Short apolipoprotein e mimetic peptides and methods of use thereof

Abstract

ApoE mimetic peptides having a length of 8-17 amino acids are provided, comprising an ApoE receptor binding region or a portion thereof, and one or more covalent linkages linking at least two discontinuous amino acids of the peptide. Also provided are methods of treating a dyslipidemia disorder, such as hypertriglyceridemia or hypercholesterolemia, or a viral infection using the peptides.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 338,654, filed on May 5, 2022, the entire contents of which are incorporated herein by reference. Field of the Invention

[0003] The present disclosure relates to apolipoprotein E (ApoE) mimetic peptides and methods of use thereof, particularly for treating dyslipidemia or viral infection.

[0004] background

[0005] Apolipoprotein E (ApoE) promotes the hepatic clearance of triglyceride-rich lipoprotein remnants by acting as a ligand for the low-density lipoprotein receptor (LDLR). In addition, it contains several amphipathic helices that can mediate cholesterol efflux. ApoE includes two domains connected by a hinge region. The N-terminal domain includes four α-helices. Helix 4 is responsible for binding to the receptor and heparan sulfate proteoglycans. The C-terminal domain includes an amphipathic α-helix that binds to lipids and mediates cholesterol efflux through the ABC Al transporter. Only lipid-bound ApoE binds to the LDL-associated receptor with high affinity because the receptor binding region is shielded in the lipid-free form.

[0006] Given the function of ApoE, many ApoE mimetic peptides have been designed as potential therapeutic drugs. These generally include a receptor binding region and a lipid binding region (Wolska et al., Cells 10: 597, 2021). Although some ApoE mimetic peptides are in early clinical trials, there is still a need for ApoE mimetic peptides with better efficacy, safety and other properties.

[0007] Overview

[0008] Disclosed herein are ApoE mimetic peptides and fusion proteins comprising ApoE mimetic peptides. In some examples, the disclosed peptides and fusion proteins have improved properties compared to previous ApoE mimetic peptides.

[0009] In some aspects, the length of ApoE mimetic peptide is 8-17 amino acid, including the receptor binding domain (such as SEQ ID NO:34) of ApoE or its part, and including one or more covalent connections of at least two non-continuous amino acids connecting the peptide.In some instances, the peptide includes an amphipathic helical domain, and the covalent connection connecting at least two non-continuous amino acids is located between two amino acids on the hydrophobic side of the amphipathic threaded domain.One or more covalent connections can be hydrocarbon staple (hydrocarbon staple), hydrocarbon stitch (hydrocarbon stitch), lactam bridge or disulfide bond. In some aspects, the covalent linkage is a hydrocarbon staple, a hydrocarbon stitch, comprising one or more of (S)-α-methyl α-pentenylglycine (S5), (S)-α-methyl α-octenylglycine (S8), dipentenylglycine (B5), (R)-α-methyl α-pentenylglycine (R5) and (R)-α-methyl α-octenylglycine (R8), or any combination thereof.

[0010] In some aspects, the disclosed ApoE mimetic peptides include one or more additional modifications. In some examples, the modifications include one or more amino acid substitutions, additions or deletions; C-terminal amidation; N-terminal acylation; one or more D-isomer amino acids; modified or unnatural amino acids; N-terminal fatty acids; C-terminal fatty acids; or a combination of two or more thereof. In a specific example, the peptide includes caprylic acid or myristic acid modifications at the N- or C-terminus.

[0011] In specific examples, the ApoE mimetic peptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 2-4, 7-12, 14-21, and 30-33, or comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 2-4, 7-12, 14-21, and 30-33.

[0012] In certain aspects, the ApoE mimetic peptide binds to lipoproteins (e.g., one or more of low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), high-density lipoprotein (HDL), and very low-density lipoprotein (VLDL), lipoprotein (a) (Lp (a)), chylomicrons, and chylomicron remnants). In other aspects, the ApoE mimetic peptide binds to heparin or heparan sulfate proteoglycans.

[0013] Also provided are fusion proteins including the disclosed ApoE mimetic peptides, such as ApoE mimetic peptides covalently linked to a second peptide. In some aspects, the fusion protein comprises a disclosed ApoE mimetic peptide linked to a human neonatal Fc receptor (FcRn) IgG binding site or an FcRn-albumin binding site. In some instances, the fusion protein comprises an amino acid sequence having at least 90% identity to an amino acid sequence of any one of SEQ ID NOs: 22-25, or the fusion protein comprises or consists of an amino acid sequence in any one of SEQ ID NOs: 22-25.

[0014] Also provided are pharmaceutical compositions comprising one or more disclosed ApoE mimetic peptides or fusion proteins and a pharmaceutically acceptable carrier. In some aspects, the composition is formulated for intravenous administration, subcutaneous administration, or oral administration.

[0015] Provided herein is a method for treating a subject with a disclosed ApoE mimetic peptide or fusion protein. In some aspects, the method includes treating a subject with dyslipidemia, for example, by administering an effective amount of a disclosed ApoE mimetic peptide or fusion protein to the subject to reduce the triglyceride level of the subject (such as a subject with hypertriglyceridemia) or reduce the cholesterol level of the subject (such as a subject with hypercholesterolemia). In other aspects, the method includes treating a viral infection of a subject (such as a subject with beta coronavirus infection, such as a subject infected by SARS-CoV-2) by administering an effective amount of a disclosed ApoE mimetic peptide or fusion protein to the subject. In a specific example, the ApoE mimetic peptide or fusion protein is administered intravenously, subcutaneously or orally to the subject.

[0016] Also provided are methods of preparing the disclosed ApoE mimetic peptides or fusion proteins.In certain aspects, the peptides or fusion proteins are produced recombinantly, such as by chemical synthesis.

[0017] Also provided is a method for reducing lipoprotein in a sample. In some respects, the method includes contacting a sample containing lipoprotein with one or more disclosed ApoE mimetic peptides under conditions sufficient to bind lipoprotein to one or more peptides, thereby forming a peptide / lipoprotein complex, and contacting a peptide / fat protein complex with glycosaminoglycans (such as dextran sulfate, heparin or heparan sulfate) under conditions sufficient to bind peptide / lipoprotein protein complex to glycosaminoglycans, thereby forming a peptide / fat protein complex. The method also includes removing peptide / lipoprotein / glycosaminoglycan complexes from the sample. In some examples, the sample is blood plasma, such as from blood plasma from a subject suffering from hypercholesterolemia.

[0018] The above and other features of the present disclosure will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic diagram of the cationic amphipathic alpha helix formed by the receptor binding domain of ApoE (SEQ ID NO: 34).

[0021] Figure 2 Schematic representation of an exemplary hydrocarbon stapled ApoE mimetic peptide (SEQ ID NO: 2) that is a portion of the ApoE receptor binding region.

[0022] Figure 3 is a schematic diagram of an exemplary hydrocarbon stapled ApoE mimetic peptide (SEQ ID NO: 14) of the ApoE receptor binding region.

[0023] Figure 4 is a schematic diagram of an exemplary hydrocarbon stitched ApoE mimetic peptide (SEQ ID NO: 15) of the ApoE receptor binding region.

[0024] Figure 5 is a schematic representation of a hydrocarbon stapled ApoE mimetic peptide (SEQ ID NO: 29) having an N-terminal myristic acid modification of ApoE amino acids 141-150.

[0025] Fig. 6A and 6B are graphs showing LDL uptake in HepG2 cells treated with the indicated peptides.

[0026] Fig. 7A and 7B VLDL uptake in HepG2 cells treated with the indicated peptides ( Fig. 7A ) and HDL uptake ( Figure 7B ).

[0027] Figure 8 are graphs showing LDL uptake in LDLR-ko HepG2 cells treated with the indicated peptides.

[0028] Figures 9A-9C Display OctaC-E139-150( Fig.9A ) or Octa-N-E139-150( Fig. 9B )-treated wild-type and LDLR-ko HepG2 cells. Fig. 9C Shown is LDL uptake in LDLR-KO HepG2 cells mediated by MyrN-E139-150 peptide.

