol % of the LNP composition. In some embodiments, the ionizable lipid ranges from about 30 mol % to about 70 mol %, from about 35 mol % to about 65 mol %, or from about 40 mol % to about 60 mol % of the LNP composition. [0085] In some embodiments, the ionizable lipids is Compound A, or a derivative or analog thereof:
Figure imgf000018_0001
In some embodiments, the ionizable lipid is Compound B, or a derivative or analog thereof:
Figure imgf000018_0002
[0087] In some embodiments, the ionizable lipid is Compound C, or a derivative or analog thereof:
Figure imgf000018_0003
[0088] In some embodiments, the ionizable lipid is Compound D, or a derivative or analog thereof:
Figure imgf000018_0004
[0089] In some embodiments, the ionizable lipid is Compound E, or a derivative or analog thereof:
Figure imgf000019_0001
[0090] In some embodiments, the ionizable lipid is Compound F, or a derivative or analog thereof:
Figure imgf000019_0002
[0091] In some embodiments, the ionizable lipid is Compound G, or a derivative or analog thereof:
Figure imgf000019_0003
[0092] In some embodiments, the ionizable lipid is Compound H, or a derivative or analog thereof:
Figure imgf000019_0004
[0093] In some embodiments, the ionizable lipids include, but are not limited to:
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
[0094] and any combination thereof.
[0095] Additional ionizable lipids of the invention can be selected from the non-limiting group consisting of 3-{didodecylamino)-Nl,Nl,4-tridodecyl-l-piperazineethanamine (KL10), N 1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4-tridodecyl- 1 ,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin- MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), (13Z,165Z)-N,N-dimethyl-3- nonydocosa-13-16-dien-l-amine (L608), 2-({8-[(3b)-cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yl oxy]propan-l -amine (Octyl-CLinDMA), (2R)- 2-({8-[(3b)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-die n- l-yloxy]propan-l -amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(3b)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien -1 -yloxy]propan-l-amine (Octyl-CLinDMA (2S)). In addition to these, an ionizable amino lipid can also be a lipid including a cyclic amine group.
[0096] In some embodiments, the ionizable lipids are one or more of the compounds described in PCT Publication Nos. WO 2015/074805, WO 2015/199952, WO 2017/112865, WO 2017/075531, and WO 2021/026358 hereby incorporated by reference in their entireties. Other ionizable lipids suitable for formulating a composition of the present disclosure can include those described in US 2015/0239834, hereby incorporated by reference in its entirety. [0097] In some embodiments, ionizable lipid comprises an amino group. In some such embodiments, the amino group is an amine head group.
[0098] In some embodiments, the ionizable lipid comprises two or more aliphatic tail groups, e.g., two to four aliphatic tail groups. In certain embodiments, the aliphatic tail groups are hydrophobic tail groups, e.g., each independently alkyl or alkenyl groups. [0099] In some embodiments, the ionizable lipid is a cationic lipid. In other embodiments, the ionizable lipid is a neutral lipid. In some embodiments, the ionizable lipid is a zwitterionic lipid.
[00100] In preferred embodiments, the ionizable lipid is protonated at physiological pH. Structural Lipids
[00101] In some embodiments, the one or more lipids comprise a structural lipid. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can include, but are not limited to, cholesterol, fecosterol, ergosterol, bassicasterol, tomatidine, tomatine, ursolic, alpha-tocopherol, and mixtures thereof.
[00102] In some embodiments, the structural lipid ranges from about 5 mol % to about 65 mol % of the LNP composition. In some embodiments, the structural lipid ranges from about about 20 mol % to about 60 mol %, from about 25 mol % to about 55 mol %, or from about 30 mol % to about 50 mol % of the LNP composition.
[00103] In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid includes cholesterol and a corticosteroid (such as, for example, prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha- tocopherol.
[00104] In some embodiments, the structural lipid is apytosterol. In some embodiments, the phytosterol is a sitosterol, a stigmasterol, a campesterol, a sitostanol, a campestanol, a brassicasterol, a fucosterol, beta-sitosterol, stigmastanol, beta-sitostanol, ergosterol, lupeol, cycloartenol, D5-avenaserol, D7-avenaserol or a D7-stigmasterol, including analogs, salts or esters thereof, alone or in combination. In some embodiments, the phytosterol component of a LNP of the disclosure is a single phytosterol. In some embodiments, the phytosterol component of a LNP of the disclosure is a mixture of different phytosterols (e.g.2, 3, 4, 5 or 6 different phytosterols). In some embodiments, the phytosterol component of an LNP of the disclosure is a blend of one or more phytosterols and one or more zoosterols, such as a blend of a phytosterol (e.g., a sitosterol, such as beta-sitosterol) and cholesterol.
[00105] In some embodiments, the one or more lipids comprise a structural lipid, optionally a steroid, such as a sterol, e.g., cholesterol.
Non-cationic Helper Lipids
[00106] In certain embodiments, the one or more lipids may comprise non-cationic helper lipids. [00107] In some embodiments, the non-cationic helper lipid ranges from about 5 mol % to about 50 mol % of the LNP composition. In some embodiments, the non-cationic helper lipid ranges from about 5 mol % to about 50 mol %, about 10 mol % to about 40 mol %, or about 10 mol % to about 30 mol % of the LNP composition. In some embodiments, the non- cationic helper lipid ranges from about 5 mol % to about 25 mol % of the LNP composition. [00108] In some embodiments, the non-cationic helper lipid is a phospholipid. In some embodiments, the non-cationic helper lipid is a phospholipid substitute or replacement. In some embodiments, the “non-cationic helper lipid” is a lipid comprising at least one fatty acid chain of at least 8 carbons in length and at least one polar head group moiety. In other embodiments, the helper lipid is not a phosphatidyl choline (PC). In certain embodiments, the non- cationic helper lipid is a phospholipid or a phospholipid substitute. In some embodiments, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. In some embodiments, the non-cationic helper lipid is a DSPC analog, a DSPC substitute, oleic acid, or an oleic acid analog. In some embodiments, a non-cationic helper lipid is a non- phosphatidyl choline (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a 1 ,2-distearoyl-i77- glycero-3- phosphocholine (DSPC) substitute.
[00109] In some embodiments, the non-cationic helper lipids are phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty' acid moieties. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). A phospholipid or an analog or derivative thereof may include choline. A phospholipid or an analog or derivative thereof may not include choline. Particular phospholipids may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell. A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2- lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. The lipid component of a lipid nanoparticle of the disclosure may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.
[00110] A phospholipid moiety' may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty' acid moiety maybe selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful in functionalizing a lipid bilayer of a LNP to facilitate membrane permeation or cellular recognition or in conjugating a LNP to a useful component such as a targeting or imaging moiety (e.g., a dye). Each possibility represents a separate embodiment of the present disclosure. Phospholipids usefill in the compositions and methods described herein may be selected from the non-limiting group consisting of 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 (cis) PC), l,2-diarachidonoyl-sn-glycero-3-phosphocholine (DAPC), 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine(22:6 (cis) PC) 1,2-diphytanoyl-sn-glycero- 3-phosphoethanolamine (4ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (PE(18:2/18:2), 1,2-dilinolenoyl- sn-glycero-3-phosphoethanolamine (PE 18:3(9Z, 12Z, 15Z), 1,2-diarachidonoyl-sn-glycero- 3-phosphoethanolamine (DAPE 18:3 (9Z, 12Z, 15Z), l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine (22:6 (cis) PE), l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), and sphingomyelin. Each possibility represents a separate embodiment of the invention. In some embodiments, a LNP includes DSPC. In certain embodiments, a LNP includes DOPE. In some embodiments, a LNP includes DMPE. In some embodiments, a LNP includes both DSPC and DOPE. In other embodiments, a non-cationic helper lipid for use in a LNP is selected from the group consisting of DSPC, DMPE, and DOPC or combinations thereof. Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
[00111 ] In certain embodiments, the one or more lipids comprise a non-cationic helper lipid. In some embodiments, the non-cationic helper lipid is a phospholipid. In some embodiments, the non-cationic helper lipid comprises a diacylglyerol group. In certain embodiments, the non-cationic helper lipid is a glycerophospholipid, such as a phosphatidylcholine. Alternatively, the non-cationic helper lipid is not a phosphatidylcholine.
[00112] In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC).
PEG Lipids
[00113] In some embodiments, the one or more lipids comprise a PEG-lipid comprising a lipid group covalently bonded to a PEG group. In some such embodiments, the lipid group is a phospholipid. In certain embodiments, the lipid group comprises a diacylglycerol, dialkylglycerol, or dialkylamine group. In certain preferred embodiments, the lipid group is a glycerophospholipid group. In some embodiments, the lipid group is dimyristoyl glycerol.
[00114] In some embodiments, the PEG lipids ranges from about 0.1 mol % to about 25 mol %, from about 1 mol % to about 25 mol %, or from about 5 mol % to about 20 mol % of the LNP composition. In some embodiments, the one or more lipids comprise a PEG-lipid comprises a lipid group covalently bonded to a PEG group. PEG lipids can affect the length of time the nanoparticles can exist in vivo (e.g., in the blood). PEG lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. PEG lipids used herein may modulate pharmacokinetic properties of the LNPs.
Typically, the PEG lipid comprises a lipid moiety and a polymer moiety based on PEG (sometimes referred to as polyethylene oxide)) (a PEG moiety). Additional suitable PEG lipids are disclosed in PCT Publication Nos. WO 2015/095340 (p. 31, line 14 to p. 37, line 6), WO 2006/007712, and WO 2011/076807 ("stealth lipids"), hereby incorporated by reference in their entireties.
[00117] In some embodiments, the lipid moiety may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about €40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. In some embodiments, the alkyl chain length comprises about CIO to C20. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. The chain lengths may be symmetrical or asymmetric.
[00118] Unless otherwise indicated, the term “PEG” as used herein means any polyethylene glycol or other polyalkylene ether polymer, such as an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety is unsubstituted. Alternatively, the PEG moiety may be substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. For example, the PEG moiety' may comprise a PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Polyethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety may be a PEG homopolymer. In certain embodiments, the PEG moiety has a molecular weight of about 130 to about 50,000, such as from about 150 to about 30,000, or even from about 150 to about 20,000. Similarly, the PEG moiety may have a molecular weight of about 150 to about 15,000, from about 150 to about 10,000, from about 150 to about 6,000, or even from about 150 to about 5,000. In certain preferred embodiments, the PEG moiety has a molecular weight of about 150 to about 4,000, from about 150 to about 3,000, from about 300 to about 3,000, from about 1,000 to about 3,000, or from about 1,500 to about 2,500.
[00119] In certain preferred embodiments, the PEG moiety is a “PEG-2K,” also termed ‘TEG 2000,” which has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula I:
Figure imgf000029_0001
[00120] wherein n is 45, meaning that the number averaged degree of polymerization comprises about 45 subunits However, other PEG embodiments known in the art may be used, including, e.g., those where the number-averaged degree of polymerization comprises about 23 subunits (n=23), and/or 68 subunits (n=68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl, such as methyl.
[00121] In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog # GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (catalog # DSPE-020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG- dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG- cholesterol (l-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- [omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]- methyl-poly(ethylene glycol)ether), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxyCpolyethylene glycol)-2000] (PEG2k-DMG) (cat. #880150P from Avanti Polar Lipids, Alabaster, Alabama, USA), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. #880120C from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), polyethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and l,2-distearyloxypropyl-3-amine-N-[rnethoxy(polyethylene glycol)- 2000] (PEG2k-DSA). In certain such embodiments, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In other embodiments, the PEG lipid may be PEG2k-DSPE. In some embodiments, the PEG lipid may be PEG2k-DMA. In yet other embodiments, the PEG lipid may be PEG2k-C-DMA. In certain embodiments, the PEG lipid may be compound S027, disclosed in WO2016/010840 (paragraphs [00240] to [00244]). In some embodiments, the PEG lipid may be PEG2k-DSA. In other embodiments, the PEG lipid may be PEG2k-Cl 1 . In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.
[00122] Other examples of PEG lipids include lipids with PEG chains such as hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHE), 1, 2-distearoyl-glycero-3- phosphoethanolamine-N-[methoxy (PEG)-2000] (DSPE-PEG2000), 1, 2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy (PEG)-2000] modified with a maleimidic group in the distal end of the chain 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (PEG)-2000], DSPE-PEG2000-MAL, l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-550] (DMPE-PEG550), 1, 2- dioleoyl-l-3-trimethylammonium propane (DOTAP), and those with a glycerol backbone e.g., DMG-PEG, DSG-PEG (DMG-PEG2000) etc.
[00123] Additional examples of PEG lipids include DMG-PEG (1,2-Dimyristoyl-sn- glycerol, methoxypolyethylene glycol-PEG), DMA-PEG (polyethylene glycol)- dimethacrylate-PEG) and DMPE-PEG550 (l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-550]), PEG), monosialoganglioside Gml, and polyamide oligomers (PAO), such as those described in U.S. Pat. No. 6,320,017. The lipid nanoparticles can include DMPE-PEG2000 or DMG-PEG which could be substituted with DMPE-PEG2000 in any of the formulations taught herein. Other suitable PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) (such as those described in U.S. Pat. No. 5,820,873), PEG-modified dialkylamines and PEG-modified l,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols and dialkylglycerols, mPEG (mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE).
Other lipid components
[00124] In some embodiments, the lipid nanopaiticles optionally comprise neutral lipids. Neutral lipids suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Neutral lipids, when present, can be any of a number of lipid species, which exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. In some embodiments, the neutral lipid component is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine). In some embodiments, the neutral lipid comprises saturated fatty acids with carbon chain lengths in the range of CIO to C20, inclusive, In some embodiments, the neutral lipid includes mono- or di-unsaturated fatty acids with carbon chain lengths in the range of CIO to C20, inclusive. Suitable neutral lipids include, but are not limited to, DPPC (Dipalmitoyl phosphatidylcholine), POPC (Palmitoyl-Oleoyl Phosphatidyl Cholin), DOPE (1,2-dioleoyl- sn-glycero-3-phosphoethanolamine), DSPC (disteroylphosphatidyl choline), egg L-alpha- phosphatidylcholine (EPC);l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); and SM (Sphingomyelin).