[0029] Fig. 10A and 10BThe total plasma cholesterol ( Fig. 10A ) and triglycerides ( Fig. 10B ). The time is before peptide administration (prebleed) or 1, 3 or 6 hours after peptide administration. *P<0.01 compared with the control group.

[0030] Fig.11 is a graph showing the reduction of mouse hepatitis virus (MHV) GFP fluorescence in HeLa mCC1a cells treated with the indicated ApoE peptides.

[0031] Fig.12 are circular dichroism (CD) spectra of the indicated peptides in 100 μM water at 20°C.

[0032] Fig.13 Solubilization of 1,2, dimyristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipid vesicles by unmodified peptides and acylated stapled peptides is shown.

[0033] Fig.14A and 14B The uptake of LDL in wild-type HepG2 cells in the presence of 10 μM peptide is shown ( Fig.14A ) and VLDL uptake in the presence of 5 μM peptide ( Fig. 14B Ac-hE18A-NH2 was included as a control (see, e.g., Datta et al., Biochemistry 39:213-220, 2000).

[0034] Fig.15 is a graph showing cell viability in the presence of the indicated peptides and concentrations.

[0035] Fig.16 Binding of LDL, MyrN-E1 41-150-amL or pre-complexed LDL / MyrN-E1 41-150-amL to a heparin-coated biolayer interferometry biosensor surface is shown.

[0036] Fig.17 Heparin chromatograms with LDL or LDL + MyrN-E141-150-amL peptide are shown. The NaCl concentration was 0.15 M at baseline and a linear gradient from 0.15 M to 2 M was applied (shown by the dotted line).

[0037] Figures 18A-18C The OctaC-E139-150 peptide ( Fig.18A and 18B ) or MyrN-E141-150-amL peptide ( Fig. 18C )-mediated heparinase treatment of wild-type HepG2 cells ( Fig.18A and 18C ) or LDL uptake by LDLR-KO HepG2 cells ( Fig.18B ).

[0038] Fig.19 Shown is the effect of heparin on LDL uptake in wild-type HepG2 cells in the presence of the MyrN-E141-150-amL peptide.

[0039] Fig. 20A and 20B The results showed that MyrN-E139-150 and OctaN-E139-150 peptides have an effect on the plasma cholesterol level in ApoE KO mice ( Fig. 20A ) and triglycerides ( Fig. 20B )’s impact.

[0040] Fig.21A and 21B Display MyrN-E141-150-amL( Fig.21A ) and OctaN-E139-150( Fig.21B ) peptide on plasma cholesterol in LDLR-KO mice.

[0041] Sequence Listing

[0042] Any nucleic acid and amino acid sequences provided herein or the attached sequence listing are shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 CFR § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.

[0043] SEQ ID NOs: 1-21 are exemplary ApoE mimetic peptides.

[0044] SEQ ID NOs: 22-25 are exemplary ApoE mimetic peptide fusion proteins.

[0045] SEQ ID NO:26 is the amino acid sequence of an exemplary mature human ApoE protein:

[0046] KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLL RDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH

[0047] SEQ ID NO:27 is an exemplary human neonatal Fc receptor (FcRn) IgG binding site:

[0048] RF (Penicillamine) TGHFG (N-methyl-glycine) (N-methyl-leucine) YPC-6-aminocaproic acid

[0049] SEQ ID NO:28 is an exemplary human neonatal Fc receptor (FcRn) albumin binding site:

[0050] VMHCFWDEEFKCDYG-6-aminocaproic acid

[0051] SEQ ID NO:29 is an exemplary myristoylated ApoE 141-150 peptide.

[0052] SEQ ID NOs: 30-33 are additional exemplary ApoE mimetic peptides.

[0053] SEQ ID NO: 34 is an exemplary ApoE receptor binding region (amino acids 134-150 of mature human ApoE protein): RVRLASHLRKLRKRLLR

[0054] SEQ ID NO:35 is a portion of the ApoE receptor binding region (amino acids 139-150 of mature human ApoE protein): SHLRKLRKRLLR

[0055] SEQ ID NO:36 is a portion of the ApoE receptor binding region (amino acids 141-150 of mature human ApoE protein): LRKLRKRLLR

[0056] SEQ ID NO:37 is a portion of the ApoE receptor binding region (amino acids 141-148 of mature human ApoE protein): LRKLRKRL

[0057] Detailed Description

[0058] ApoE mimetic peptides are provided herein. ApoE peptides bind to lipoproteins via the hydrophobic side of the helix (also containing one or more hydrocarbon staples) and / or the acyl chains attached to the N or C termini. At the same time, the positively charged residues on the hydrophilic side of the helix interact with heparan sulfate proteoglycans (HSPGs) and receptors, such as LDLR and possible LDLR-related protein-1 (LRP1).

[0059] In some instances, the disclosed peptides provide advantages over previous ApoE mimetics (such as those described in Wolska et al., Cells 10:597, 2021). Advantages include one or more of the following: shorter length, stronger potency, more similar to the native sequence of ApoE, increased proteolytic resistance, increased plasma half-life, and increased oral bioavailability. As described herein, the disclosed ApoE mimetics reduce plasma total cholesterol and triglycerides in ApoE-ko mice, and also reduce plasma total cholesterol in LDLR-ko mice. Therefore, they can be used to treat dyslipidemia, such as hypercholesterolemia or hypertriglyceridemia. In addition, the disclosed peptides reduce the replication of mouse hepatitis virus, a prototype of beta coronavirus (such as SARS-CoV-2), demonstrating their usefulness as potential antiviral agents.

[0060] I. Terminology

[0061] Unless otherwise indicated, technical terms are used conventionally. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms "a", "an", and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "peptide" includes singular or plural peptides and can be considered equivalent to the phrase "at least one peptide". As used herein, the term "comprising" means "including". It should also be understood that, unless otherwise indicated, any and all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for descriptive purposes.

[0062] Although many methods and materials similar or equivalent to the methods and materials described herein can be used, specific suitable methods and materials are described herein. In the event of a conflict, the present specification (including term explanations) shall prevail. In addition, the materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0063] To facilitate review of various aspects of the present disclosure, the following explanations of specific terms are provided:

[0064] Alkyl: refers to a group having 1 to 25 (C 1-25 ) or more carbon atoms, for example, 1 to 10 (C 1-10 ) carbon atoms, 1 to 4 (C 1-4 ) carbon atoms, 1 to 14 (C 1-14 ) carbon atoms or 2 to 22 (C 2-22 ) carbon atoms or 6 to 18 (C 6-18 ) carbon atoms. The alkyl portion may be substituted or unsubstituted. For example, the term includes straight and branched hydrocarbon groups such as methyl (CH3), ethyl (-CH2CH3), n-propyl (-CH2CH2CH3), isopropyl (-CH(CH3)2), n-butyl (-CH2-CH2CH2CH3), isobutyl (-CH2CH2(CH3)2), sec-butyl (-CH(CH3)(CH2CH3)), tert-butyl (-C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), neopentyl (-CH2C(CH3)3), hexyl (CH6H 13 ), heptyl (C7H 15 ), octyl (C8H 17 ), decyl (C 10 H 21 ), dodecyl (C 12 H 25 ), tetradecyl (C 14 H 29 ), hexadecyl (C 16 H 33 ), octadecyl (C 18 H 37 ) or eicosyl (C 20 H 41 ).

[0065] Amphipathic: An amphipathic molecule contains hydrophobic (non-polar) and hydrophilic (polar) groups. An example of an amphipathic molecule is an amphipathic peptide. An amphipathic peptide can also be described as a helical peptide having hydrophilic amino acid residues on one face of the helix and hydrophobic amino acid residues on the opposite face. In some examples, the peptides described herein will form an amphipathic helix in vitro or in vivo under appropriate conditions.

[0066] Apolipoprotein E (ApoE): A 299 amino acid protein that plays a role in the clearance of triglyceride-rich lipoproteins and cholesterol efflux. There are three different isoforms of ApoE (ApoE2, ApoE3, and ApoE4) that differ at amino acid positions 112 and 158. ApoE2 is associated with type III hyperlipoproteinemia, while ApoE4 is a risk factor for Alzheimer's disease. ApoE mutations are associated with familial dysbetalipoproteinemia or type III hyperlipoproteinemia, which is characterized by elevated plasma cholesterol and triglycerides due to impaired clearance of chylomicrons and lipoproteins.

[0067] Unless the context clearly indicates otherwise, the term ApoE includes any ApoE gene, cDNA, mRNA or protein from any organism. The nucleic acid and protein sequences of ApoE are publicly available. For example, GenBank Accession No. NM_000041.4 discloses human ApoE nucleic acid sequences, and GenBank Accession No. NP_000032.1 discloses human ApoE protein sequences, both of which are incorporated herein by reference as provided by GenBank on May 5, 2022. In other examples, ApoE includes a mature (processed) form of the protein, for example, lacking a signal sequence. SEQ ID NO: 26 is the amino acid sequence of an exemplary mature human ApoE protein.

[0068] Coronavirus: A large family of positive-sense, single-stranded RNA viruses that can infect humans and non-human animals. Coronaviruses get their name from the crown-like spikes on their surface. The viral envelope consists of a lipid bilayer containing the viral membrane (M), envelope (E), and spike (S) proteins. Most coronaviruses cause mild to moderate upper respiratory tract illnesses, such as the common cold.

[0069] Betacoronaviruses are a genus of coronaviruses. Three betacoronaviruses have emerged that can cause severe illness and death in humans: severe acute respiratory syndrome coronavirus (SARS-CoV), SAR-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Other betacoronaviruses that infect humans include human coronavirus HKU1 (HKU1-CoV) and human coronavirus OC43 (OC43-CoV).

[0070] Domain: A protein domain is a part or fragment of a protein that has common structural, physicochemical and functional characteristics; for example, hydrophobic, polar, globular, helical domains or properties, such as an alpha helical domain, such as an amphipathic helical domain; or a receptor binding domain (such as the receptor binding domain of ApoE).

[0071] Dyslipidemic conditions: conditions associated with altered levels of any or all lipids or lipoproteins in the blood. Dyslipidemic conditions include, for example, hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, HDL deficiency, apoe deficiency, apoA-I deficiency, and cardiovascular disease (e.g., coronary artery disease, atherosclerosis, and restenosis).

[0072] Familial Hypercholesterolemia (FH): An inherited condition that results in very high plasma LDL levels. FH affects approximately 1 in 200-250 people. Mutations in the LDLR, the primary receptor in the liver responsible for LDL uptake, are the primary cause of FH. Mutations that affect LDLR function result in decreased clearance of LDL from the circulation. FH patients with very high LDL levels may require LDL-apheresis.

[0073] Fusion: Joined by a covalent bond. In some instances, "fusion" refers to making two polypeptides into one continuous polypeptide molecule by recombinant means. For example, a fusion protein is a protein that includes two separate polypeptides joined together.

[0074] Hydrocarbon staple or stitch: A carbon-carbon bond between at least two moieties in a peptide that, in some instances, stabilizes the alpha helical structure. Hydrocarbon staples are all hydrocarbon crosslinks formed by α,α-disubstituted non-natural amino acids, including terminal olefin chains. In some instances, the non-natural amino acids are discontinuous, for example, separated by 3 or 6 amino acids. Hydrocarbon stitching refers to the formation of two hydrocarbon staples with a common attachment point in the middle position (e.g., two staples are formed using three non-natural amino acids). Exemplary non-natural amino acids that can be used to form hydrocarbon staples or stitches include (S)-α-methyl, α-pentenylglycine (S5), (S)-α-methyl, α-octenylglycine (S8), (R)-α-methyl, α-pentenylglycine (R5), and (R)-α-methyl, α-octenylglycine (R8), in any combination. Additionally, dipentenylglycine (B5) can be used to form hydrocarbon stitches, for example, allowing the formation of two staples with B5 as a common attachment point (see Figure 4Examples of hydrocarbon stitches in . For example, Walensky and Bird, J. Med. Chem. 57:6275-6288, 2014 and Hilinski et al., J. Am. Chem. Soc. 136:12314-12322, 2014.

[0075] Inhibit or treat disease: Inhibit the overall development of a disease, disorder, or condition, for example, in a subject at risk for a disease such as a dyslipidemia disorder or viral infection. "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. As used herein, the term "improvement," with respect to a disease, pathological condition, or symptom, refers to any observable beneficial effect of a treatment. A beneficial effect may be demonstrated, for example, by a delayed onset of clinical symptoms of a disease in a susceptible subject, a reduction in the severity of some or all of the clinical symptoms of a disease, a slowing of disease progression, a reduction in the number of relapses of a disease, an improvement in the overall health or well-being of a subject, or by other parameters specific to a particular disease.

[0076] Isolated: An "isolated" biological component (such as a nucleic acid molecule or protein) is one that is substantially separated or purified from other biological components in the environment in which the component occurs (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles). Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids or proteins.

[0077] Linker or Linkage: A molecule that connects two other molecules by a covalent bond, or by an ionic bond, a Van der Waals bond, or a hydrogen bond. In certain examples, the linker comprises a hydrocarbon staple or a hydrocarbon stitch. In other examples, the linker is a peptide bond.

[0078] Lipoprotein: A biochemical composition containing proteins and lipids, with proteins bound to them to enable fats to move between water inside and outside cells. There are five major classes of lipoprotein particles, from largest to smallest in molecular size: chylomicrons, very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). HDL has the highest ratio of protein to cholesterol; its most abundant apolipoproteins are apoA-I and apoA-II. LDL contains apolipoprotein B, and its core is composed of linoleate, including esterified and nonesterified cholesterol molecules. The diameter of an LDL particle is about 22 nm, and its mass is about 3 million daltons. Lipoprotein a (Lp(a)) is a subclass of lipoprotein; Lp(a) consists of an LDL-like particle and a specific apolipoprotein(a) (apo(a)), which is covalently bound to the apolipoprotein B of the LDL-like particle.

[0079] Peptide, polypeptide or protein: A polymer in which the monomers are amino acid residues linked together by, for example, amide bonds. When the amino acid is an α-amino acid, either the L-optical isomer or the D-optical isomer can be used. The terms "peptide" or "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences. The term "peptide" is specifically intended to encompass naturally occurring peptides as well as recombinantly or synthetically produced peptides. The term "amino acid" refers to naturally occurring α amino acids, non-natural α amino acids, natural β amino acids and non-natural β amino acids, as well as modified amino acids (e.g., modified by adding alkyl groups, hydroxyl groups, phosphate groups, fatty acid groups, linkers or functional groups). The term "residue" or "amino acid residue" includes amino acids incorporated into peptides, polypeptides or proteins.

[0080] Unnatural amino acids include, but are not limited to, 4-hydroxyproline, desmosine, γ-aminobutyric acid, β-cyanoalanine, norvaline, 4-(R)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine, 1-amino-cyclopropanecarboxylic acid, 1-amino-2-phenyl-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidinylacetic acid, 4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminopimelic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta-, and para-substituted phenylalanines, disubstituted phenylalanines, substituted tyrosines, and statins. Additionally, amino acids may be derivatized to include hydroxylated, phosphorylated, sulfonated, acylated, or glycosylated amino acid residues.

[0081] In some aspects, the modification involves substituting one or more amino acids for amino acids with similar physicochemical and / or structural properties (e.g., "conservative" substitutions). Examples of conservative substitutions are shown below.

[0082]

[0083] Conservative substitutions generally preserve (a) the structure of the peptide backbone in the area of ​​the substitution, for example, in a helical conformation, (b) the charge or hydrophobicity of the target site molecule, or (c) a substantial portion of the side chains.

[0084] In other aspects, one or more substitutions may be non-conservative, such as changes in which (a) a hydrophilic residue, such as a seryl or threonyl group replaces (or is replaced by) a hydrophobic residue, such as a leucyl, isoleucyl, phenylalanyl, valyl, or alanyl group; (b) a cysteine ​​or proline group replaces (or is replaced by) any other residue; (c) a residue having a positively charged side chain, such as a lysyl, arginyl, or histidine group replaces (or is replaced by) an electronegative residue, such as a glutamyl or aspartyl group; or (d) a residue having a bulky side chain, such as a phenylalanine group replaces (or is replaced by) a residue having no side chain, such as a glycine.