[00125] In some embodiments, the lipid nanoparticles optionally comprise cationic lipids. Cationic lipids suitable for use in a lipid composition of the disclosure include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N- dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), l,2-Dioleoyl-3-Dimethylammonium -propane (DODAP), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),
1.2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP), l,2-Dilineoyl-3- Dimethylammonium-propane (DLINDAP), dilauryl(C12:0) trimethyl ammonium propane (DLTAP), Dioctadecylamidoglycyl spermine (DOGS), DC-Choi, Dioleoyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA),
1.2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), 3- Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12- octadecadienoxy)propane (CLinDMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 2-[5,-(cholest-5-en-3[beta]-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9,,l-2'- octadecadienoxy) propane (CpLinDMA), N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and l,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP). In certain embodiments, the cationic lipid is DOTAP or DLTAP.
[00126] A number of cationic lipids, and methods for making them, are described in, for example, U.S. Pat. Numbers 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; 5,785,992; 5,830,430; 6,056,938; 7,893,302; 7,404,969; 8,034,376; 8,283,333; and 8,642,076; U.S. Pat. Publ. Nos. 2006/0083780 and 2006/0240554; as well as PCT Publ. Numbers WO 2010/054406, WO 2010/054401, WO 2010/054405, WO 2010/054384, WO 2012/040184, WO 2011/153120, WO 2011/149733, WO 2011/090965, WO 2011/043913, WO 2011/022460, WO 2012/061259, WO 2012/054365, WO 2012/044638, WO 2010/080724, WO 2010/21865, and WO 2008/103276, hereby incorporated by reference in their entireties.In some embodiments, the lipid nanoparticles optionally comprise anionic lipids. Anionic lipids can also include, but are not limited to, fetty acids (e.g., oleic, linoleic, linolenic acids); cholesteryl hemisuccinate (CHEMS); l,2-di-0-tetradecyl-sn-glycero-3- phospho-(l ’-rac-glycerol) (Diether PG); l,2-dimyristoyl-sn-glycero-3-phospho-(l '-rac- glycerol) (sodium salt); 1 ,2-dimyristoyl-sn-glycero- 3-phospho-L-serine (sodium salt); 1- hexadecanoyl,2-(9Z, 12Z)-octadecadienoyl-sn-glycero-3- phosphate; 1,2-dioleoyl-sn-glycero- 3-[phospho-rac-(l -glycerol)] (DOPG); dioleoylphosphatidic acid (DOPA); 1,2-dioleoyl-sn- glycero-3-phospho-L-serine (DOPS); and derivatives thereof. Other examples of suitable anionic lipids include, but are not limited to fatty acids, such as oleic, linoleic, and linolenic acids; and cholesteryl hemisuccinate. Such lipids can be used alone or in combination, for a variety of purposes, such as to attach ligands to the liposome surface.
[00128] In some embodiments, the lipid nanoparticles described herein further comprise one or more compounds that are capable of enhancing the cellular uptake or cytosolic distribution of the lipid nanoparticle and/or its encapsulated composition (e.g., small molecule drug, protein-based drug, or nucleic acid-based drug etc.). Compounds that can enhance the cellular uptake can include levodopa, naphazoline hydrochloride, acetohexamide, niclosamide, diprophylline, and isoxicam, or a combination thereof. Compounds that can enhance the cytosolic distribution can include azaguanine-8, isoflupredone acetate, chloroquine, trimethobenzamide, hydrochloride, isoxsuprine hydrochloride, and diphemanil methylsulfete, or a combination thereof. [00129] In some embodiments, the lipid nanoparticles comprise lipid bilayers encapsulating one or more agents encompassed by the present disclosure, such as small molecule drug, protein-based drug, or nucleic acid-based drug. In some embodiments, the lipid nanoparticles are formulated to facilitate an uptake into cells. In some embodiments, the lipid nanoparticles are formulated to facilitate uptake into monocytes, dendritic cells, and/or macrophages. [00130] The lipid nanoparticle can, in some embodiments, further comprise additional agents. In some embodiments, the lipid nanoparticle further comprises one or more antioxidants. The antioxidant can help stabilize the lipid nanoparticle and prevent, decrease, and/or inhibit degradation of the cationic lipids and/or active agents encapsulated in the lipid nanoparticle. In some embodiments, the antioxidant is a hydrophilic antioxidant, a lipophilic antioxidant, a metal chelator, a primary antioxidant, a secondary antioxidant, or salts or mixtures thereof. In some embodiments, the antioxidant comprises EDTA, or a salt thereof. In some embodiments, the lipid nanoparticle further comprises EDTA in combination with one, two, three, four, five, six, seven, eight, or more additional antioxidants (e.g., primary' antioxidants, secondary antioxidants, or other metal chelators). Examples of antioxidants include, but are not limited to, hydrophilic antioxidants, lipophilic antioxidants, and mixtures thereof. Non-limiting examples of hydrophilic antioxidants include chelating agents (e.g., metal chelators) such as ethylenediaminetetraacetic acid (EDTA), citrate, ethylene glycol tetraacetic acid (EGTA), l,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), diethylene triamine pentaacetic acid (DTPA), 2,3-dimercapto-l-propanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA), cc-lipoic acid, salicylaldehyde isonicotinoyl hydrazone (SIR), hexyl thioethylamine hydrochloride (HTA), desferrioxamine, salts thereof, and mixtures thereof. Additional hydrophilic antioxidants include ascorbic acid, cysteine, glutathione, dihydrolipoic acid, 2 -mercaptoethane sulfonic acid, 2 -mercaptobenzimidazole sulfonic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, sodium metabisulfite, salts thereof, and mixtures thereof. Non-limiting examples of lipophilic antioxidants include vitamin E isomers such as a-, β-, y-, and δ-tocopherols and α-, β-, y-, and 5-tocotrienols; polyphenols such as 2-tert-butyl-4-methyl phenol, 2-tert-butyl-5-methyl phenol, and 2-tert-butyl-6-methyl phenol; butylated hydroxyanisole (BHA) (e.g., 2-teri-butyl- 4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole); butylhydroxytoluene (BHT); tert- butylhydroquinone (TBHQ); ascorbyl palmitate; rc-propyl gallate; salts thereof; and mixtures thereof. [00131] In some embodiments, the lipid-based particle is a pH-sensitive nanoparticle. Such pH-sensitive nanoparticles (PNSDS) can be beneficial for the delivery of biologically active agents (e.g, siRNA molecules (Tang et al., SiRNA Crosslinked Nanoparticles for the Treatment of Inflammation-induced Liver Injury, Advanced Science, 2016, 4(2), el 600228)). [00132] In some embodiments, the lipid nanoparticle (LNP) is formulated to encapsulate an agent, such as a small molecule drug, protein-based drag, or nucleic acid-based drag, using a spontaneous vesicle formation formulation procedure as previously described in Semple et al. (2010) Nat. Biotechnol. 28172-28176.
[00133] In some embodiments, lipid nanoparticles can be engineered to alter the surface properties of particles so the lipid nanoparticles can penetrate the mucosal barrier. Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
[00134] Lipid nanoparticles engineered to penetrate mucus can comprise, but are not limited to, a polymeric material (i.e., a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer. The polymeric material can include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material can be biodegradable and/or biocompatible. The polymeric material can additionally be irradiated. As a non-limiting example, the polymeric material can be gamma irradiated (e.g., PCT Publ. No. WO 2012/082165, hereby incorporated by reference in its entirety)- Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone)? poly(D,L-lactide-co- caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co- PPO-co-D,L-lactide), polyalkyl cyanoacralaie, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene. polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as polyethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co- caprolactone), and trimethylene carbonate, polyvinylpyrrolidone. The lipid nanoparticle can be coated or associated with a co-polymer such as, but not limited to, a block co-polymer, and (polyethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see, e.g., U.S. Pat. Publ. Numbers 2012/0121718 and 2010/0003337; and U.S. Pat. No. 8,263,665). The co-polymer can be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle can be in such a way that no new chemical entities are created. For example, the lipid nanoparticle can comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. (2011) Angew. Chem. Int. Ed. 50:2597-2600). [00135] In some embodiments, agents encompassed by the present disclosure can be sustained release formulations, such as encapsulated into a nanoparticle or a rapidly eliminated nanoparticle and the nanoparticles or a rapidly eliminated nanoparticle can then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art. As a non-limiting example, the polymer, hydrogel or surgical sealant can be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL). Nanoparticles can be encapsulated into any polymer known in the art that can form a gel when injected into a subject. As a non-limiting example, the nanoparticle can be encapsulated into a polymer matrix that can be biodegradable.
[00136] In some embodiments, compositions encompassed by the present disclosure can be formulated as controlled release nanoparticles. In one example, the nanoparticle formulation for controlled release and/or targeted delivery can further include at least one controlled release coating. Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®). In another example, the controlled release and/or targeted delivery formulation can comprise at least one degradable polyester which can contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
[00137] In some embodiments, compositions encompassed by the present disclosure can be formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other conjugate-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of therapeutic agents (Aleku et al. (2008) Cancer Res. 68: 9788-9798; Strumberg et al. (2012) Int. J. Clin. Pharmacol. Then (2012) 50:76-78; Santel et al. (2006) Gene Ther. 13:1222-1234; Santel et al. (2006) Gene Ther. 13: 1360-1370; Gutbier et al. (2010) Pulm. Pharmacol. Ther. 23:334-344; Kaufinann et al. (2010) Microvasc. Res. 80:286-293; Weide et al. (2009) J. Immunother. 32:498-507; Weide et al. (2008) J. Immunother. 31:180-188; Pascolo (2004) Exp. Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al. (2011) J. Immunother. 34:1-15; Song et al. (2005) Nature Biotechnol. 23:709-717; Peer et al. (2007) Proc. Nail. Acad. Sci. U.S.A. 6:4095-4100; and deFougerolles (2008) Hum. Gene Ther. 19:125-132).
[00138] In some embodiments, therapeutic agents and compositions encompassed by the present disclosure can be encapsulated in, linked to and/or associated with synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in International Pub. Nos. WO 2010/005740, WO 2010/030763, WO 2012/13501, WO 2012/149252, WO 2012/149255, WO 2012/149259, WO 2012/149265, WO 2012/149268, WO 2012/149282, WO 2012/149301, WO 2012/149393, WO 2012/149405, WO 2012/149411, and WO 2012/149454, and U.S. Pat. Publ. Numbers 2011/0262491, 2010/0104645, 2010/0087337, and 2012/0244222, hereby incorporated by reference in their entireties. The synthetic nanocarrier formulations can be lyophilized, such as by methods described in PCT Publ. No. WO 2011/072218 and U.S. Pat No. 8,211,473, hereby incorporated by reference in their entireties.
[00139] The synthetic nanocarriers can contain reactive groups to release the conjugates described herein (e.g., PCT Publ. No. WO 2012/0952552 and U.S. Pat. Publ. No.
2012/0171229, hereby incorporated by reference in their entireties). Synthetic nanocarriers can be formulated for targeted release, e.g., to release the therapeutic agents at a specified pH and/or after a desired time interval. As a non-limiting example, the synthetic nanoparticle can be formulated to release the conjugates after 24 hours and/or at a pH of 4.5 (e.g., PCT Publ. Numbers WO 2010/138193 and WO 2010/138194 and U.S. Pai. Publ. Numbers
2011/0020388 and 2011/0027217, hereby incorporated by reference in their entireties). In some embodiments, the synthetic nanocarriers can be formulated for controlled and/or sustained release of conjugates described herein. As a non-limiting example, the synthetic nanocarriers for sustained release can be formulated by methods known in the art, described herein and/or as described in PCT Publ. No. WO 2010/138192 and U.S. Pat. Publ. No. 2010/0303850, hereby incorporated by reference in their entireties.
Size of LNP s
[00140] LNP size may be measured by various analytical methods known in the art. In some embodiments, LNP size may be measured using Asymetric-Flow Field Flow Fractionation - Multi-Angle Light Scattering (AF4-MALS). In certain embodiments, LNP size may be measured by separating particles in the composition by hydrodynamic radius, followed by measuring the molecular weights, hydrodynamic radii and root mean square radii of the fractionated particles. In some embodiments, LNP size and particle concentration may be measured by nanoparticle tracking analysis. In certain embodiments, LNP samples are diluted appropriately and injected onto a microscope slide. A camera records the scattered light as the particles are slowly infused through field of view. After the movie is captured, the Nanoparticle Tracking Analysis processes the movie by tracking pixels and calculating a diffusion coefficient. This diffusion coefficient can be translated into the hydrodynamic radius of the particle. Such methods may also count the number of individual particles to give particle concentration. In some embodiments, LNP size, morphology, and structural characteristics may be determined by cryo-electron microscopy (“cryo-EM”). [00141] The LNPs of the LNP compositions disclosed herein have a size (e.g., Z-average diameter) of about 1 to about 500 nm. In some embodiments, the LNPs have a size of about 10 to about 250 nm. In further embodiments, the LNPs have a size of about 50 to about 200 nm. In some embodiments, the LNPs have a size of about 75 to about 200 nm or about 100 to 200 nm. In some embodiments, the LNPs have a size of about 75 to about 150 nm.
[00142] In some embodiments, the mean lipid particle diameter is greater than 300 nm. In some embodiments, the lipid particle has a diameter of about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less, e.g., fiom about 50 to about 150 nm. Smaller particles generally exhibit increased circulatory lifetime in vivo compared to larger particles. Smaller particles have an increased ability to reach tumor sites than larger nanoparticles. In certain such embodiments, the lipid particle has a diameter fiom about 15 to about 50 nm.
Cargo
[00143] The cargo delivered via LNP composition may be a biologically active agent. In certain embodiments, the cargo is or comprises one or more biologically active agent, such as an antibody (e.g., monoclonal, chimeric, humanized, nanobody, and fragments thereof etc.), cholesterol, hormone, peptide, protein, chemotherapeutic and other types of antineoplastic agent, mRNA, low molecular weight drag, vitamin, co-fector, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex forming oligonucleotide, antisense DNA or RNA composition, chimeric DNA:RNA composition, allozyme, aptamer, ribozyme, decoys and analogs thereof, plasmid and other types of vectors, and small nucleic acid molecule, RNAi agent, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA) and "self-replicating RNA" (encoding a replicase enzyme activity and capable of directing its own replication or amplification in vivo) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), and iRNA (asymmetrical interfering RNA). The above list of biologically active agents is exemplary only, and is not intended to be limiting. Such compounds may be purified or partially purified, may be naturally occurring or synthetic, and may be chemically modified.