[0085] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers that can be used in the present disclosure are known to those of ordinary skill in the art. For example, Remington: The Science and Practice of Pharmacy, Adejare (Ed.) Academic Press, London, United Kingdom, 23 rd Edition (2021), which describes compositions and formulations suitable for drug delivery of the peptides disclosed herein.

[0086] In general, the property of the carrier will depend on the specific mode of administration adopted. For example, parenteral preparations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solution, dextrose aqueous solution, glycerol, etc. as carriers. For solid compositions (such as powders, pills, tablets or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biological neutral carriers, pharmaceutical compositions to be administered can also contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0087] Phospholipids: Phospholipids consist of a water-soluble polar head connected to two water-insoluble nonpolar tails (via negatively charged phosphate groups). Both tails are composed of fatty acids, and each tail is about 14 to about 24 carbon groups long. When placed in an aqueous environment, phospholipids form a bilayer or micelle with the hydrophobic tails aligned against each other. This forms a membrane with hydrophilic heads on both sides. In some cases, phospholipids are a type of lipid that is a major component of animal cell membranes.

[0088] Purified: The term purified does not require absolute purity; rather, it is a relative term. Thus, for example, a purified compound preparation is one in which the compound is more enriched than in the environment in which it is produced, such as within a cell or a biochemical reaction chamber. In some aspects, a compound preparation is purified such that the compound comprises at least 50% of the content of the preparation.

[0089] Recombinant: A recombinant nucleic acid or protein is one that has a non-natural sequence or is an artificial combination of two separate sequence fragments. This artificial combination can be achieved by chemical synthesis or by artificial manipulation of separate nucleic acid fragments, such as through genetic engineering techniques. In some instances, a recombinant protein is a protein encoded by a recombinant nucleic acid molecule.

[0090] Subject: Living multicellular vertebrates. This category includes humans, veterinary and laboratory subjects, including human and non-human mammals.

[0091] Therapeutically effective amount: The amount of a given agent (or combination of agents) sufficient to achieve a desired effect in a subject treated with the agent, e.g., an amount sufficient to prevent, treat, reduce and / or ameliorate the symptoms and / or underlying causes of a condition or disease. In certain aspects, an "effective amount" refers to an amount sufficient to reduce plasma cholesterol and / or triglyceride levels in a subject and / or treat or inhibit dyslipidemia or viral infection.

[0092] II. ApoE mimetic peptides

[0093] ApoE mimetic peptides are disclosed herein. The peptide includes all or part of the ApoE receptor binding region, is 8-17 amino acids in length, and includes one or more covalent connections of at least two non-continuous amino acids connecting the peptide. In some aspects, the ApoE mimetic peptide increases cellular lipoprotein uptake, reduces serum triglyceride levels in a subject, reduces serum total cholesterol levels in a subject, or a combination of two or more thereof.

[0094] The disclosed ApoE mimetic peptides include the receptor binding region of ApoE (e.g., SEQ ID NO: 34), a modified ApoE receptor binding region (e.g., SEQ ID NO: 34 amino acid sequence, wherein one or more amino acids are substituted by non-natural amino acids) or a portion thereof. In some aspects, the length of the peptide is 8 to 17 amino acids (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids long). In a specific example, the length of the peptide is 8, 10, 12 or 17 amino acids. In some aspects, the length of the peptide is 17 amino acids and includes SEQ ID NO: 34 amino acids or a modified form thereof. In other aspects, the length of the peptide is 12 amino acids and includes SEQ ID NO: 35 or a modified form thereof. In another aspect, the length of the peptide is 10 amino acids and includes SEQ ID NO: 36 amino acids or a modified form thereof. In another aspect, the length of the peptide is 8 amino acids and includes SEQ ID NO: 37 amino acids or a modified form thereof. In any of the disclosed peptides, one or more (eg, 1, 2, 3, 4 or more) amino acids in the sequence may be substituted with an unnatural amino acid capable of forming a hydrocarbon staple or stitch.

[0095] Peptide includes one or more (such as 1,2,3 or 4) covalent connections between two discontinuous amino acids in the peptide. In some aspects, peptide includes a covalent connection between a pair of discontinuous amino acids in the peptide. In other aspects, peptide includes two covalent connections, and each covalent connection is between one of two pairs of different discontinuous amino acids in the peptide. In further aspects, the peptide includes two covalent connections between three discontinuous amino acids in the peptide, wherein one discontinuous amino acid has a covalent connection with each of the other two discontinuous amino acids. In some aspects, covalent connection is hydrocarbon staple (hydrocarbon staple), hydrocarbon stitching (hydrocarbon stitch), disulfide bond or lactam bridge (for example, formed by lysine ε-amino and glutamic acid or aspartic acid side group carboxyl cyclization), or other chemical bonds formed by stable spirals.

[0096] When there are two or more covalent linkages, the covalent linkages can be identical or different covalent linkages. In some specific examples, the disclosed peptides include a hydrocarbon staple, two hydrocarbon staples, a hydrocarbon stitch (including two hydrocarbon stitches) or a hydrocarbon staple and a lactam bridge. In some instances, covalent linkage provides one or more improved properties for the peptide, including one or more of the stabilization of the alpha helical structure, the improvement of binding to lipoproteins and the resistance to proteolysis.

[0097] In specific aspects, the disclosed peptides include one or more hydrocarbon staples or hydrocarbon stitches. Hydrocarbon staples are all hydrocarbon crosslinks formed between two parts, such as α, α-disubstituted non-natural amino acids including terminal olefin chains. Hydrocarbon stitches are composed of two hydrocarbon staples with a common attachment point in the middle position. Therefore, in some instances, at least two (e.g., at least 2, 3, 4 or more) non-continuous amino acids in the ApoE peptide are replaced by non-natural amino acids that are capable of forming hydrocarbon staples when connected. In some instances, non-continuous amino acids are separated by 3 or 6 amino acids.

[0098] Exemplary non-natural amino acids that can be used to form hydrocarbon staples or stitches include (S)-α-methyl, α-pentenylglycine (S5), (S)-α-methyl, α-octenylglycine (S8), (R)-α-methyl, α-pentenylglycine (R5), (R)-α-methyl, α-octenylglycine (R8), bis-pentenylglycine (B5), (S)-pentenylalanine, (S)-octenylalanine, (R)-pentenylalanine and (R)-octenylalanine, any combination thereof. In some examples, the non-natural amino acid is selected so that it can form a hydrocarbon staple. Thus, for example, if two non-natural amino acids are separated by 2 or 3 amino acids, hydrocarbon staple is "short", and the connection is between two pentenyl-modified amino acids (e.g., between S5 and S5, between R5 and R5, between S5 and R5, or between R5 and S5). If two non-natural amino acids are separated by 6 amino acids, hydrocarbon staple is "long", and the connection is between the amino acid modified by the pentenyl group and the amino acid modified by the octenyl group (e.g., between R8 and S5 or between S8 and R5). In other examples, non-natural amino acids are selected to enable them to form hydrocarbon stitching (hydrocarbon stitch). In a specific example, the formation of hydrocarbon stitching (hydrocarbon stitch) includes that the amino acid modified by the octenyl group is separated by 6 amino acids from the dipentenylglycine residue, which is separated by 3 amino acids (e.g., R8-B5-R5, R8-B5-S5, S8-B5-S5, or S8-B5-R5) from the pentenyl-modified amino acid. In other examples, the formation of hydrocarbon stitching includes a pentynyl modified amino acid separated by 3 amino acids from a dipentenyl glycine residue, which is separated by 6 amino acids from an octenyl modified amino acid (e.g., S5-B5-S8, S5-B5-R8, R5-B5-R8, or R5-B5-S8). In another example, the formation of hydrocarbon stitching includes an octenyl modified amino acid separated by 6 amino acids from a dipentenyl glycine residue, which is separated by 6 amino acids from a second octenyl modified amino acid (e.g., R8-B5-S8, S8-B5-R8, R8-B5-R8, or S8-B5-S8). In another example, the formation of a hydrocarbon stitch includes a pentynyl modified amino acid separated from a dipentenylglycine residue by 3 amino acids, which in turn is separated from a pentynyl modified amino acid by 3 amino acids (e.g., R5-B5-S5, R5-B5-R5, S5-B5-R5, or S5-B5-S5).

[0099] The present disclosure also encompasses one or more additional modified peptides in addition to the above-mentioned covalent connection and any modification required to produce such connection. In some aspects, the peptide includes one or more modifications, including adding, deleting and / or replacing one or more amino acids in the ApoE mimetic peptide with another naturally occurring amino residue compared to the naturally occurring ApoE amino acid sequence, or compared to non-naturally occurring amino acids. In some instances, leucine (e.g., leucine corresponding to leucine 149 of SEQ ID NO: 26) can be replaced with α-methyl-leucine, N-methyl-leucine or β-leucine. In other instances, arginine (e.g., arginine corresponding to arginine 150 of SEQ ID NO: 26) can be replaced with homoarginine. In a further example, one or more lysine residues in the peptide can be replaced with arginine or homoarginine. In some instances, substitution can make the peptide more resistant to proteolysis, increase plasma half-life, increase bioavailability, or a combination of two or more thereof.

[0100] In addition to naturally occurring, gene-encoded amino acids, one or more amino acid residues in the disclosed peptides may be substituted with naturally occurring non-encoded amino acids and / or synthetic amino acids. In some aspects, the substitutions may increase lipid binding, increase resistance to proteolysis, increase oral bioavailability, or a combination thereof. In some examples, such amino acids include acylation of the ε-amino group of lysine; N-alkylation of arginine, histidine, or lysine; alkylation of the carboxyl group of glutamic acid or aspartic acid; deamidation of glutamine or asparagine; β-alanine and other omega-amino acids, such as 3-aminopropionic acid, 2,3-diaminopropionic acid, 4-aminobutyric acid, etc.; α-aminoisobutyric acid; ε-aminocaproic acid; δ-aminovaleric acid; N-methylglycine or sarcosine; ornithine; citrulline; penicillamine; tert-butylalanine; tert-butylglycine; N-methylisoleucine; phenylglycine; cyclohexylalanine; norleucine; naphthylalanine; 4-chlorophenylalanine; 2-fluorophenylalanine; 3 -fluorophenylalanine; 4-fluorophenylalanine; α-methyl-leucine; N-methyl-leucine; β-leucine; 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; β-2-thienylalanine; methionine sulfoxide; homoarginine; N-acetyl lysine; α-methyl-lysine; 2,4-diaminobutyric acid; 2,3-diaminobutyric acid; p-aminophenylalanine; N-methyl valine; homocysteine; homophenylalanine; homoserine; hydroxyproline; homoproline; N-methylated amino acids; peptoids (in which the side chain group is attached to the nitrogen atom of the peptide backbone instead of the α-carbon); and β-peptides (in which the amino group is attached to the β carbon instead of the α carbon). In a specific example, one or more leucine residues in the peptide (e.g., the leucine corresponding to amino acid 149 of SEQ ID NO: 26) are substituted with α-methyl-leucine or N-methyl-leucine.

[0101] Although in some aspects, the amino acids of the disclosed ApoE mimetic peptides are L-amino acids, in other aspects, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or all) L-amino acids are replaced by D-amino acids (e.g., L-Leu→D-Leu). In some instances, including one or more D-amino acids can make the peptide more resistant to proteolysis, increase plasma half-life, and / or increase oral bioavailability. In some aspects, all amino acids in the peptide are D-amino acids. In other aspects, one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) are D-amino acids. In some instances, L-Lys becomes D-Arg or D-homoarginine, or L-Arg becomes D-Lys or D-homoarginine. In some instances, at least 1, 2, or 3 C-terminal amino acids are D-amino acids. Specific non-limiting examples include peptides of any one of SEQ ID NOs: 7-10, 32, and 33. In further examples, the modified or non-naturally occurring amino acid, non-encoded amino acid, or synthetic amino acid (such as those described above) can be a D-amino acid.

[0102] In further aspects, the disclosed peptides may include N-terminal modifications, C-terminal modifications, or both. In some instances, the peptides include N-terminal acylation, C-terminal amidation, or both. In other instances, the modification includes N-terminal or C-terminal fatty acids, for example, to increase the binding of the peptide to lipoproteins. In some instances, the fatty acid is a C6-C18 fatty acid (from caproic acid to stearic acid). In a specific instance, the fatty acid is caprylic acid (also referred to as caprylic acid) or myristic acid.

[0103] In other examples, N-terminal modifications include deamination, N-low alkyl, N-di-low alkyl, constrained alkyl (e.g., branched, cyclic, fused, adamantyl) and N-acyl modifications. Exemplary C-terminal modifications include amide, low alkyl amide, constrained alkyl (e.g., branched, cyclic, fused, adamantyl) alkyl, dialkyl amide and low alkyl ester modifications. Low alkyl is C1-C4 alkyl.

[0104] In some aspects, the disclosed ApoE mimetic peptides include an amino acid sequence having at least 90% sequence identity (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity) to any one of the amino acid sequences of SEQ ID NOs: 2-4, 7-12, 14-21 and 30-33. In other examples, the amino acid sequence of the ApoE mimetic peptide includes or consists of any one of the amino acid sequences of SEQ ID NOs: 2-4, 7-12, 14-21 and 30-33. Exemplary hydrocarbon stapled or stitched ApoE mimetic peptides are provided in Table 1.

[0105] Table 1. Exemplary ApoE peptides

[0106]

[0107]

[0108] Ac: N-terminal acylation

[0109] NH2: C-terminal amidation

[0110] S5: (S)-α-methyl, α-pentenylglycine

[0111] R8: (R)-α-methyl, α-octenylglycine

[0112] S8: (S)-α-Methyl, α-octenylglycine

[0113] R5: (R)-α-methyl, α-pentenylglycine

[0114] B5: Dipentenylglycine

[0115] Lowercase letters represent D-amino acids

[0116] Fusion protein is also provided, which includes a disclosed ApoE mimetic peptide connected or fused with another peptide. In some aspects, the ApoE mimetic peptide is connected to a peptide or modified peptide that can improve oral bioavailability and / or increase peptide plasma half-life. Extra peptides can be fused to the N-terminal of the ApoE mimetic peptide or the C-terminal of the ApoE mimetic polypeptide. In some aspects, the ApoE mimetic peptide and another peptide are continuous (for example, directly connected). In other aspects, the ApoE mimetic peptide and additional peptide are connected by a joint, such as a peptide joint. In some instances, the peptide joint is 1,2,3,4 or more amino acids long, such as 1-3 glycine residues or proline residues. In other instances, the joint is a bifunctional joint (such as succinimide), a disulfide bond, 6-aminocaproic acid or a non-coding amino acid, such as isoaminobutyric acid.

[0117] IgG and albumin have extremely long plasma half-lives, while most other human proteins exhibit rapid blood clearance. This long half-life of IgG and albumin is the result of interaction with the neonatal Fc receptor (FcRn), which forms an intracellular protein pool, prevents lysosomal degradation and is subsequently recycled to the extracellular space. Genetic fusions between therapeutic proteins and the Fc domain of IgG, albumin, or long, flexible polypeptide stretches have been introduced to extend protein circulation. FcRn can bind IgG at pH 6, but not at physiological pH (7.4), and this pH dependence may be the key to the mechanism by which FcRn extends the half-life of IgG. It is believed that after IgG is taken up into cells, FcRn can bind to IgG in acidic endosomes, thereby avoiding degradation in lysosomes. The IgG molecules then return to the cell surface by exocytosis and are released back into circulation because FcRn has the least affinity for IgG at extracellular pH 7.4.

[0118] In some examples, the ApoE mimetic peptide is linked to a human neonatal Fc receptor (FcRn) binding sequence, such as an IgG binding site (e.g., SEQ ID NO: 27), an albumin binding site (e.g., SEQ ID NO: 28), or a non-IgG / non-albumin FcRn binding site. For example, see U.S. Patent Nos. 9,527,890; 9,012,603; 9,574,190; 10,046,023; 10,316,073; 10,588,935; Mezo et al., Proc. Natl. Acad. Sci. USA 105: 2337-2342, 2008.