Methods of Making Lipid Nanoparticles [00144] In some embodiments, LNPs are formed by mixing an aqueous biologically active agent solution with an organic solvent-based lipid solution. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol. For example, the organic solvent may be 100% ethanol. A pharmaceutically acceptable buffer, e.g., for in vivo administration of LNPs, may be used. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 6.5. In certain embodiments, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 7.0. In certain embodiments, the composition has a pH ranging from about 7.2 to about 7.7. In additional embodiments, the composition has a pH ranging from about 7.3 to about 7.7 or ranging from about 7.4 to about 7.6. In further embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of a composition may be measured with a micro pH probe.
[00145] In some embodiments, a cryoprotectant is mixed with the composition comprising LNPs. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. The composition comprising LNPs may include up to 10% cryoprotectant, such as, for example, sucrose. In certain embodiments, the composition comprising LNPs may comprise tris saline sucrose (TSS). In certain embodiments, the composition comprising LNPs may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% cryoprotectant. In certain embodiments, the composition comprising LNPs may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% sucrose. In some embodiments, the composition comprising LNPs may include a buffer. In some embodiments, the buffer may comprise a phosphate buffer (PBS), a Tris buffer, a citrate buffer, and mixtures thereof. In certain exemplary- embodiments, the buffer comprises NaCl. In certain embodiments, the buffer lacks NaCl. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer.
Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary' embodiments of the LNP compositions contain 5% sucrose and 45 mM NaCl in Tris buffer. In other exemplary- embodiments, the composition comprising LNPs may contain sucrose in an amount of about 5% w/v, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The salt, buffer, and cryoprotectant amounts may be varied such that the osmolality of the overall composition is maintained. For example, the final osmolality may be maintained at less than 450 mOsm/L. In further embodiments, the osmolality is between 350 and 250 mOsm/L. Certain embodiments have a final osmolality of 300 +/- 20 mOsm/L or 310 +/- 40 mOsm/L.
[00146] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing of the aqueous RNA solution and the lipid solution in an organic solvent is used. In certain such embodiments, flow rates, junction size, junction geometry', junction shape, tube diameter, solutions, and/or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be concentrated or purified, e.g., via dialysis, centrifugal filter, tangential flow filtration, or chromatography. The LNPs may be stored as a suspension, an emulsion, or a lyophilized powder, for example. In some embodiments, an LNP composition is stored at 2-8° C. In certain embodiments, the LNP compositions are stored at room temperature. In additional embodiments, an LNP composition is stored frozen, for example at -20° C or -80° C. In other embodiments, an LNP composition is stored at a temperature ranging from about 0° C to about -80° C. Frozen LNP compositions may be thawed before use, for example on ice, at room temperature, or at 25° C.
[00147] In some embodiments, LNP compositions encompassed by the present disclosure can also be formulated using natural and/or synthetic polymers to reduce or inhibit the attachment of unwanted substances, such as bacteria, to the LNPs, or to enhance drug deliver of the LNPs. Non-limiting examples of polymers which can be used for drug delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, CA) formulations from MIRUS® Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGY™ (Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers, RONDELTM (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, CA) and pH responsive co-block polymers such as, but not limited to, PHASERXTM (Seattle, WA). For example, agents and compositions encompassed by the present disclosure can be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof. [00148] The polymers used in the present disclosure may have undergone processing to reduce or inhibit the attachment of unwanted substances, such as bacteria, to the LNPs. The polymer can be processed by methods known and/or described in the art and/or described in PCT Publ. No. WO 2011/50467, hereby incorporated by reference in its entirety.
Apo E-mimicking peptides
[00149] The LNP compositions disclosed herein comprise apolipoprotein E-mimicking peptides. The ApoE-mimicking peptides can be single domain or dual domain peptides. [00150] Disclosed are ApoE-mimicking peptides comprising a receptor binding domain of ApoE and a lipid-associating peptide. In some embodiments, the ApoE-mimicking peptide further comprises an amino hexanoic acid, such as an acetylated amino hexanoic acid (Ac- Aha).
[00151] In certain embodiments, the Ac- Aha is at the N-terminus of the peptide. In other embodiments, the Aha can be inserted between the lipid-associating peptide comprises a class A amphipathic-helical domain.
[00152] In certain embodiments, the ApoE-mimicking peptide comprises an Ac-Aha, wherein the lipid-associating peptide comprises a class A amphipathic-helical domain. For example, the class A amphipathic-helical domain is DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1), DWLRAFYDKVAEKLREAF (SEQ ID NO: 2), DWLRALYDKVAEKLREAL (SEQ ID NO: 3), DLLRALYDKVAEKLREAW (SEQ ID NO: 4), or FAEKLKEAVKDYFAKLWD (SEQ ID NO: 5).
[00153] In certain embodiments, the ApoE-mimicking peptide comprises an Ac-Aha, wherein the lipid-associating peptide comprises a class A amphipathic-helical domain, wherein the receptor binding domain of ApoE can be covalently linked to the lipid- associating peptide.
[00154] In certain embodiments, the ApoE-mimicking peptide comprises an Ac-Aha, w'herein said peptide is protected using an amide group at the C-terminus.
[00155] In certain embodiments, the ApoE-mimicking peptide comprises an Ac-Aha, wherein the receptor binding domain of ApoE can be LRKLRKRLLR (SEQ ID NO: 6), LRRLRRRLLR (SEQ ID NO: 7), LRKMRKRLMR (SEQ ID NO: 8), or RLTRKRGLK (SEQ ID NO: 9). The receptor binding domain of ApoE can also be, but is not limited to, LRKLRKRFFR (SEQ £D NO: 17), LRKLPKRLLR (SEQ ID NO: 18), LRNVRKRLVR (SEQ ID NO: 19), MRKLRKRVLR (SEQ ID NO: 20), LRRLRRRLLR (SEQ ID NO: 7), LRKLRKRFFR (SEQ ID NO: 17), LRKLRKRLLR (SEQ ID NO: 6), or LRKMRKRLMR (SEQ ID NO: 8).
[00156] In certain embodiments, the ApoE-mimicking peptide comprises an Ac-Aha, wherein the ApoE-mimicking peptide can be Ac-Aha-hElSA-NH2 or Ac-Aha-[R]hEl 8A- NFb. The ApoE-mimicking peptide of Ac-Aha-hElSA-NH2 is Ac-Aha- LRKLRKRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 21). The ApoE- mimicking peptide of Ac-Aha-[R]hE18A-NH2 is Ac-Aha- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NHb (SEQ ID NO: 22).
[00157] In certain embodiments, the ApoE-mimicking peptide comprises a fatty acid moiety, co-amino fatty acid moiety, or an acetylated co-amino fatty acid moiety.
[00158] In certain embodiments, the receptor binding domain of ApoE can be covalently linked to the lipid-associating peptide.
[00159] In certain embodiments, the ApoE-mimicking peptide is protected using an amide group at the C-terminus.
[00160] In certain embodiments, the ApoE-mimicking peptide can be: butanoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
23); hexanoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
24); octanoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 11; decanoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NHz (SEQ ID NO:
25); lauroyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 26); myristoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
12); palmitoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
13); stearoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 27); palmitoleoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 28); arachidoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
29); behenoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
30); oleoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 14); ricinoleoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
31); linolenoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
32); vacceoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
33); gadoleoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
34); erucoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 35); cetoleoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
36); nervonoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
37); adrenoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
38); a-linolenoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
39); y-linolenoyl-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO:
40);
EPA-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 41); or DHA-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 42).
In the foregoing, the fatty acid moiety is shown at the left side and is linked to the peptide LRRLRRRLLR (SEQ ID NO: 7). “EPA” indicates a moiety derived from 5,8,11,14, 17- eicosapentaenoic acid; and “DHA” indicates a moiety- derived from 4,7,10,13,16,19- docosahexaenoic acid.
[00161] In certain embodiments, the ApoE-mimicking peptide comprises a fatty acid moiety derived from a natural oil or fat, e.g., fish oil, wherein the ApoE-mimicking peptide can be:
(fish oil)-LRRLRRRLLR-DWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 43).
In the foregoing “(fish oil)” indicates that the fatty acids in fish oil, including, but not limited to, fish oil components such as EPA and DHA, are linked to linked to the peptide LRRLRRRLLR (SEQ ID NO: 7). Thus, the ApoE-mimicking peptide is a mixture of peptides comprising fatty acid groups derived from the fish oil used to prepare them. [00162] In certain embodiments, the ApoE-mimicking peptide can be any of the disclosed peptides comprising a fatty acid.
[00163] In certain embodiments, the ApoE-mimicking peptide can be any of the disclosed peptides comprising an acetylated fatty acid.
[00164] In certain embodiments, the ApoE-mimicking peptide comprises an Ac-Aha, wherein the receptor binding domain of apolipoprotein E is scrambled. Examples of scrambled receptor binding domains of ApoE are provided below.
[00165] In certain embodiments, the receptor binding domain is covalently linked to said lipid-associating peptide, wherein both the receptor binding domain of apolipoprotein E and the lipid-associating peptide are scrambled. Examples of scrambled receptor binding domains of ApoE and scrambled lipid-associating peptides are provided below.
[00166] Apolipoprotein E-mimicking peptides have both direct cholesterol lowering effects by providing an alternative ligand for receptors on the liver to clear atherogenic Apolipoprotein B containing lipoproteins (LDL, VLDL, and β-VLDL), and direct beneficial effects on the artery wall. The Apo E-mimicking peptides can enhance the removal of cholesterol from the artery- wall, working in conjunction with HDL, increasing the formation of lipid poor preP-HDL that accept cholesterol from macrophages. The Apo E-mimicking peptides can stimulate macrophage-mediated clearance of dead and dying cells in the artery wall (eflferocytosis), improve the quality of HDL by increasing PON-1 levels and bringing down plasma lipid hydroperoxide levels, decrease macrophage content in atherosclerotic lesions resulting in more stable lesions, and decrease inflammation in the artery wall. As a result, the Apo E-mimicking peptides reduce the size of atherosclerotic lesions more rapidly than apoA-I mimetic peptides and more rapidly than the statins (HMG-CoA reductase inhibitors). Atherosclerotic lesion regression persists in Apo E-mimicking peptides treated animals even when cholesterol levels are the same as in saline treated animals.
Apolipoprotein E
[00167] Apolipoprotein E (Apo E) plays an important role in the metabolism of triglyceride- rich lipoproteins, such as very low density lipoprotein (VLDL) and chylomicrons. Apolipoprotein E mediates the high affinity binding of Apo E-containing lipoproteins to the low density lipoprotein (LDL) receptor (Apo B, E receptor) and the members of its gene family, including LDL receptor related protein (LRP), very low density lipoprotein receptor (VLDLR) and the Apo E2 receptor (Apo E2R).
[00168] Apo E is a protein that binds lipid and has two major domains (Mahley, R.W., et al. J. Lipid Res. 1999, 40:622-630). The 22 kDa amino terminal domain has been shown by X- ray crystallographic studies to be a 4-helix bundle (Wilson, C., et al. Science 1991;252: 1817- 1822) and to contain a positively-charged receptor binding domain. For this region to mediate very low-density lipoprotein (VLDL) binding to its receptors, the apolipoprotein must associate with the lipoprotein surface; this is enabled by the C -terminal amphipathic helical region. If the 4-helix bundle that contains the positively charged receptor-binding domain does not open up on the lipoprotein surface, then the VLDL is defective in binding to receptors. Thus, the positively charged arginine (Arg)-rich cluster domain of the Apo E and the C-terminal amphipathic helical domain, are both required for the enhanced uptake of atherogenic Apo E-containing lipoproteins.
[00169] Apo E is secreted as a 299 amino acid residue protein with a molecular weight of 34,200. Based on thrombin cleavage of Apo E into two fragments, a two-domain hypothesis was initially suggested to explain the fact that the C-terminal region of Apo E (192-299) is essential for its binding to hypertriglyceridemic VLDL and the N-terminal 22 kDa domain (1- 191), binds to the LDL-R.
Fatty Acids
[00170] The LNP compositions disclosed herein can be linked to a fatty' acid moiety, an co- amino fatty acid moiety, or an acetylated co-amino fatty acid moiety. In various embodiments, the fatty acid moiety, the co-amino fatty acid moiety, or the acetylated co-amino fatty acid moiety is linked to a disclosed peptide via the N-terminal amino group of the peptide.
[00171] In certain embodiments, the linkage between the fatty acid moiety, the co-amino fatty acid moiety, or the acetylated co-amino fatty acid moiety and the N-terminal amino group of the peptide has the a structure represented by the following formulas II-IV, respectively:
Figure imgf000045_0001
wherein A is an aliphatic group have 2-32 carbon atoms.. In preferred embodiments, the aliphatic group is an alkyl group. In certain embodiments, the aliphatic group comprises 0-3 double bonds. In certain such embodiments, the aliphatic group is an alkenyl group.
[00172] In certain embodiments, the fatty acid moiety linked to the disclosed peptide is derived from a purified fatty acid. In some embodiments, the fatty acid moiety linked to the disclosed peptide is derived from a saturated fatty acid. In a other embodiments, the fatty acid moiety linked to the disclosed peptide is derived from an unsaturated fatty acid, such as a polyunsaturated fatty acid with two or more double bonds.
[00173] Exemplary fatty acids from which a fatty acid moiety is derived include, without limitation, butyric acid, caproic acid, caprylic acid, capric acid, decanoic acid, lauric acid, myristic acid, palmitic acid, pentadecanoic acid, stearic acid, arachidic acid, behenic acid, erucic acid, lignoceric acid, margaric acid, myristoleic acid, palmitoleic acid, oleic acid, gadoleic acid, ricinoleic acid, vaccenic acid, linoleic acid, linolenic acid, alpha-linolenic acid, gamma-linolenic acid, licanic acid, margaroleic acid, arachidic acid, gadoleic acid, nervonic acid, arachidonic acid, docosapentaenoic (DPA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and the like.
[00174] Exemplary saturated fatty acids include, but are not limited to, propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid, nonacosanoic acid, triacontanoic acid, henatriacontanoic acid, dotriacontanoic acid, tritriacontanoic acid, tetratriacontanoic acid, pentatriacontanoic acid, and hexatriacontanoic acid.
[00175] Exemplary unsaturated fatty acids include, but are not limited to, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA), erucic acid, docosahexaenoic acid (DHA), and docosapentaenoic acid.
[00176] In certain embodiments, the tatty acid moiety linked to the disclosed peptide is derived from an unpurified fatty acid or mixture of fatty acids such as natural oil or fat. Typically, a natural oil or fat is a heterogeneous mixture of generally hydrophobic compounds comprising one or more fatty acids. The tatty acid source may comprise a natural oil or fat, such as (but not limited to) animal fits, biological oils, or vegetable oils such as soya bean oil, coconut oil, palm oil, palm kernel oil, rapeseed oil, cottonseed oil, linseed oil, sunflower oil, fish oil, algae oil, and the like.