[0119] Fusion proteins can include any modification discussed above about ApoE mimetic peptides. In a specific example, the fusion protein includes one or more of N-terminal acylation, C-terminal amidation, N-terminal fatty acid modification (such as caprylic acid or myristic acid) and C-terminal fatty acid modification (such as caprylic acid and myristic acid).

[0120] In some aspects, the fusion protein includes an amino acid sequence having at least 90% sequence identity (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to the amino acid sequence of any one of SEQ ID NOs: 22-25. In other examples, the amino acid sequence of the fusion protein includes or consists of the amino acid sequence of any one of SEQ ID NOs: 22-25. Exemplary fusion proteins include those provided in Table 2.

[0121] Table 2. Exemplary ApoE mimetic peptide fusion proteins

[0122]

[0123] 1: Penicillamine

[0124] 2: N-methyl-glycine

[0125] 3: N-methyl-leucine

[0126] 4: 6-aminocaproic acid

[0127] Ac: N-terminal acylation

[0128] NH2: C-terminal amidation

[0129] S5: (S)-α-methyl, α-pentenylglycine

[0130] R8: (R)-α-methyl, α-octenylglycine

[0131] In some aspects, disclosed ApoE mimetic peptide or fusion protein can be separated from various sources.For example, disclosed peptide or fusion protein can be by chemical synthesis (such as solid phase or solution phase synthesis), then react under appropriate conditions to produce one or more covalently connected between two or more non-continuous amino acids of peptide.In some instances, covalent connection is hydrocarbon staple (hydrocarbon staple) or hydrocarbon stitch (hydrocarbon stitch), and hydrocarbon staple (hydrocarbon staple) or hydrocarbon stitch (hydrocarbon stitch) is produced by ruthenium catalyzed metathesis or dicyclic closure metathesis.

[0132] In some aspects, the ApoE mimetic peptides or fusion proteins disclosed herein retain one or more activities of ApoE, including lipoprotein binding activity, LDLR binding activity, LRP1 binding activity, heparan sulfate proteoglycan (HSPG) binding activity, mediating cellular lipoprotein uptake, reducing serum triglyceride levels of subjects, reducing total serum cholesterol levels of subjects and / or reducing viral replication in cells or subjects. In some instances, the disclosed peptides or fusion proteins increase cellular lipoprotein uptake (such as LDL, IDL, HDL, Lp (a), chylomicrons, chylomicron remnants or VLDL uptake) by at least about 5 to 40 times compared to a control (e.g., a corresponding peptide having a native ApoE sequence that does not include a covalent link or no peptide / buffer). In further examples, when administered to ApoE-ko mice, the disclosed peptides or fusion proteins reduce serum triglycerides by at least about 50% (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more) within one hour compared to a control group (e.g., a corresponding peptide having a native ApoE sequence that does not contain a covalent link or no peptide / buffer). In additional examples, when administered to ApoE-ko mice, the disclosed peptides or fusion proteins reduce serum cholesterol by at least about 10% (e.g., at least about 10%, at least about 15%, at least about 20%, at least about 25% or more) within 1-6 hours compared to a control group (e.g., a corresponding peptide having a native ApoE sequence that does not contain a covalent link or no peptide / buffer). In further examples, the disclosed peptides or fusion proteins reduce viral replication in cells by at least about 50% compared to a control (eg, a corresponding peptide having a native ApoE sequence not comprising a covalent linker or no peptide / buffer).

[0133] III. Pharmaceutical Compositions and Methods of Use

[0134] In exemplary aspects, a composition comprising one or more ApoE mimetic peptides or fusion proteins disclosed herein is applied to a subject with dyslipidemia (e.g., hypertriglyceridemia, hypercholesterolemia, or cardiovascular disease) in a therapeutically effective amount. In other examples, a therapeutically effective amount of ApoE mimetic peptides or fusion proteins disclosed herein is applied to a subject with an ApoE genetic defect or a subject with abnormal beta lipoproteinemia (e.g., a subject with ApoE2 / E2 alleles). In another example, a therapeutically effective amount of ApoE mimetic peptides or fusion proteins is applied to a subject with familial hypercholesterolemia due to LDLR mutation. In other aspects, a composition comprising one or more ApoE mimetic peptides or fusion proteins disclosed herein is applied to a subject with a viral infection or disease. The effective dose for these uses will depend on the severity of the disease and the overall health of the subject and other factors. The therapeutically effective amount of the compound is to provide subjective relief of one or more symptoms or an objectively identifiable improvement indicated by a clinician or other qualified observer.

[0135] ApoE mimetic peptides or fusion proteins can be administered by any means known to those skilled in the art (see, e.g., Banga, Therapeutic Peptides and Proteins, Third Edition, Taylor & Francis Group, 2015), such as by intramuscular, subcutaneous or intravenous injection, or oral, nasal, inhalation or anal administration. In one aspect, it is administered by intravenous injection. On the other hand, the administration is oral. In order to extend the time that the ApoE mimetic peptide or fusion protein can be used to inhibit or treat dyslipidemia or viral infection, the peptide can be provided as an implant, an oily injection or a particle system. The particle system can be a microparticle, a microcapsule, a microsphere, a nanocapsule or a similar particle. In a specific aspect, the administered ApoE mimetic peptide includes an amino acid sequence of any one of SEQ ID NOs: 2-4, 7-12, 14-21 and 30-33. In other aspects, a fusion protein comprising an ApoE mimetic peptide is administered to the subject, such as any one of SEQ ID NOs: 22-25.

[0136] In some instances, the method comprises administering a therapeutically effective amount of a disclosed ApoE mimetic peptide or fusion protein to a subject in need thereof. In some instances, the subject is a human or veterinary subject. In a specific example, the subject is a human.

[0137] In some instances, the subject suffers from hypertriglyceridemia. The normal or ideal serum triglyceride of a human subject is less than 150 mg / dL. The critical high triglyceride range is 150-199 mg / dL, the high triglyceride range is 200-499 mg / dL, and the extremely high triglyceride range is 500 mg / dL or higher. Therefore, in some instances, the serum triglyceride of the subject receiving treatment is 150 mg / dL or higher. A therapeutically effective amount of one or more disclosed peptides or fusion proteins is administered to the subject, such as an amount that causes a reduction in serum triglyceride levels of about at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more), for example, compared to the triglyceride level before the peptide or fusion protein is administered. In other examples, a therapeutically effective amount of a disclosed peptide or fusion protein lowers serum triglycerides in a subject to less than 150 mg / dL.

[0138] In some instances, the subject suffers from hypercholesterolemia, including but not limited to familial hypercholesterolemia. The normal or ideal total cholesterol of an adult subject is less than 200 mg / dL (children are less than 170 mg / dL). The critical high total cholesterol range of an adult is 200-239 mg / dL, and the range of high total cholesterol is 240 mg / dL or higher (children are 170-199 mg / dL and 200 mg / dL or higher). Therefore, in some instances, the subject receiving treatment is an adult and has a total cholesterol level of 200 mg / dL or higher serum. One or more disclosed peptides or fusion proteins of a therapeutically effective amount are administered to the subject, such as causing a reduction in serum total cholesterol levels by about at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% or more), for example, compared to the total cholesterol level before administering the peptide or fusion protein. In other examples, a therapeutically effective amount of a disclosed peptide or fusion protein reduces serum total cholesterol in a subject to less than 200 mg / dL.

[0139] In other aspects, the subject suffers from or is suspected of having a viral infection or disease. In some instances, the subject is infected or suspected of being infected with an enveloped virus. Exemplary enveloped viruses include influenza virus, herpes virus (such as cytomegalovirus), poxvirus, hepadnavirus (such as hepatitis B virus), flavivirus (such as dengue virus or West Nile virus), coronavirus (such as beta coronavirus), orthomyxovirus, paramyxovirus (such as measles or mumps), filovirus (such as Ebola virus or Marburg virus) and retrovirus (such as HIV). In some instances, the subject is infected (or suspected of being infected) with beta coronavirus, such as SARS-CoV-2. On the one hand, a therapeutically effective amount of peptide is administered to the subject, such as an amount required to inhibit viral replication by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% (compared to no treatment). On the other hand, a therapeutically effective amount is the amount required to reduce the viral titer in a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% (compared to no treatment). In other examples, a therapeutically effective amount can also be the amount required to reduce or eliminate the symptoms of one or more viral infections, such as the amount required to reduce or eliminate fever, cough, or shortness of breath, such as in the case of respiratory viruses such as beta coronaviruses.