[00177] In certain such embodiments, the natural oil or fat is one that contains or is enriched for one or more omega-3 fatty acids, for example, marine oil, for example, fish oil, krill oil and algae oil. Any oil containing DHA and/or EP A can be used. In certain embodiments, the natural oil or fat contains at least 70% or about 70%, by weight, DHA, for example, at least 75% or about 75%, at least 80% or about 80%, at least 85% or about 85%, or at least 90% or about 90%, by weight, DHA. In some embodiments, the natural oil or fat contains between 5% or about 5% and 15% or about 15% EPA, for example, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15%, by weight, EPA. In certain embodiments, the natural oil or fat contains not more than 10% or about 10% EPA or less than 10% or about 10%, EPA.
[00178] In certain embodiments, the fatty acid moiety is derived from an omega-3 fatty acid. As used herein, the term “omega-3 polyunsaturated fatty acid(s)” or “omega-3 fatty acid” refers to a family of unsaturated fatty carboxylic acids that have in common a carbon-carbon bond in the n-3 position (i.e., the third bond from the methyl end of the molecule). Typically, they contain from about 16 to about 24 carbon atoms and from three to six carbon-carbon double bonds. Omega-3 polyunsaturated fatty acids can be found in nature, and these natural omega-3 polyunsaturated forty acids frequently have all of their carbon-carbon double bonds in the cis-configuration.
[00179] Exemplary omega-3 fatty acids include, but are not limited to, 7,10,13- hexadecatrienoic acid (sometimes abbreviated as 16:3 (n-3)); 9,12,15-octadecatetrienoic acid (a-linolenic acid (ALA), 18:3 (n-3)); 6,9,12,15-octadecatetraenoic acid (stearidonic acid (STD), 18:4 (n-3)); 11,14,17-eicosatrienoic acid (eicosatrienoic acid (ETE), 20:3 (n-3)); 8,11,14,17-eicosatetraenoic acid (eicosatetraenoic acid (ETA), 20:4 (n-3)); 5,8,11,14,17- eicosapentaenoic acid (eicosapentaenoic acid (EPA), (20:5 (n-3)); 7,10,13,16,19- docosapentaenoic acid (docosapentaenoic acid (DPA), 22:5 (n-3)); 4,7,10,13,16,19- docosahexaenoic acid (docosahexaenoic acid (DHA), 22:6 (n-3)); 9,12,15,18,21- tetracosapentaenoic acid (tetracosapentaenoic acid, 24:5 (n-3)); and 6,9,12,15,18,21- tetracosahexaenoic acid (tetracosahexaenoic acid, 24:6 (n-3)). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are found in nature in fish oils, and have been used in a variety of dietary/therapeutic compositions. [00180] Various lengths of fatty acids are contemplated. In certain embodiments, a fatty acid comprises a chain length between C6 and C24, CIO and C24, CIO and C28, or CIO and C32, including synthetic fatty acids with odd carbon numbers. In certain such embodiments, a fatty acid comprises a chain length selected from: CIO, C12, C14, C16, C18, C20, C20, C22 and C24, preferably fiom C14, C16 and C18. In other embodiments, the fetty acid has a chain length selected from C13, C15 and C17. In certain embodiments, the fatty acid has between 4 and 28 carbons.
[00181] In certain embodiments of the present disclosure, the fatty acid aliphatic chain comprises 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 carbon atoms.
[00182] In certain embodiments, the fatty' acid is a naturally-occurring fatty acid. In certain such embodiments, the fetty acid is a short chain fatty acid (e.g., less than six carbons), a medium chain fatty' acid (e.g., 6-12 carbons), long chain fatty acids (e.g., longer than 12 carbons), or a ven- long chain fatty acid (e.g., longer than 22 carbons). In some embodiments, the fatty acid is an unsaturated fatty acid in the cis configuration. In other embodiments, the fatty acid is an unsaturated fatty' acid in the trans configuration.
[00183] In certain embodiments, the ApoE-mimicking peptide comprises a co-amino fatty acid moiety.
[00184] Exemplary- co-amino-fatty acid moieties are derived from co-amino-fatty acids including, without limitation, 4-amino-butyric acid, 6-amino-caproic acid, 8-amino-caprylic acid, 10-amino-capric acid (10-amino-decanoic acid), 12-amino-lauric acid (12-amino- dodecanoic acid), 14-amino-myristic acid (14-amino-tetradecanoic acid), 14-amino- myristoleic acid, 16-amino-palmitic acid (16-amino-hexadecanoic acid), 18-amino-stearic acid, 18-amino-oleic acid, 16-amino-palmitoleic acid, 18-amino-linoleic acid, 18-amino- linolenic acid and 20-amino-arachidonic acid. In certain embodiments, the co-amino-fetty' acid moieties are derived from 6-amino-caproic acid.
[00185] In other embodiments, the co-amino fatty acid moiety is 4-amino-butanoyl, 6-amino- caproyl, 8-amino-octanoyl, 10-amino-decanoyl, 12-amino-lauroyl, 14-amino-myristoyl, 14- amino-myristoleoyl, 16-amino-palmiteoyl, 18-amino-stearoyl, 18-amino-oleoyl, 16-amino- palmitoleoyl, 18-amino-linoleoyl, 18-amino-linolenoyl, or 20-amino-arachidonoyl. In certain preferred embodiments, co-amino fatty acid moiety is 6-amino-caproyl (or alternatively referred to as 6-amino hexanoyl).
[00186] In certain embodiments, the co-amino-fatty acid moiety' is derived from a co-amino- fatty acid having the structure:
Figure imgf000049_0001
wherein A is an aliphatic group have 2-32 carbon atoms. In certain embodiments, the aliphatic group is an alkyl group. In some embodiments, the aliphatic group comprises 0-3 double bonds. In some such embodiments, the aliphatic group is an alkenyl group. In certain preferred embodiments, A is -(CH2)5-.
[00187] In certain embodiments, the co-amino-fatty acid moiety is linked to the peptide via the N-terminal amino group of the peptide, and following linking to the peptide, the co-amino- fatty acid moiety has the structure:
Figure imgf000049_0002
wherein A is an aliphatic group have 2-32 carbon atoms.. In certain embodiments, the aliphatic group is an alkyl group. In some embodiments, the aliphatic group comprises 0-3 double bonds. In some such embodiments, the aliphatic group is an alkenyl group. In certain preferred embodiments
Figure imgf000049_0005
[00188] In some embodiments, the ApoE-mimicking peptide comprises an acetylated co- amino fatty acid moiety. In certain embodiments, the disclosed peptides can be linked to any of the disclosed co-amino-fatty acids, and then further comprise an acetyl moiety' on the co- amino group.
[00189] In certain embodiments, the co-amino-fatty acid moiety is linked to the peptide via the N-terminal amino group of the peptide, and following linking to the peptide, the co-amino group is acetylated, and the co-amino-fatty acid moiety has the structure:
Figure imgf000049_0003
wherein A is an aliphatic group have 2-32 carbon atoms.. In certain embodiments, the aliphatic group is an alkyl group. In some embodiments, the aliphatic group comprises 0-3 double bonds. In some such embodiments, the aliphatic group is an alkenyl group. In certain preferred embodiments
Figure imgf000049_0004
[00190] The fatty acids from which the fatty acid moiety is derived are commercially available and can be prepared by different chemical methods (Recent Developments in the Synthesis of Fatty Acid Derivatives, Editors: Knothe G and Derksen JTB, AOCS Press 1999, ISBN 1-893997-00-6.) Receptor binding domain peptides
[00191] The receptor binding domain peptide for the Apo E mimetics can be a human receptor binding domain peptide of Apo E. For example, receptor binding domain peptide of the disclosed Apo E mimetics can comprise the amino acid sequence of LRKLRKRLLR (SEQ ID NO: 6), LRRLRRRLLR (SEQ ID NO: 7), or LRKLRKRFFR (SEQ ID NO: 17). The receptor binding domain peptide of such Apo E mimetics can also be from a species selected from the group consisting of mouse, rabbit, monkey, rat, bovine, pig and dog.
[00192] Examples of receptor binding domain peptides that can be used in the disclosed Apo E mimetics are provided in Table 1.
Figure imgf000050_0001
[00193] The italicized residues in Table 1 indicate changes from the human sequence; however, the property of the amino acid is conserved. The bold-italicized residues in Table 1 indicate the difference from the human sequence at that position.
[00194] The receptor binding domain peptide for the Apo E mimetics can also be the LDL receptor (LDLR) binding domain of apolipoprotein B (ApoB). The LDL receptor (LDLR) binding domain of ApoB can have the sequence RLTRKRGLK (SEQ ID NO: 9). ApoB- 100 is a 550,000 Da glycoprotein with nine amino acids (3359-3367) serving as the binding domain for the LDL receptor (Segrest et al., J. Lipid. Res. 42, pp. 1346-1367 (2001)). Upon binding to LDLR in clathrin coated pits, LDL is internalized via endocytosis and moves into the endosome where a drop in pH causes the receptor to dissociate from the LDL. The receptor is recycled back to the surface of the cell while the LDL is moved into the lysosome where the particle is degraded (Goldstein et al., Ann. Rev. Cell Biol. 1, pp. 1-39 (1985)). The LDL receptor (LDLR) binding domain of ApoB when used with the disclosed peptides can also be altered and/or modified as described throughout this application for Apo E. For example, LDL receptor (LDLR) binding domain of ApoB can be used with the disclosed lipid-associating peptides, wherein the LDL receptor (LDLR) binding domain of ApoB is covalently linked to said lipid-associating peptide. In addition, the LDL receptor (LDLR) binding domain of ApoB can be scrambled, reverse-oriented, can be part of a domain switched peptide as described below.
Linid-Associating Peptides
[00195] Lipid-associating peptides can be used alone or in combination with the Apo E- mimicking peptides. The lipid associating peptide for these Apo E mimetics can be, but are not limited to, class A amphipathic helical peptides, class A amphipathic helical peptide mimetics of apoA-I having aromatic or aliphatic residues in the non-polar face, small peptides including pentapeptides, tetrapeptides, tripeptides, dipeptides and pairs of amino acids, Apo-J (G* peptides), and peptide mimetics, e.g., as described below. а. Class A Amphipathic Helical Peptides
[00196] In certain embodiments, the lipid-associating peptides for use in the disclosed methods include class A amphipathic helical peptides, e.g., as described in U.S. Pat. No. б,664,230, and PCT Publications WO 2002/15923 and WO 2004/034977, hereby incorporated by reference in their entireties It was discovered that peptides comprising a class A amphipathic helix ("class A peptides"), are capable of mitigating one or more symptoms of atherosclerosis as well as treating other disorders.
[00197] Class A peptides are characterized by formation of an a-helix that produces a segregation of polar and non-polar residues thereby forming a polar and a nonpolar face with the positively charged residues residing at the polar-nonpolar interface and the negatively charged residues residing at the center of the polar face (see, e.g., Anantharamaiah (1986) Meth. Enzymol, 128: 626-668). It is noted that the fourth exon of apo A-I, when folded into 3.667 residues/tum produces a class A amphipathic helical structure.
[00198] One class A peptide, designated 18A (see, e.g., Anantharamaiah (1986) Meth. Enzymol, 128: 626-668) was modified as described herein to produce peptides orally administrable and highly effective at inhibiting or preventing one or more symptoms of atherosclerosis and/or other indications described herein. Without being bound by a particular theory, it is believed that the disclosed peptides can act in vivo by picking up seeding molecule(s) that mitigate oxidation of LDL.
[00199] Increasing the number of Phe residues on the hydrophobic face of 18A can increase lipid affinity as determined by the computation described by Palgunachari et al. (1996) Arteriosclerosis, Thrombosis, & Vascular Biol. 16: 328-338. Theoretically, a systematic substitution of residues in the nonpolar face of 18A with Phe could yield six peptides.
Peptides with an additional 2, 3 and 4 Phe would have theoretical lipid affinity
Figure imgf000052_0002
values of 13, 14 and 15 units, respectively. However, the /.values jumped four units if the additional Phe were increased from 4 to 5 (to 19 X units). Increasing to 6 or 7 Phe would produce a less dramatic increase (to 20 and 21
Figure imgf000052_0001
units, respectively).
[00200] A number of these class A peptides were made including, the peptide designated 4F, D4F, 5F, and D5F, and the like. Various class A peptides inhibited lesion development in atherosclerosis-susceptible mice and rabbits. In addition, the peptides show varying, but significant degrees of efficacy in mitigating one or more symptoms of the various pathologies described herein. A number of such peptides are illustrated in Table 2.
Figure imgf000052_0003
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
* Linkers are underlined; NMA is N-Methyl Anthranilyl
[00201] In certain embodiments, the peptides include variations of 4F (D-W-F-K-A-F-Y -D- K-V-A-E-K-F-K-E-A-F in Table 2 (SEQ ID NO: 47)), also known as L-4F, where all residues are L form amino acids) or D-4F where one or more residues are D form amino acids). In any of the peptides described herein, the C-terminus, and/or N-terminus, and/or internal residues can be blocked with one or more blocking groups as described herein. [00202] While various peptides of Table 2 are illustrated with an acetyl group or an N- methylanthranilyl group protecting the amino terminus and an amide group protecting the carboxyl terminus, any of these protecting groups may be eliminated and/or substituted with another protecting group as described herein. The peptides can comprise one or more D-form amino acids as described herein. In certain embodiments, every amino acid (e.g., every chiral amino acid) of the peptides of Table 2 is a D-form amino acid.
[00203] It is also noted that Table 2 is not folly inclusive. Using the teachings provided herein, other suitable class A amphipathic helical peptides can routinely be produced (e.g., by conservative or semi-conservative substitutions (e.g., D replaced by E), extensions, deletions, and the like). Thus, for example, truncations may be made of any one or more of peptides shown herein (e.g., peptides identified as 2F, 3F, 3F14, 4F, 5F, 6F, or 7F— in Table 2). Thus, for example, A-F-Y-D-K-V-A-E-K-L-K-E-A-F (SEQ ID NO: 62) illustrates a peptide comprising 14 amino acids from the C-terminus of 18A comprising one or more D amino acids, while others illustrate other truncations.
[00204] Longer peptides are also suitable. Such longer peptides may entirely form a class A amphipathic helix, or the class A amphipathic helix (helices) can form one or more domains of the peptide. In addition, this invention contemplates multimeric versions of the peptides (e.g., concatamers). Thus, for example, the peptides illustrated herein can be coupled together (directly or through a linker (e.g., a carbon linker, or one or more amino acids) with one or more intervening amino acids). Illustrative polymeric peptides include 18A-Pro-18A and the peptides in the following table (Table 2B), in certain embodiments comprising one or more D amino acids, more preferably with every amino acid a D amino acid as described herein and/or having one or both termini protected.
Figure imgf000057_0001
Figure imgf000058_0006
b. Class A Amphipathic Helical Peptide Mimetics of apoA-I Having Aromatic or Aliphatic Residues in the Non-Polar Face
[00205] The LNP compositions disclosed herein comprise modified class A amphipathic helix peptides. Certain preferred peptides incorporate one or more aromatic residues at the center of the nonpolar face, e.g.
Figure imgf000058_0002
, , (as present in 4F), or with one or more aliphatic residues at the center of the nonpolar fece, e.g. see, e.g., Table 3. Without being bound
Figure imgf000058_0003
to a particular theory', the central aromatic residues on the nonpolar fece of the peptide
Figure imgf000058_0001
due to the presence of it electrons at the center of the nonpolar fece can allow water molecules to penetrate near the hydrophobic lipid alkyl chains of the peptide-lipid complex, which in turn would enable the entry of reactive oxygen species (such as lipid hydroperoxides) shielding them from the cell surface. The peptides with aliphatic residues at the center of the nonpolar face, e.g. can act similarly but not quite as effectively as
Figure imgf000058_0004
Figure imgf000058_0005
[00206] In certain embodiments, the peptides can convert pro-inflammatory HDL to anti- inflammatory HDL or make anti-inflammatory HDL more anti-inflammatory, and/or decrease LDL-induced monocyte chemotactic activity generated by artery wall cells equal to or greater than D4F or other peptides shown in Table 2.
Figure imgf000058_0007
c. Other class A and some Class Y Amphipathic Helical Peptides. [00207] Class A amphipathic helical peptides that have an amino acid composition identical to one or more of the class A amphipathic helical peptides described above. Thus, for example, in certain embodiments this invention contemplates peptides having an amino acid composition identical to 4F. Thus, in certain embodiments, this invention includes active agents that comprise a peptide that consists of 18 amino acids, where the 18 amino acids consist of 3 alanines (A), 2 aspartates (D), 2 glutamates (E), 4 phenylalanines (F), 4 lysines (K), 1 valine (V), 1 tryptophan (W), and 1 tyrosine (Y); and where the peptide forms a class A amphipathic helix; and protects a phospholipid against oxidation by an oxidizing agent. In certain embodiments, the peptides comprise least one "D" amino acid residue; and in certain embodiments, the peptides comprise all "D” form amino acid residues. A variety of such peptides are illustrated in Table 4. Reverse (retro-), inverse, retro-inverso-, and circularly permuted forms of these peptides are also contemplated. Table 4 provides the sequences and identifier names for representative 18 amino acid length class A amphipathic helical peptides with the amino acid composition comprising 3 alanines (A), 2 aspartates (D), 2 glutamates (E), 4 phenylalanines (F), 4 lysines (K), 1 valine (V), 1 tryptophan (W), and 1 tyrosine (Y). [00208] In Table 4, the bold underlined residues indicate the changes made to each analog. For example, the changes may be switching D-E, reversing W-2, F-3 positions, or switching F-6 and Y-7 positions.
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
[00209] A person skilled in the art may readily identify biologically active and usefill peptides. Thus, for example, the following peptides have been accurately identified as active: 3F1; 3F2; 4F the reverse (retro) forms thereof and the retro-inverso forms thereof. Lipid- associating peptides can comprise a peptide that is 18 amino acids in length and forms a class A amphipathic helix. In some embodiments, the peptide comprises the following amino acid composition: 2 aspartates, 2 glutamates, 4 lysines, 1 tryptophan, 1 tyrosine, no more than one leucine, no more than 1 valine, no less than 1 and no more than 3 alanines, and with 3 to 6 amino acids from the group: phenylalanine, alpha-naphthalanine, beta-naphthalanine, histidine. In some embodiments, the peptide comprises either 9 or 10 amino acids on the polar face in a helical wheel representation of the class A amphipathic helix. In some embodiments, the peptide comprises 4 amino acids with positive charge at neutral pH. In some embodiments, two of the positively charged residues reside at the interlace between the polar and non-polar faces. In some embodiments, two of the four positively charged residues reside on the polar face that are contiguous. In some embodiments, two of the amino acid residues on the non-polar face are from the group: phenylalanine, alpha-naphthalanine, beta- naphthalanine, histidine. In some embodiments, two of the amino acid residues on the non- polar face are are also contiguous, and if there are 4 or more amino acids from this group on the non-polar face, there are also at least 2 residues from this group that are not contiguous. In some instances, all of the acidic amino acids are glutamic acid rather than having two aspartic acids and two glutamic acids. In some embodiments, the lipid associating peptide can be 18A, wherein each of the acidic amino acids of 18A are Glu residues.
[00210] Certain class Y as well as class A amphipathic helical peptides are disclosed. Class Y amphipathic helical peptides are known to those of skill in the art (see, e.g., Segrest et al. (1992) J. Lipid Res. 33: 141-166; Oram and Heinecke (2005) Physiol Rev. 85: 1343-1372, and the like). These peptides include, but are not limited to, an 18 amino acid peptide that forms a class A amphipathic helix or a class Y amphipathic helix described by formula V: D X X KY X X D K XY D K X K D Y X (V) where the D's are independently Asp or Glu; the Ks are independently Lys or Arg; the Xs are independently Leu, nor Leu, Vai, lie, Trp, Phe, Tyr
Figure imgf000069_0001
, or a-Nal and all X residues are on the non-polar face (e.g., when viewed in a helical wheel diagram) except for one that can be on the polar face between two K residues; the Y's are independently Ala, His, Ser, Gin, Asn, or Thr non-polar face (e.g., when viewed in a helical wheel diagram) and the Y's are independentiy one Ala on the polar face, one His, one Ser, one Gin one Asn, or one Thr on the polar face (e.g., when viewed in a helical wheel diagram), where no more than two K are be contiguous (e.g., when viewed in a helical wheel diagram); and where no more than 3 D's are contiguous (e.g., when viewed in a helical wheel diagram) and the fourth D is be separated from the other D's by a Y. Representative peptides of this kind which include peptides with histidine, and/or alpha- and/or beta-napthalanine are shown in Table 5. Reverse (retro-), inverse, retro-inverso-, and circularly permuted forms of these peptides are also contemplated.
Figure imgf000069_0002
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0002
[00211] Examples of class A 4F and Rev 4F analogs with beta-Nph are shown below.
Similarly, alpha-Nph analogs can be designed. Furthermore, His can be incorporated to Nph analogs. D>E analogs, E>D analogs and D-E switch analogs are additional possibilities..
Figure imgf000072_0001
[00212] As described above for 4Nph, a minimum of 7 additional analogs for each of the analogs given below.
Figure imgf000073_0001
[00213] For each of the analog described below, a minimum of 7 additional analogs are possible as described above by switching D-E, D>E and E>D and single D or E analogs.
Figure imgf000073_0002
[00214] For the analogs described below, additional analogs are possible by incorporating His or alpha-Nph and beta-Nph.
Figure imgf000074_0001
Figure imgf000075_0001
[00215] For each of the analogs below, additional H and Nph analogs are possible using the examples described above. Each analog can yield 7 analogs with the changes described above.
Rev3F-2
Figure imgf000075_0002
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
[00216] It is also noted that any of the peptides described herein can comprise non-natural amino acids in addition to or instead of the corresponding the natural amino acids identified herein. Such modifications include, but are not limited to acetylation, amidation, fbrmylation, methylation, sulfation, and the like. Illustrative non-natural amino acids include, but are not limited to Ornithine, norleucine, norvaline, N-methylvaline, 6-N-methyllysine, N- methylisoleucine, N-methylglycine, sarcosine, inosine, allo-isoleucine, isodesmolysine, 4- hydroxyproline, 3-hydroxyproline, allo-hydroxylysine, hydoxylisine, N-ethylasparagine, N- ethylglycine, 2,3-diaminopropionic acid, 2,2'-diaminopropionic acid, desmosine, 2,4- diaminobutyric acid, 2-aminopimelic acid, 3-aminoisobutyric acid, 2 -aminoisobutyric acid, 2- aminoheptanoic acid, 6-aminocaproic acid, 4-aminobutyric acid, 2-aminobutyric acid, beta- alanine, 3-aminoadipic acid, 2-aminoadipic acid, and the like. In certain embodiments and one or more of the natural amino acids of the peptides described herein, can be substituted with the corresponding non-natural amino acid (e.g., as described above).
[00217] In certain embodiments, this invention contemplates particularly the use of modified lysines. Such modifications include, but are not limited to, biotin modification of epsilon lysines and/or methylation of the epsilon lysines. Illustrative peptide comprising epsilon methylated lysines include, but are not limited to: Ac-D-W-F-K(eCH3)2-A-F-Y-D-K(eCH3)2-
Figure imgf000082_0001
Other modified amino acids include but are not limited to ornithine analogs and homoaminoalanine analogs (instead of for Haa
Figure imgf000082_0002
and for Om] and the like. It is noted that these modifications are illustrative
Figure imgf000082_0003
and not intended to be limiting. Illustrative 4F analogues that possess modified amino acids are shown in Table 6.
Figure imgf000082_0004
Figure imgf000083_0001
Figure imgf000084_0001
Single Domain Peptides
[00218] Disclosed are single-domain Apo E mimetics. The single-domain Apo E mimetics can comprise a receptor binding domain of Apo E or a lipid-associating peptide.
Dual Domain Peptides
[00219] Dual domain peptides are also disclosed. Dual domain peptides can be Apo E- mimicking peptides, comprising of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide.
[00220] Also disclosed are Apo E-mimicking peptides, comprising of a combination of the disclosed receptor binding domains of apolipoprotein B and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide. Non-limiting examples of the disclosed Apo E-mimicking peptides are provided in Table 7. The disclosed Apo E-mimicking peptides can also be N-terminally protected using acetyl and amino groups. Table 7 provides non-limiting representative examples of the disclosed Apo E-mimicking peptides comprising a dual domain.
Figure imgf000084_0002
Figure imgf000085_0001
Domain switched peptides
[00221] Also disclosed are Apo E mimetics, comprising of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a domain switched orientation. Also disclosed are Apo E mimetics, comprising of a combination of the disclosed receptor binding domains of apotipoprote in B and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a domain switched orientation. These peptides can be referred to as “domain switched” “switched domain”, or “switched” peptides. For example, disclosed are Apo E mimetics, consisting of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a domain switched orientation to those described above and in Table 7. Specifically, the lipid-associating peptide is covalently linked to the receptor binding domain of apolipoprotein E such that the lipid-associating peptide is at the N-terminus of the apolipoprotein E-mimicking peptide.
Table 8 provides non-limiting examples of the disclosed Apo E mimetics comprising a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a domain switched orientation.
Figure imgf000086_0001
[00222] In certain embodiments, the domain switched Apo E mimetics can also be N- terminally protected using acetyl and amino groups.
Peptides with reverse orientation
[00223] The LNP compositions disclosed herein comprise Apo E-mimicking peptides, comprising of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a reversed orientation. For example, disclosed are Apo E-mimicking peptides, comprising of a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein either the sequence of the receptor binding domain or the sequence of the lipid- associating peptide or both sequences are in the reversed orientation. Also disclosed are Apo
E-mimicking peptides, comprising of a combination of the disclosed receptor binding domains of apolipoprotein B and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a reversed orientation. Table 9 provides non-limiting examples of the disclosed Apo E-mimicking peptides comprising a combination of the disclosed receptor binding domains of apolipoprotein E and the disclosed lipid-associating peptides, wherein said receptor binding domain is covalently linked to said lipid-associating peptide in a reversed orientation.
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Scrambled peptides
[00224] The LNP compositions disclosed herein comprise Apo E-mimicking peptides, comprise of: a receptor binding domain of apolipoprotein E and a lipid-associating peptide, wherein said receptor binding domain is covalently linked to said lipid-associating peptide, wherein the receptor binding domain of apolipoprotein E is scrambled. For example, disclosed is a apolipoprotein E-mimicking peptide, comprising of: a receptor binding domain of apolipoprotein E comprising the amino acid sequence of D-W-L-K-A-F-V-Y-D-K-V-F- K-L-K-E-F-F (SEQ ID NO: 94); and a lipid-associating peptide, w'herein said receptor binding domain is covalently linked to said lipid-associating peptide. Also disclosed are Apo E-mimicking peptides, comprising of: a receptor binding domain of apolipoprotein B and a lipid-associating peptide, wherein said receptor binding domain is covalently linked to said lipid-associating peptide, wherein the receptor binding domain of apolipoprotein B is scrambled.
[00225] In certain embodiments, the Apo E-mimicking peptides comprise of: a receptor binding domain of apolipoprotein E and a lipid-associating peptide, wherein said receptor binding domain is covalently linked to said lipid-associating peptide, wherein the lipid- associating peptide is scrambled. For example, disclosed herein is a Apo E-mimicking peptides, comprising: a lipid binding domain of apolipoprotein E comprising the amino acid sequence of E-W-L-K-A-F-V-Y-E-K-V-F-K-L-K-E-F-F (SEQ ID NO: 95) and a receptor binding domain peptide, wherein said lipid binding domain is covalently linked to said receptor binding domain peptide.
[00226] In certain embodiments, the Apo E mimetics comprise of: a receptor binding domain of apolipoprotein E and a lipid-associating peptide of apolipoprotein E, wherein receptor binding domain is covalently linked to said lipid-associating peptide, wherein both the receptor binding domain and the lipid-associating peptide are scrambled. Table 10 provides non-limiting examples of the disclosed scrambled Apo E mimetics comprising a receptor binding domain of apolipoprotein E and a lipid-associating peptide, wherein said receptor binding domain is covalently linked to said lipid-associating peptide, wherein the receptor binding domain of apolipoprotein E is scrambled. Table 10: Scrambled Domain Peptides.
Name Receptor Binding Linid-Assodating Pentides Domains of Ano E hE-Scl8A (hE with Sc 18 A LRKLRKRLLR KAFEEVLAKKFYDKALWD also referred to (SEQ ID NO: 6) (SEQ ID NO: 666) as Sc2F)
LRLLRKLKRR DWLKAFYDKVAEKLKEAF
SchE-18A (SEQ ID NO: 665) (SEQ ID NO: 1)
[00227] The disclosed scrambled Apo E mimetics can also be N-terminally and C-terminally protected using acetyl and amide groups. The disclosed scrambled Apo E mimetics can also be reverse-oriented as described above.