[0140] One or more of the provided ApoE mimetic peptides or fusion proteins can be combined with one or more pharmaceutically acceptable carriers or excipients for administration to human or animal subjects. Remington: The Science and Practice of Pharmacy, Adejare (Ed.), Academic Press, London, United Kingdom, 23 rdEdition (2021), describes compositions and formulations suitable for drug delivery of one or more therapeutic compounds or molecules, such as one or more peptides or fusion proteins alone or in combination with another agent. Examples of suitable pharmaceutically acceptable carriers, vehicles or excipients include sterile aqueous or non-aqueous solutions, suspensions and / or emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's oil or fixed oil. Intravenous carriers include liquid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, etc., may also be present. Other pharmaceutically acceptable carriers include carbohydrates (eg, glucose, sucrose or dextran), antioxidants (eg, ascorbic acid or glutathione), chelating agents, low molecular weight proteins, lipids, wetting agents, emulsifiers, dispersing agents and preservatives.

[0141] In general, the preparation is prepared by uniformly and closely combining one or more disclosed peptides or fusion proteins with a pharmaceutically acceptable carrier or excipient. Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents and solutes to make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. The preparation can be contained in unit dose or multi-dose containers, such as sealed ampoules and vials, which can be stored under freeze-dried (lyophilized) conditions, and only sterile liquid carriers, such as water for injection or saline, need to be added directly before use. Instant injection solutions and suspensions can be prepared by sterile powders, granules and tablets commonly used by those of ordinary skill in the art.

[0142] In some aspects, the disclosed peptides or fusion proteins are formulated into complexes with one or more phospholipids. In one aspect, before administration to a subject, one or more disclosed peptides or fusion proteins are pre-complexed with phospholipids or other lipids into disc-shaped or spherical particles. In one example, the peptide is pre-complexed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Methods for preparing the disclosed peptide-phospholipid complexes include Schwendeman et al., J. Lipid Res. 56: 1727-1737, 2015; Yuan et al., Nanomedicine: Nanotechnology, Biology and Medicine 48: 102646, 2023; and Shu et al., Aterioscler Thromb Vase Biol. 30: 2504-2509, 2010.

[0143] Peptides, fusion proteins and pharmaceutical compositions provided herein, including those for treating dyslipidemia or viral diseases, can be administered by different routes, such as oral (including oral and sublingual), rectal, parenteral (including intravenous or intraperitoneal), aerosol, nasal, intramuscular, subcutaneous, intradermal and topical. They can be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles and liposomes.

[0144] The amount of peptide, fusion protein or pharmaceutical composition that is effective depends on the nature of the disease or condition to be treated, and the stage of the disease or condition. The effective amount can be determined by standard clinical techniques. The precise dose used in the formulation also depends on the route of administration and should be determined according to the judgment of the healthcare practitioner and the circumstances of each subject. An example of such a dosage range is about 0.1 mg / kg to about 200 mg / kg body weight (e.g., about 0.1 to about 10 mg / kg, about 1 to about 25 mg / kg, about 5 to about 50 mg / kg, about 25 to about 75 mg / kg, about 50 to about 100 mg / kg, about 75 to about 150 mg / kg, or about 100 to about 200 mg / kg, such as about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, or about 200 mg / kg), administered in single or divided doses. Another example of dosage range is that single or divided dose is 1.0 to 100mg / kg body weight. In some aspects, disclosed peptide, fusion protein or pharmaceutical composition is used twice a day, every day, every other day, every week or with lower frequency. In a specific example, once a week, the subject is intravenously injected with peptide, fusion protein or composition. In another specific example, once a day, the subject is orally administered with peptide, fusion protein or composition. Treatment may continue indefinitely, or can be stopped when reaching the target clinical parameter, such as normal range of triglyceride or total cholesterol level or removal of viral infection (for example, symptom relief or no viremia).

[0145] The specific dosage level and frequency of administration for any particular subject may vary and will depend on many factors, including the specific disease or condition, the activity of the specific compound, the metabolic stability and duration of action of the compound, the age, weight, general health, sex, diet, mode and timing of administration, rate of excretion, drug combination, and the severity of the condition of the subject being treated.

[0146] The disclosed peptide, fusion protein or pharmaceutical composition can be administered with approximately the same dosage, ascending dosage regimen or loading dosage regimen (e.g., wherein the loading dose is about two to five times the maintenance dose) throughout the treatment period. In some aspects, the dosage varies during the course of treatment according to the condition of the subject being treated, the severity of the disease or condition, the apparent response to treatment and / or other factors determined by those of ordinary skill in the art.

[0147] In other aspects, the disclosed peptides are used in vitro or ex vivo methods to promote or increase the removal of VLDL, LDL, Lp (a) and / or lipoprotein X (LpX) from a sample such as plasma. In some aspects, the disclosed peptides are used in methods of LDL-apheresis (e.g., for subjects with familial hypercholesterolemia). In some aspects, the method includes contacting a sample containing lipoproteins (e.g., a biological sample from a subject, such as plasma) with one or more disclosed peptides under conditions sufficient to bind lipoproteins (e.g., LDL and / or VLDL) to one or more peptides, thereby forming a peptide / lipoprotein complex. Then, under conditions sufficient to bind the peptide / lipoprotein complex to the glycosaminoglycan, the peptide / lipoprotein complex is contacted with a glycosaminoglycan (e.g., dextran sulfate, heparin or heparan sulfate), thereby forming a peptide / lipoprotein / glycosaminoglycan complex. In a specific example, the glycosaminoglycan is dextran sulfate. The peptide / lipoprotein / glycosaminoglycan complex is removed from the sample to produce lipoprotein-depleted plasma. In some aspects, glycosaminoglycans are fixed on a solid support (e.g., a column) or in a slurry form. In some instances, the method reduces the amount of lipoprotein (e.g., LDL) in the sample by at least about 30% (e.g., at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more). In other instances, the inclusion of the disclosed peptide or fusion protein increases the amount of lipoprotein (e.g., LDL) removed from the sample compared to the use of glycosaminoglycans alone.

[0148] In some examples, the method can be used to remove lipoproteins (e.g., LDL) from the plasma of a subject (e.g., a subject with familial hypercholesterolemia) by LDL apheresis. LDL apheresis systems include cascade filtration (or lipid filtration), immunoadsorption, heparin-induced LDL precipitation, dextran sulfate LDL adsorption, and LDL hemoperfusion. In some aspects, these methods utilize heparin-induced LDL precipitation apheresis. In some examples, a blood processing system (e.g., System; Kaneka Pharma America LLC) removes blood from a subject and separates plasma from blood cells. The separated plasma is contacted with one or more disclosed peptides or fusion proteins under conditions sufficient to bind lipoproteins (e.g., LDL and / or VLDL) to one or more peptides or fusion proteins, thereby forming peptide / lipoprotein complexes. The plasma containing the peptide / lipoprotein complexes is then passed through a column containing dextran sulfate chains. The peptide / lipoprotein complexes are retained on the column and the amount of lipoproteins in the plasma is reduced. The blood cells are added back to the plasma and the blood is returned to the subject. Example

[0149] The following examples are provided to illustrate certain features of various aspects of the present disclosure. These examples should not be construed as limiting the present disclosure to the specific features described.