Linkages
[00228] In certain embodiments, the ApoE-mimicking peptides may comprise a linker to connect the receptor binding domain of ApoE and the lipid-associating peptide together. Any suitable linker can be used in accordance with the present disclosure. The peptide linkages can be selected from the group consisting of
Figure imgf000090_0007
Figure imgf000090_0009
etc. by methods known
Figure imgf000090_0008
in the art and further described in the following references: Spatola (1983) p. 267 in Chemistry' and Biochemistry' of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York; Spatola (1983) Vega Data 1(3) Peptide Backbone Modifications, (general review); Morley (1980) Trends Pharm Sci pp. 463-468 (general review); Hudson et al. (1979) int J PeptProt Res 14: 177-185 Spatola et al. (1986) Life
Figure imgf000090_0006
Sci 38:1243-1249
Figure imgf000090_0005
Hann, (1982) J ChemSoc Perkin Trans 1 307-314
Figure imgf000090_0004
cis and trans); Almquist et al. (1980) J Med. Chem. 23: 1392-1398
Figure imgf000090_0003
Jennings- White et al. (1982) Tetrahedron Lett. 23:2533
Figure imgf000090_0011
Szelke et al., European Appln. EP 45665 (1982) CA: 97:39405 (1982 Holladay et al. (1983) Tetrahedron
Figure imgf000090_0010
Lett 24:4401-4404 and Hruby (1982) Life Sci., 31:189-199
Figure imgf000090_0002
Figure imgf000090_0012
[00229] One particularly preferred non-peptide linkage is
Figure imgf000090_0001
Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), reduced antigenicity, and others. [00230] In certain embodiments, the linker is a cleavable linker. To give but a few examples, cleavable linkers include protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, pH sensitive linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, x-ray cleavable tinkers, etc.
Variants
[00231 ] The receptor binding domain or the lipid-assodating peptide can be modified or altered as described above. For example, the receptor binding domain or the lipid-associating peptide can be mutated, scrambled, and/or reverse-oriented. Any other modifications or alterations disclosed herein for the dual-domain polypeptides can also be used for the single- domain peptides.
[00232] Numerous other variants or derivatives of the peptides disclosed herein are also contemplated. For example, scrambled peptides can also be reverse-oriented, or can be in a switched orientation. Additionally, reverse-oriented peptides can be in a switched orientation. All other combinations of the disclosed peptides are also contemplated. Non- limiting examples of the peptides have been described herein (see Tables 1-5, for example). As used herein, the term “analog” is used interchangeably with “variant” and “derivative.” Variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. Such, amino acid sequence modifications typically fall into one or more of three classes: substantial; insertional; or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily are smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final derivative or analog. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with Tables 11 and 12 and are referred to as conservative substitutions.
[00233] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 11, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties are those in which: (a) the hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; tryptophan, tyrosinyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or hystidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case; or (e) by increasing the number of sites for sulfation and/or glycosylation.
Figure imgf000092_0001
Figure imgf000093_0001
[00234] In certain embodiments, the Apo E mimetics and other proteins or peptides herein disclosed which have at least, 70% or at least 75% or at least 80% or at least 85% or at least 90% or at least 95% homology to the Apo E mimetics specifically recited herein. Those of skill in the art readily understand how to determine the homology of two proteins.
[00235] The polypeptides can be modified by either natural processes, such as post- translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins - Structure and Molecular Properties 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).
[00236] Variants can also include peptidomimetics. Peptidomimetics typically are short sequences of amino acids that in biological properties mimic one or more fimction(s) of a particular protein. Peptide analogs enhance some property of the original peptide, such as increases stability, increased efficacy, enhanced delivery-, increased half-life, etc. Methods of making peptidomimetics based upon a known polypeptide sequence is described, for example, in U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. The present disclosure encompasses peptidomimetics having a bond, a peptide backbone, or an amino acid component replaced with a suitable mimic. Some non-limiting examples of unnatural amino acids which may be suitable amino acid mimics include
Figure imgf000094_0002
0 alanine, L -amino butyric acid, L
Figure imgf000094_0004
amino butyric acid, L-
Figure imgf000094_0005
amino isobutyric acid, L
Figure imgf000094_0003
amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N- Boc-N-
Figure imgf000094_0001
Figure imgf000094_0006
CBZ-L-lysine, N-
Figure imgf000095_0001
-Boc-N-a-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L- norvaline, N-a-Boc-N-
Figure imgf000095_0002
-L-omithine, N- -Boc-N-a-CBZ-L-omithine, Boc-p-nitro-L-
Figure imgf000095_0003
phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.
Nucleic Acids
[00237] The nucleic acids that can encode the polypeptide sequences described herein are also disclosed. This would include all degenerate sequences related to a specific polypeptide sequence, i.e. all nucleic acids having a sequence that encodes one particular polypeptide sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in feet disclosed and described herein through the disclosed polypeptide sequences.
Blocking/Protecting Groups and D Residues
[00238] The LNP compositions provided herein can comprise an acetyl group followed by a protecting group. The protecting group can be, but is not limited to, a fatty acid. The fatty acids can be saturated, unsaturated or essential fetty acids. Fatty acids can include but are not limited to DHA, EPA, linoleic acid, or any other saturated amino acid such as myristic acid. [00239] In certain embodiments, the LNP compositions provided herein can bear one, two, three, four, or more protecting groups. The protecting groups can be coupled to the C- and/or N-terminus of the peptide(s) and/or to one or more internal residues comprising the peptide(s) (e.g., one or more R-groups on the constituent amino acids can be blocked). Thus, for example, in certain embodiments, any of the peptides described herein can bear, e.g., an acetyl group protecting the amino terminus and/or an amide group protecting the carboxyl terminus. One example of such a “dual protected peptide” is Ac- LRKLRKRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 10) with blocking groups), either or both of these protecting groups can be eliminated and/or substituted with another protecting group as described herein. Without being bound by a particular theory, it was a discovery of this invention that blockage, particularly of the amino and/or carboxyl termini of the subject peptides of this invention can improve oral deliver}' and can also increase serum half-life.
[00240] A wide number of protecting groups are suitable for this purpose. Such groups include, but are not limited to acetyl, amide, and alkyl groups with acetyl and alkyl groups being particularly preferred for N-terminal protection and amide groups being preferred for carboxyl terminal protection. For example, the protecting groups can include, but are not limited to alkyl chains as in fatty acids, propeonyl, formyl, and others. Carboxyl protecting groups include amides, esters, and ether-forming protecting groups can also be used. For example, an acetyl group can be used to protect the amino terminus and an amide group can be used to protect the carboxyl terminus. These blocking groups enhance the helix-forming tendencies of the peptides. Additional blocking groups include alkyl groups of various lengths, e.g., groups having the formula:
Figure imgf000096_0001
where n ranges from about 1 to about 20, preferably from about 1 to about 16 or 18, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
[00241] Additionally, the protecting groups include, but are not limited to alkyl chains as in fatty7 acids, propeonyl, formyl, and others. For example, carboxyl protecting groups can include amides, esters, and ether-forming protecting groups. These blocking groups can enhance the helix-forming tendencies of the peptides. Blocking groups can include alkyl groups of various lengths, e.g., groups having the formula:
Figure imgf000096_0002
where n ranges from about 3 to about 20, preferably from about 3 to about 16, more preferably from about 3 to about 13, and most preferably from about 3 to about 10.
[00242] Other protecting groups include, but are not limited to Fmoc, t-butoxycarbonyl (t- BOC), 9-fhioreneacetyl group, 1 -fluorenecarboxylic group, 9-florenecaiboxylic group, 9- fluorenone-1 -carboxylic group, benzyloxycarbonyl, xanthyl (Xan), trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), tosyl (Tos), 2,2,5 ,7,8- pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), benzyloxy' (BzlO), benzyl (Bzl), benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), l-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6- DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-C1-Z), 2-bromobenzyloxy-carbonyd (2-Br-Z), benzyloxymethyl (Bom), cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t- butyl (tBu), acetyl (Ac), and trifluoroacetyl (TFA).
[00243] Protecting/blocking groups are well known to those of skill as are methods of coupling such groups to the appropriate residue(s) comprising the peptides of this invention (see, e.g., Greene etal., (1991) Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc. Somerset, N.J.). For example, acetylation can be accomplished during the synthesis when the peptide is on the resin using acetic anhydride. Amide protection can be achieved by the selection of a proper resin for the synthesis.
[00244] The compositions disclosed herein can also comprise one or more D-form (dextro rather than levo) amino acids as described herein. For example, at least two enantiomeric amino acids, at least 4 enantiomeric amino acids or at least 8 or 10 enantiomeric amino acids can be in the “D” form amino acids. Additionally, every' other, or even every amino acid (e.g., every enantiomeric amino acid) of the peptides described herein is a D-form amino acid.
[00245] Additionally, at least 50% of the enantiomeric amino acids can be “D” form, at least 80% of the enantiomeric amino acids are “D” form, at least 90%, or even all of the enantiomeric amino acids can be in the “D” form amino acids.
[00246] FMOC-Aha can be added to the growing chain as the last amino acid using the normal amino acid chain extension procedure (use of HOBt+DCC or HBTU as condensing agents). After the removal of the FMOC group using 20% piperidine in DMF, the NH2 can be acetylated using either excess of acetic anhydride under basic conditions or by condensing acetic acid using amino acid condensing agents used for peptide chain elongation.
Pharmaceutical Compositions
[00247] In certain aspects, provided herein are pharmaceutical compositions comprising a lipid nanoparticle (LNP) comprising one or more lipids and an apolipoprotein E (ApoE)- mimicking peptide, wherein the ApoE-mimicking peptide comprises a receptor-binding domain of ApoE and a lipid-associating domain.
[00248] In certain embodiments, the compositions and methods provided herein can be utilized to treat a subject in need thereof. In certain embodiments, the subject is a mammal such as a human, or a non-human mammal. When administered to a subject, such as a human, the composition is preferably administered as a pharmaceutical composition and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In certain embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery- system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop.
[00249] In certain embodiments, the pharmaceutical compositions provided herein comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a composition. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug deliverysystem or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a therapeutic compound. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
[00250] In certain embodiments, the pharmaceutical compositions provided herein can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The composition may also be formulated for inhalation. In certain embodiments, a composition may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973; 5,763,493; 5,731,000; 5,541,231; 5,427,798; 5,358,970; and 4,172,896, as well as in patents cited therein.
[00251] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
[00252] Methods of preparing these formulations or compositions include the step of bringing into association an active composition with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a composition with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
[00253] Formulations suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water- in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a composition as an active ingredient. Compositions may also be administered as a bolus, electuary or paste.
[00254] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the tike), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
[00255] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered composition moistened with an inert liquid diluent. [00256] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredients) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients. [00257] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
[00258] Besides inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfinning, and preservative agents.
[00259] Suspensions, in addition to the active compositions, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
[00260] Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active composition with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active composition.
[00261] Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.
[00262] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
[00263] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active composition may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
[00264] The ointments, pastes, creams and gels may contain, in addition to an active composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
[00265] Powders and sprays can contain, in addition to an active composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary' propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
[00266] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compositions in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
[00267] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
[00268] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. [00269] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
[00270] Injectable depot forms are made by forming microencapsulated matrices of the subject compositionsin biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drag to polymer, and the nature of the particular polymer employed, the rate of drag release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drag in liposomes or microemulsions that are compatible with body tissue.
[00271] In certain embodiments, active compositionscan be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
[00272] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery' of drags, including proteinacious biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compositional a particular target site.
[00273] In certain embodiments, compounds can be used alone or conjointly administered with another type of therapeutic agent (e.g., an antiviral agent).
[00274] In certain embodiments, conjoint administration of therapeutic compositions with one or more additional therapeutic agent(s) (e.g., one or more additional chemotherapeutic agent(s)) provides improved efficacy relative to each individual administration of the composition or the one or more additional therapeutic agent(s). In certain such embodiments, the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the therapeutic composition and the one or more additional therapeutic agent(s). [00275] Pharmaceutically acceptable salts of compositionin the methods provided herein. In certain embodiments, contemplated salts include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts include, but are not limited to, Na, Ca, K, Mg, Zn, copper, cobalt, cadmium, manganese, or other metal salts.
[00276] In certain embodiments, the compositions described herein may comprise wetting agents, emulsifiers and/or lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants.
[00277] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfete, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Dosing
[00278] A therapeutically effective dose of a composition comprising a lipid nanoparticle (LNP) comprising one or more lipids and an apolipoprotein E (ApoE)-mimicking peptide is about 0.01 mg/kg to about 500 mg/kg, about 0.05 mg/kg to about 250 mg/kg, about 0.1 mg/kg to about 100 mg/kg, about 0.5 mg/kg to about 50 mg/kg, or about 1 mg/kg to about 25 mg/kg.
[00279] A therapeutically effective dose of a composition comprising a lipid nanoparticle (LNP) comprising one or more lipids and an apolipoprotein E (ApoE)-mimicking peptide is about 0.01 mg/kg to about 500 mg/kg. In some embodiments, the therapeutically effective dose of a composition comprising a lipid nanoparticle (LNP) is preferably about 0.01 mg/kg to 100 mg/kg. [00280] The lipid nanoparticle can be administered, for example, once daily, twice daily, once every two weeks, or once monthly. The duration of administration of the lipid nanoparticle can be dependent on the severity of a disease or the stage of disease a subject is afflicted with. For example, the lipid nanoparticle can be administered for at least 1 or 2 weeks. Longer durations of administration are also contemplated.
[00281] Actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
[00282] If desired, the effective daily dose of the active compositioncan be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments, the active compositionis administered two or three times daily. In some embodiments, the active compositionis administered once daily.
Methods of treating disease
[00283] In certain aspects, the compositions and methods provided herein are useful for the treatment or prevention of a disease, disorder, or condition.
Immune Disorders
[00284] In some embodiments, the compositions and methods described herein relate to the treatment or prevention of a disease or disorder associated a pathological immune response, such as an autoimmune disease, an allergic reaction and/or an inflammatory disease. In some embodiments, the disease or disorder is an inflammatory bowel disease (e.g., Crohn’s disease or ulcerative colitis). In some embodiments, the disease or disorder is psoriasis. In some embodiments, the disease or disorder is atopic dermatitis.
[00285] The compositions and methods described herein can be used to treat any subject in need thereof.
[00286] The pharmaceutical compositions described herein can be used, for example, as a pharmaceutical composition for preventing or treating an autoimmune disease, such as chronic inflammatory bowel disease, systemic lupus erythematosus, psoriasis, muckle-wells syndrome, rheumatoid arthritis, multiple sclerosis, or Hashimoto's disease; an allergic disease, such as a food allergy, pollenosis, or asthma; an infectious disease, such as an infection with Clostridium difficile; an inflammatory' disease such as a TNF-mediated inflammatory disease (e.g., an inflammatory disease of the gastrointestinal tract, such as pouchitis, a cardiovascular inflammatory condition, such as atherosclerosis, or an inflammatory lung disease, such as chronic obstructive pulmonary disease, fibrotic disease, or cystic fibrosis); a pharmaceutical composition for suppressing rejection in organ transplantation or other situations in which tissue rejection might occur; a supplement, food, or beverage for improving immune functions; or a reagent for suppressing the proliferation or function of immune cells.
[00287] In some embodiments, the compositions and methods provided herein are usefill for the treatment of inflammation. In certain embodiments, the pharmaceutical compositions described herein can be used for preventing or treating inflammation of any tissue and organs of the body, including musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, ocular inflammation, inflammation of the reproductive system, and other inflammation, as discussed below.
[00288] Immune disorders of the musculoskeletal system include, but are not limited, to those conditions affecting skeletal joints, including joints of the hand, wrist, elbow, shoulder, jaw, spine, neck, hip, knew, ankle, and foot, and conditions affecting tissues connecting muscles to bones such as tendons. Examples of such immune disorders, which may be treated with the compositions and methods described herein include, but are not limited to, arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendonitis, synovitis, tenosynovitis, bursitis, fibrositis (fibromyalgia), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, osteitis pubis, and osteitis fibrosa cystic).
[00289] Ocular immune disorders refers to an immune disorder that affects any structure of the eye, including the eye lids. Examples of ocular immune disorders which may be treated with the compositions and methods described herein include, but are not limited to, blepharitis, blepharochalasis, conjunctivitis, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis.
[00290] Examples of nervous system immune disorders which may be treated with the compositions and methods described herein include, but are not limited to, encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis and schizophrenia. Examples of inflammation of the vasculature or lymphatic system which may be treated with the compositions and methods described herein include, but are not limited to, arthrosclerosis, arthritis, phlebitis, vasculitis, and lymphangitis.
[00291] Examples of digestive system immune disorders which may be treated with the compositions and methods described herein include, but are not limited to, cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease, ileitis, and proctitis. Inflammatory bowel diseases include, for example, certain art- recognized forms of a group of related conditions. Several major forms of inflammatory bowel diseases are known, with Crohn's disease (regional bowel disease, e.g., inactive and active forms) and ulcerative colitis (e.g., inactive and active forms) the most common of these disorders. In addition, the inflammatory' bowel disease encompasses irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis. Other less common forms of IBD include indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet’s disease, sarcoidosis, scleroderma, IBD- associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis.
[00292] Examples of reproductive system immune disorders which may be treated with the compositions and methods described herein include, but are not limited to, cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis, and vulvodynia.
[00293] The compositions and methods described herein may be used to treat autoimmune conditions having an inflammatory' component. Such conditions include, but are not limited to, acute disseminated alopecia universalise, Behcet's disease, Chagas' disease, chronic fatigue syndrome, dysautonomia, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, diabetes mellitus type 1, giant cell arteritis, goodpasture's syndrome. Grave's disease, Guillain-Barre syndrome, Hashimoto's disease, Henoch-Schonlein purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, Muckle-Wells syndrome, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, ord's thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjogren's syndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmune haemolytic anemia, interstitial cystitis, Lyme disease, morphea, psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.
[00294] The compositions and methods described herein may be used to treat T-cell mediated hypersensitivity diseases having an inflammatory component. Such conditions include, but are not limited to, contact hypersensitivity, contact dermatitis (including that due to poison ivy), uticaria, skin allergies, respiratory allergies (hay fever, allergic rhinitis, house dustmite allergy) and gluten-sensitive enteropathy (Celiac disease).
[00295] Other immune disorders which may be treated with the compositions and methods described herein include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, percarditis, peritonoitis, pharyngitis, pleuritis, pneumonitis, prostatistis, pyelonephritis, and stomatisi, transplant rejection (involving organs such as kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel, skin allografts, skin homografts, and heart valve xengrafts, sewrum sickness, and graft vs host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sexary's syndrome, congenital adrenal hyperplasis, nonsuppurative thyroiditis, hypercalcemia associated with cancer, pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensistivity reactions, allergic conjunctivitis, keratitis, herpes zoster ophthalmicus, iritis and oiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis, fulminating or disseminated pulmonary tuberculosis chemotherapy, idiopathic thrombocytopenic purpura in adults, secondary thrombocytopenia in adults, acquired (autoimmune) haemolytic anemia, leukaemia and lymphomas in adults, acute leukaemia of childhood, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, solid organ transplant rejection, sepsis. Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, Type 1 diabetes, asthma, inflammatory' bowel disease, systemic lupus erythematosus, psoriasis, chronic obstructive pulmonary disease, and inflammation accompanying infectious conditions (e.g., sepsis).
Metabolic Disorders
[00296] In some embodiments, the compositions and methods described herein relate to the treatment or prevention of a metabolic disease or disorder a, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), Nonalcoholic Steatohepatitis (NASH) or a related disease. In some embodiments, the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema. In some embodiments, the compositions and methods described herein relate to the treatment of Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH). [00297] The compositions and methods described herein can be used to treat any subject in need thereof.
Cardiovascular Disease
[00298] In some embodiments, the compositions and methods described herein relate to the treatment of hypertension, coronal}' artery disease, stroke, cardiovascular disease, peripheral artery- disease, cerebral vascular disease, diabetes-derived cardiovascular diseases, or congestive heart failure.
[00299] In some embodiments, the compositions described herein relate to the treatment of heart diseases, vascular diseases and/or cardiovascular diseases or disease of the cardiovascular system, e.g., acute and chronic heart failure, arterial hypertension, coronary heart disease, stable and instable angina pectoris, myocardial ischemia, myocardial infarction, coronary microvascular dysfunction, microvascular obstruction, no-reflow-phenomenon, shock, atherosclerosis, coronary artery disease, peripheral artery disease, peripheral arterial disease, intermittent claudication, severe intermittent claudication, limb ischemia, critical limb ischemia, hypertrophy of the heart, cardiomyopathies of any etiology (such as, e.g., dilatative cardiomyopathy, restrictive cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy), fibrosis of the heart, atrial and ventricular arrhythmias, transitory and/or ischemic attacks, apoplexy, ischemic and/or hemorrhag ic stroke, preeclampsia, inflammatory cardiovascular diseases, metabolic diseases, diabetes, type-I- diabetes, type-11- diabetes, diabetes mellitus, peripheral and autonomic neuropathies, diabetic neuropathies, diabetic microangiopathies, diabetic retinopathy, diabetic ulcera at the extremities, gangrene, CREST-syndrome, hypercholesterolemia, hypertriglyceridemia, lipometabolic disorder, metabolic syndrome, increased levels of fibrinogen and low-density lipoproteins (i.e. LDL), increased concentrations of plasminogen-activator inhibitor 1 (PAI-1), as well as peripheral vascular and cardiac vascular diseases, peripheral circulatory disorders, primary' and secondary Raynaud syndrome, disturbances of the microcirculation, arterial pulmonary' hypertension, spasms of coronary and peripheral arteries, thromboses, thromboembolic diseases, edema- formation, such as pulmonary edema, brain-edema, renal edema, myocardial edema, myocardial edema associated with heart failure, restenosis after i.e. thrombolytic therapies, percutaneous-transluminal angioplasties (PTA), transluminal coronary angioplasties (PTCA), heart transplantations, bypass-surgeries as well as micro- and macrovascular injuries (e.g., vasculitis), reperfusion-damage, arterial and venous thromboses, microalbuminuria, cardiac insufficiency, endothelial dysfunction. In the light of the present disclosure, heart failure includes more specific or related kinds of diseases such as acute decompensated heart failure, right heart feilure, left heart failure, global insufficiency, ischemic cardiomyopathy, dilataiive cardiomyopathy, congenital heart defects), valve diseases, heart feilure related to valve diseases, mitral valve stenosis, mitral valve insufficiency', aortic valve stenosis, aortic valve insufficiency, tricuspid valve stenosis, tricuspid valve insufficiency, pulmonary valve stenosis, pulmonary valve insufficiency, combined valvular defects, inflammation of the heart muscle (myocarditis), chronic myocarditis, acute myocarditis, viral myocarditis, bacterial myocarditis, diabetic heart feilure, alcohol-toxic cardiomyopathy, cardiac storage diseases, heart feilure with preserved ejection fraction (HFpEF), diastolic heart feilure, heart feilure with reduced ejection fraction (HFrEF), systolic heart failure. In the context of the present disclosure, the terms atrial arrhythmias and ventricular arrhythmias also include more specific and related disease-entitites, such as: Atrial fibrillation, paroxysmal atrial fibrillation, intermittent atrial fibrillation, persistent atrial fibrillation, permanent atrial fibrillation, atrial flutter, sinus arrhythmia, sinus tachycardia, passive heterotopy, active heterotopy, replacement systoles, extrasystoles, disturbances in the conduction of impulses, sick-sinus syndrome, hypersensitive carotis-sinus, tachycardias, AV- node re-entry tachycardias, atrioventricular re-entry tachycardia, WPW-syndrome (Wolff - Parkinson-White syndrome), Mahaim-tachycardia, hidden accessory' pathways/tracts, permanent junctional re-entry tachycardia, focal atrial tachycardia, junctional ectopic tachycardia, atrial re-entry tachycardia, ventricular tachycardia, ventricular flutter, ventricular fibrillation, sudden cardiac death. In the context of the present disclosure, the term coronary heart disease also include more specific or related diseases entities, such as: Ischemic heart disease, stable angina pectoris, acute coronary syndrome, instable angina pectoris, NSTEMI (non-ST-segement-elevation myocardial infarction), STEM1 (ST-segement-elevation myocardial infarction), ischemic damage of the heart, arrhythmias, and myocardial infarction.
Cancer
[00300] In some embodiments, the compositions and methods described herein relate to the treatment of cancer. In some embodiments, any cancer can be treated using the methods described herein. Examples of cancers that may treated by compositions and methods described herein include, but are not limited to, cancer cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be one of the following histological types, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coh; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory' carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory- neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fimgoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; or hairy cell leukemia.
[00301] In some embodiments, the cancer comprises breast cancer (e.g., triple negative breast cancer).
[00302] In some embodiments, the cancer comprises colorectal cancer (e g., microsatellite stable (MSS) colorectal cancer).
[00303] In some embodiments, the cancer comprises renal cell carcinoma.
[00304] In some embodiments, the cancer comprises lung cancer (e.g., non-small cell lung cancer).
[00305] In some embodiments, the cancer comprises bladder cancer.
[00306] In some embodiments, the cancer comprises gastroesophageal cancer. [00307] In some embodiments, the compositions and methods provided herein relate to the treatment of a leukemia. The term "leukemia" includes broadly progressive, malignant diseases of the hematopoietic organs/systems and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Non-limiting examples of leukemia diseases include acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia. Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, undifferentiated cell leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, and promyelocytic leukemia.
[00308] In some embodiments, the compositions and methods provided herein relate to the treatment of a carcinoma. The term "carcinoma" refers to a malignant growth made up of epithelial cells tending to infiltrate the surrounding tissues, and/or resist physiological and non-physiological cell death signals and gives rise to metastases. Non-limiting exemplary types of carcinomas include, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infentile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, and carcinoma scroti.
[00309] In some embodiments, the compositions and methods provided herein relate to the treatment of a sarcoma. The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance. Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing' s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
[00310] Additional exemplary neoplasias that can be treated using the compositions and methods described herein include Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary' tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, plasmacytoma, colorectal cancer, rectal cancer, and adrenal cortical cancer.
[00311] In some embodiments, the cancer treated is a melanoma. The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Non-limiting examples of melanomas are Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
[00312] Particular categories of tumors that can be treated using compositions and methods described herein include lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, kidney cancer, as well as metastases of all the above. Particular types of tumors include hepatocellular carcinoma, hepatoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, pulmonary squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), bronchioloalveolar carcinoma, renal cell carcinoma, hypernephroma, hypemephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma including small cell, non-small and large cell lung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma, rectal carcinoma, hematopoietic malignancies including all types of leukemia and lymphoma including: acute myelogenous leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin* s lymphoma, non-Hodgkin* s lymphoma, plasmacytoma, colorectal cancer, and rectal cancer.
[00313] Cancers treated in certain embodiments also include precancerous lesions, e.g., actinic keratosis (solar keratosis), moles (dysplastic nevi), acitinic chelitis (farmer's lip), cutaneous horns, Barrett's esophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, actinic (solar) elastosis and cervical dysplasia.
[00314] Tumors treated in some embodiments include non-cancerous or benign tumors, e.g., of endodermal, ectodermal or mesenchymal origin, including, but not limited to cholangioma, colonic polyp, adenoma, papilloma, cystadenoma, liver cell adenoma, hydatidiform mole, renal tubular adenoma, squamous cell papilloma, gastric polyp, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, nevus, meningioma, and ganglioneuroma.
Liver Disease
[00315] In some embodiments, the compositions and methods described herein relate to the treatment of liver diseases. Such diseases include, but are not limited to, Alagille Syndrome, Alcohol-Related Liver Disease, Alpha- 1 Antitrypsin Deficiency, Autoimmune Hepatitis, Benign Liver Tumors, Biliary Atresia, Cirrhosis, Galactosemia, Gilbert Syndrome, Hemochromatosis, Hepatitis A, Hepatitis B, Hepatitis C, Hepatic Encephalopathy, Intrahepatic Cholestasis of Pregnancy (ICP), Lysosomal Acid Lipase Deficiency (LAL-D), Liver Cysts, Liver Cancer, Newborn Jaundice, Primary Biliary Cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Reye Syndrome, Type I Glycogen Storage Disease, and Wilson Disease.
Neurodegenerative Disease
[00316] The compositions and methods and/or solid dosage forms described herein may be used to treat neurodegenerative and neurological diseases. In certain embodiments, the neurodegenerative and/or neurological disease is Parkinson’s disease, Alzheimer’s disease, prion disease, Huntington’s disease, macular degeneration, motor neuron diseases (MND), spinocerebellar ataxia, spinal muscular atrophy, dystonia, idiopathicintracranial hypertension, epilepsy, nervous system disease, central nervous system disease, movement disorders, multiple sclerosis, encephalopathy, peripheral neuropathy or post-operative cognitive dysfunction. Delivery and internalization of LNPs by cells
[00317] In certain aspects, the compositions and methods provided herein are usefol for the delivery and internalization of LNPs by cells.
[00318] In some embodiments, the compositions and methods described herein are useful for delivering cargo (e.g., a biologically active agent) to liver cells (e.g., a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a liver sinusoidal cell) or to a cancer cell or a tumor cell (e.g., a primary tumor or metastatic cancer cells). In certain embodiments, the compositions and methods are usefol for delivering cargo (e.g., a biologically active agent) to the skin, adipose, muscle, or lymph nodes (e.g., by subcutaneous dosing). In other embodiments, the compositions and methods are usefol for delivering cargo (e.g., a biologically active agent) to a brain cell, splenic cell (e.g., a splenocytes), ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell, a skeletal muscle cell, a non-immune cell, T-cell, a stem cell(e.g., a hematopoietic cell), a lung cell, or a kidney cell.
[00319] In some embodiments, the liver or liver cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, portal vein injection, catheterization, stenting) to facilitate delivery of cargo (e.g., a biologically active agent).
[00320] In certain embodiments, the kidney or kidney cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stenting) to facilitate delivery of cargo (e.g., a biologically active agent).
[00321] In some embodiments, the tumor or tumor cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stenting), to facilitate delivery of cargo (e.g., a biologically active agent).
[00322] In certain embodiments, the CNS or CNS cells (e.g., brain cells and/or spinal cord cells) are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheterization, stenting, osmotic pump administration (e.g., intrathecal or ventricular)), to facilitate delivery of cargo (e.g., a biologically active agent).
[00323] In some embodiments, the PNS or PNS cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection), to facilitate delivery of cargo (e.g., a biologically active agent).
[00324] In certain embodiments, the lung or lung cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., pulmonary administration directly to lung tissues and cells), to facilitate delivery' of cargo (e.g., a biologically active agent).
[00325] In some embodiments, the vasculature or vascular cells are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., clamping, catheterization, stenting), to facilitate delivery of cargo (e.g., a biologically active agent).
[00326] In certain embodiments, the skin or skin cells (e.g., dermis cells and/or follicular cells) are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct dermal application, iontophoresis), to facilitate delivery of cargo (e.g., a biologically active agent).
[00327] In some embodiments, the eye or ocular cells (e.g., macula, fovea, cornea, retina) are contacted with the composition described herein via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eyedrops, implants), to facilitate delivery' of cargo (e.g., a biologically active agent).
[00328] In certain embodiments, the ear or cells of the ear (e.g., cells of the inner ear, middle ear and/or outer ear) are contacted with the composition described herein as is generally known in the art, such as via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection), to facilitate delivery-.
[00329] In some embodiments, cells of the immune system (e.g., antigen-presenting cells, including professional antigen presenting cells) are contacted with the composition described herein intramuscularly, after which immune cells can infiltrate the delivery site and process delivered RNA and/or process encoded antigen produced by non-immune cells, such as muscle cells. Such immune cells can include macrophages (e.g., bone marrow derived macrophages), dendritic cells (e.g., bone marrow derived plasmacytoid dendritic cells and/or bone marrow derived myeloid dendritic cells), monocytes (e.g., human peripheral blood monocytes), etc. (for example, WO2012/006372, incorporated herein by reference in its entirety.
Incorporation by Reference
[00330] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Equivalents
[00331] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS We claim:
1. A lipid nanoparticle (LNP) comprising one or more lipids and an apolipoprotein E (ApoE)-mimicking peptide, wherein the ApoE-mimicking peptide comprises a receptor- binding domain of ApoE and a lipid-associating domain.
2. The LNP of claim 1, wherein the LNP is an ionizable LNP.
3. The LNP of claim 1 or 2, wherein the one or more lipids comprise an ionizable lipid.
4. The LNP of claim 3, wherein the ionizable lipid comprises an amino group.
5. The LNP of claim 4, wherein the amino group is an amine head group.
6. The LNP of any one of claims 3-5, wherein the ionizable lipid comprises two or more aliphatic tail groups.
7. The LNP of claim 6, wherein the ionizable lipid comprises two to four aliphatic tail groups.
8. The LNP of claim 6 or 7, wherein the aliphatic tail groups are hydrophobic tail groups.
9. The LNP of any one of claims 6-8, wherein the aliphatic tail groups are each independently alkyl or alkenyl groups.
10. The LNP of any one of claims 3-9, wherein the ionizable lipid is a cationic lipid.
11. The LNP of any one of claims 3-9, wherein the ionizable lipid is a neutral lipid.
12. The LNP of any one of claims 3-11, wherein the ionizable lipid is a zwitterionic lipid.
13. The LNP of any one of claims 3-12, wherein the ionizable lipid is protonated physiological pH.
14. The LNP of any one of the preceding claims, wherein the one or more lipids comprise a structural lipid.
15. The LNP of claim 14, wherein the structural lipid is a steroid.
16. The LNP of claim 15, wherein the steroid is a sterol.
17. The LNP of claim 16, wherein the sterol is cholesterol.
18. The LNP of any one of the preceding claims, wherein the one or more lipids comprise a non-cationic helper lipid.
19. The LNP of claim 18, wherein the non-cationic helper lipid is a phospholipid.
20. The LNP of claim 18 or 19, wherein the non-cationic helper lipid comprises a diacylglyerol group.
21. The LNP of any one of claims 18-20, wherein the non-cationic helper lipid is a glycerophospholipid.
22. The LNP of any one of claims 18-21, wherein the non-cationic helper lipid is a phosphatidylcholine.
23. The LNP of any one of claims 18-21, wherein the non-cationic helper lipid is not a phosphatidylcholine.
24. The LNP of claim 18, wherein the phospholipid is distearoylphosphatidylcholine (DSPC).
25. The LNP of any one of the preceding claims, wherein the one or more lipids comprise a PEG-lipid comprising a lipid group covalently bonded to a PEG group.
26. The LNP of claim 25, wherein the lipid group is a phospholipid.
27. The LNP of claim 25 or 26, wherein the lipid group comprises a diacylglycerol, dialkylglycerol, or dialkylamine group.
28. The LNP of any one of claims 25-27, wherein the lipid group is a glycerophospholipid group.
29. The LNP of claim 25, wherein the lipid group is dimyristoyl glycerol.
30. The LNP of any one of the preceding claims, wherein the lipid-associating domain comprises a class A amphipathic-helical domain.
31. The LNP of claim 30, wherein the class A amphipathic-helical domain is DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1), DWLRAFYDKVAEKLREAF (SEQ ID NO: 2), DWLRALYDKVAEKLREAL (SEQ ID NO: 3), DLLRALYDKVAEKLREAW (SEQ ID NO: 4), or FAEKLKEAVKDYFAKLWD (SEQ ID NO: 5).
32. The LNP of claim 30, wherein the class A amphipathic-helical domain is DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1).
33. The LNP of any one of the preceding claims, wherein the receptor-binding domain of ApoE is capable of binding to an LDL receptor.
34. The LNP of any one of the preceding claims, wherein the receptor-binding domain of ApoE is LRKLRKRLLR (SEQ ID NO: 6), LRRLRRRLLR (SEQ ID NO: 7), LRKMRKRLMR (SEQ ID NO: 8), or RLTRKRGLK (SEQ ID NO: 9).
35. The LNP of claim 34, wherein the receptor-binding domain of ApoE is
LRKLRKRLLR (SEQ ID NO: 6) or LRRLRRRLLR (SEQ ID NO: 7).
36. The LNP of claim 34, wherein the receptor-binding domain of ApoE is LRRLRRRLLR (SEQ ID NO: 7).
37. The LNP of any one of the preceding claims, wherein the receptor-binding domain of ApoE is covalently bonded to the lipid-associating domain.
38. The LNP of claim 37, wherein the C-terminus of the receptor-binding domain of ApoE is covalently bonded to the N-terminus of the lipid-associating domain.
39. The LNP of any one of the preceding claims, wherein an amino group is bonded to the C-terminus of the lipid-associating domain.
40. The LNP of any one of the preceding claims, wherein an acyl group is bonded to the N-terminus of the receptor-binding domain of ApoE.
41. The LNP of any one of the preceding claims, wherein the ApoE-mimicking peptide comprises a fatty' acid moiety.
42. The LNP of any one of claims 1-39, wherein the ApoE-mimicking peptide comprises a fatty acid moiety, and the fatty acid moiety is bonded to the N -terminus of the receptor- binding domain of ApoE.
43. The LNP of claim 41 or 42, wherein the fatty acid moiety comprises a chain of 4 to 20 carbon atoms.
44. The LNP of any one of claims 41-43, wherein the fatty acid moiety' is a saturated fatty' acid moiety'.
45. The LNP of any one of claims 41-43, wherein the fatty acid moiety comprises one, two, or three alkene groups.
46. The LNP of claim 41 or 42, wherein the fatty acid moiety is butanoyl, caproyl, octanoyl, decanoyl, lauroyl, myristoyl, myristoleoyl, palmitoyl, stearoyl, oleoyl, palmitoleoyl, linoleoyl, linolenoyl, or arachidonoyl.
47. The LNP of claim 46, wherein the fatty acid moiety is octanoyl, myristoyl, palmitoyl, or oleoyl.
48. The LNP of any one of claims 41-45, wherein the fatty acid moiety' comprises an co- amino group.
49. The LNP of claim 48, w'herein the fatty acid moiety is 4-amino-butanoyl, 6-amino- caproyl, 8-amino-octanoyl, 10-amino-decanoyl, 12-amino-lauroyl, 14-amino-myristoyL 14- amino-myristoleoyl, 16 -amino-palmitoyl, 18-amino-stearoyl, 18 -amino -oleoyl, 16-amino- palmitoleoyl, 18-amino-linoleoyl, 18-amino-linolenoyl, or 20-amino-arachidonoyl.
50. The LNP of claim 48 or 49, wherein the co-amino group comprises an acyl group.
51. The LNP of claim 50, wherein the fatty acid moiety is Ac-Aha.
52. The LNP of any one of claims 1-29, wherein the ApoE-mimicking peptide is Ac- LRKLRKRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 10); octanoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 11); myristoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 12); palmitoyl-
LRRLRRRLLRDWLKAFYDKVAEKLKEAF-NH2 (SEQ ID NO: 13); or oleoyl- LRRLRRRLLRDWLKAFYDKVAEKLKEAF (SEQ ID NO: 14).
Figure imgf000124_0001
53. The LNP of any one of claims 1-29, wherein the ApoE-mimicking peptide is Ac-Aha- or Ac-Aha-[R]hEl 8A-NH2.
Figure imgf000124_0002
54. The LNP of any one of the preceding claims, further comprising a payload.
55. The LNP of claim 54, wherein the payload comprises a drug.
56. The LNP of claim 54 or 55, wherein the payload comprises a peptide, polypeptide, protein, or nucleic acid.
57. The LNP of claim 56, wherein the payload comprises a nucleic acid selected from RNA, mRNA, dsRNA, siRNA, antisense RNA, ribozyme, CRISPR/Cas9, ssDNA, and DNA.
58. The LNP of claim 57, wherein the payload comprises an mRNA encoding a secreted protein, membrane-bound protein, intracellular protein, antibody molecule, or enzyme.
59. The LNP of any one of the preceding claims, wherein the LNP is formulated for systemic delivery.
60. The LNP of any one of the preceding claims, wherein the LNP is formulated for parenteral, e.g., intravenous, intramuscular, subcutaneous, intrathecal, or intradermal; or enteral, e.g., oral, rectal, or sublingual, delivery.
61. The LNP of any one of the preceding claims, wherein the LNP is not in an organism.
62. A pharmaceutical composition comprising an LNP according to any one of the preceding claims and a pharmaceutically acceptable carrier.
63. A method of treating a disease, comprising administering an LNP according to any one of claims 1-61, or a pharmecutical composition according to claim 62, to a subject.
64. The method of claim 63, wherein the disease is coronary artery disease, rheumatoid arthritis, diabetes, neurodegenerative disease, Alzheimer’s disease, peripheral artery disease, cerebral vascular disease, diabetes-derived cardiovascular diseases, macular degeneration, congestive heart failure, systemic lupus, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, cardiovascular, renovascular diseases, metabolic diseases, immune disorders, fibrotic disease, inflammation, and/or infectious disease.
65. The method of claim 63 or 64, wherein the LNP is systemically delivered to the subject.
66. The method of any one of claims 63-65, wherein the LNP is parenterally, e.g., intravenously, intramuscularly, subcutaneously, intrathecally, or intradermally; or enterally, e.g., orally, rectally, or sublingually, delivered to the subject.
67. The method of any one of claims 63-66, wherein the LNP is administered in an amount of about 0.01 mg/kg to about 500 mg/kg.
68. The method of any one of claims 63-67, wherein the LNP is internalized by a cell.
69. The method of claim 68, wherein the LNP increases internalization by a cell compared to the same LNP without the ApoE-mimicking peptide.
70. The method of claim 68 or 69, wherein the cell is a liver cell, e.g., a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a liver sinusoidal cell.
71. The method of claim 68 or 69, wherein the cell is a brain cell.
72. The method of claim 68 or 69, wherein the cell is a splenic cell, e.g., a splenocyte.
73. The method of claim 68 or 69, wherein the cell is an ovarian cell, a lung cell, an intestinal cell, a heart cell, a skin cell, an eye cell, or a skeletal muscle cell.
74. The method of any one of claims 68-73, wherein the cell is a non-immune cell.
75. The method of any one of claims 68-74, wherein the cell is a cancer cell.
76. The method of any one of claims 68-75, wherein the cell is a T-cell.
77. The method of any one of claims 68-76, wherein the cell is a stem cell.
78. The method of claim 77, wherein the stem cell is a hematopoietic cell.
79. The method of any one of claims 68-78, wherein the cell is a lung cell.
80. The method of any one of claims 68-79, wherein the cell is a kidney cell.
81. The method of any one of claims 63-80, wherein the total plasma concentration of cholesterol in the subject is lowered.
82. The method of any one of claims 63-81, wherein the plasma LDL concentration, the plasma VLDL concentration, or both in the subject are lowered.
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