[0150] Example 1 In vitro testing of ApoE mimetic peptides

[0151] Lipid uptake in wild-type and LDLR-ko HepG2 cells was assessed as described by Lucero et al. (J. Lipid Res. 63(1), Article 100160, 2022). Briefly, HepG2 cells or HepG2LDLR ko cells were seeded in 96-well plates (25,000 cells per well) in medium containing DMEM, 10% (v / v) FBS, 100 IU / ml penicillin G, and 100 pg / ml streptomycin 48 hours before the experiment. Proteins in LDL, HDL, or VLDL were stained with the amino-reactive fluorescent dye Alexa Fluor TM 568 NHS ester (succinimidyl ester) (Invitrogen) labeled. Alexa568-LDL (or -HDL or -VLDL) with a final total protein concentration of 50 μg / ml was mixed with different apoE peptides at different concentrations or with the same amount of buffer in DMEM / 0.1% BSA. The Alexa568-lipoprotein / peptide mixture or Alexa568 lipoprotein was then preincubated at 37°C for 30 minutes. The cells were washed with PBS, and the Alexa568-lipoprotein / peptide mixture or Alexa568-lipoprotein in DMEM / 0.1% BSA was added to the cells on a 96-well plate (in triplicate) and incubated for 1 hour or 4 hours. The cells were washed with PBS, dissociated from the wells with trypsin, and resuspended in ice-cold PBS / 0.5% BSA / 2.5mM EDTA. Cellular uptake of LDL / HDL / VLDL was assessed by measuring fluorescence by fluorescence activated cell sorting (FACS) flow cytometry (BD LRS Fortessa (BD Biosciences, Franklin Lakes, NJ)).

[0152] Compared with the control peptide, the ApoE mimetic peptide enhanced the uptake of LDL by wild-type HepG2 cells ( Fig. 6A and 6B These peptides also enhanced the uptake of VLDL and HDL in wild-type HepG2 cells ( Fig. 7A and 7B ). At higher peptide concentrations, LDL uptake increased regardless of the presence of LDLR. However, the difference in uptake at lower peptide concentrations may indicate that apoE peptide also mediates LDL uptake via the LDLR pathway ( Figure 8 and Figures 9A-9C ).

[0153] Example 2 In vivo testing of ApoE mimetic peptides

[0154] The effects of OctaC-E139-150 and OctaN-E139-150 peptides were tested in ApoE-ko mice. The mice were maintained on a normal diet. At 3.5 months of age, 15 mice were divided into three groups of 5 mice each, as follows: 1) control group; 2) OctaC-E139-150 peptide group; and 3) OctaN-E139-150 peptide group. The peptides were dissolved in PBS at a concentration of 1.5 mg / ml (810 μM OctaC-E139-150; 849 μM OctaN-E139-150). The mice were weighed one day before peptide administration and the amount of peptide was calculated to receive 4.6 mg / kg of peptide (2.5 μmol / kg) per mouse. The calculated amount of 1.5 mg / ml peptide was placed in a syringe and standardized to 80 μl with PBS. Mice received an intravenous injection of PBS (control) or peptide. Blood samples were collected immediately before injection, 1 hour, 3 hours and 6 hours after injection. The blood was centrifuged at 3000 rpm for 20 minutes at 4°C, and plasma (20-30 μl) was separated and stored at 4°C. The next day, plasma triglycerides and total cholesterol were measured using the L-triglyceride M assay and cholesterol E kit (Wako, FUJIFILM Medical Systems USA).

[0155] Octa-E139-150 and OctaN-E139-150 peptides significantly reduced plasma total cholesterol in mice for at least 6 hours after administration ( Fig. 10A In addition, the peptide significantly reduced plasma triglyceride levels, although this effect was transient ( Fig. 10B ).

[0156] Example 3 Effect of ApoE mimetic peptide on viral replication

[0157] Mouse hepatitis virus (MHV) is a beta coronavirus that infects mouse cells through the mouse CEACAM1 receptor (mCC1a receptor). HeLa (human epithelial) cells modified to express the mCC1a receptor can be infected by MHV. The recombinant MHV used in this experiment has been modified to contain an additional nucleocapsid (N) protein fragment that is fused to a green fluorescent protein tag (GFP) at the N terminus (see Verheije et al., J. Virol. 84: 11575-1579, 2010). When cells are infected with MHV, MHV replicates to produce more GFP and enhances the fluorescent signal.

[0158] HeLa mCC1a cells were cultured in 96-well plates. Cells were infected with MHV-GFP for 2 hours at 37°C. The viral stock solution was diluted 40 times. The stock solution was prepared by infecting HeLa cells with MHV-GFP for 3 days and collecting the cell culture medium. The cells were washed with PBS and treated with medium (DMEM plus 10% FBS, 100 IU / ml penicillin G, 100 μg / ml streptomycin, 0.5 mg / ml G418) containing ApoE peptide (15 μM) or medium without peptide and incubated at 37°C for 16 hours. The cells were washed with PBS and the total GFP fluorescence (ex / em 488 / 520) in the cells was measured on the plate using a fluorescence plate reader.

[0159] like Fig.11 As shown, treatment with OctaC-E139-150 or stitched E134-150 peptides showed a significant decrease in MHV-GFP fluorescence, indicating reduced viral replication in the treated cells.

[0160] Example 4 Additional in vitro characterization of ApoE mimetic peptides

[0161] The effect of hydrocarbon stapling on the physical properties of ApoE mimetic peptides was studied. Circular dichroism of 100 μM peptide in aqueous solution at 20°C showed that the hydrocarbon-stapled peptide was α-helical, while the unmodified peptide was disordered ( Fig.12 ).

[0162] To evaluate the lipid binding properties of the unmodified peptides and the acylated stapled peptides, a DMPC vesicle solubilization assay was performed. The unmodified linear peptide E139-150 did not solubilize DMPC phospholipid vesicles, whereas the acylated stapled peptides solubilized DMPC phospholipid vesicles ( Fig.13 ). This suggests that hydrocarbon stapling and peptide acylation increase the lipid binding affinity of the peptide.

[0163] Fluorescently labeled LDL or VLDL (Alexa Fluor 568-NHS ester) at a final protein concentration of 50 μg / ml was preincubated with or without apoE peptide for 30 min at 37°C. The final peptide concentration was 10 μM for LDL uptake and 5 μM for VLDL uptake studies. Wild-type HepG2 cells were then incubated with the lipoprotein / peptide mixture for 1 h at 37°C. Lipoprotein uptake was assessed by flow cytometry. Fig.14A , 14B As shown in Figures 6B and 6B, hydrocarbon stapled peptides, especially acylated stapled peptides, increased the uptake of LDL and VLDL by HepG2 cells.

[0164] Since cationic amphiphilic peptides may be cytotoxic, the peptides were also tested for potential cytotoxic effects on cells. Cell Counting Kit-8 (Dojindo) was used to assess the effect of increasing peptide concentrations on the viability of wild-type HepG2 cells. None of the peptides were cytotoxic to WT HepG2 cells under the conditions used in the lipid uptake studies ( Fig.15 ).

[0165] Example 5 ApoE mimetic peptide promotes the binding of LDL to heparin

[0166] It was observed that the ApoE mimetic peptide promoted the binding of LDL to heparin. This was confirmed by biolayer interferometry (BLI) and heparin chromatography. Biotinylated heparin was immobilized on a BLI streptavidin (SA) biosensor (Sartorius). Binding of 100 nM LDL, 5 μM MyrN-E141-150-amL or pre-complexed 100 nM LDL and 5 μM MyrN-E14-150-amL to heparin was measured in PBS, 0.1% BSA ( Fig.16 ). 400 μl of 1 μM LDL or 1 μM LDL and 20 μM MyrN-E141-150-amL pre-complex was injected into a 1 ml HiTrap Heparin HP column. The column was washed with 10 ml buffer (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4), and LDL or LDL / peptide complex was eluted with a linear gradient of NaCl (10 mM sodium phosphate, pH 7.4, 0.15 M-2 M NaCl gradient) ( Fig.17 ).

[0167] The involvement of heparan sulfate proteoglycans (HSPGs) in peptide-mediated LDL uptake was tested. WT HepG2 cells were pretreated with a mixture of heparanase I, II, and III (IBEX Pharmaceuticals) to degrade heparan sulfate chains on the cell surface. Next, LDL uptake studies were performed using OctaC-E139-150-S and MyrN-E141-150-amL peptides ( Figures 18A-18C ). LDL uptake without peptide addition was not affected by heparinase treatment, but heparinase treatment attenuated the effect of low-dose peptide on LDL uptake ( Fig.18A ). At higher peptide concentrations, heparinase treatment was less effective and partial uptake was restored. Similarly, heparinase treatment attenuated MyrN-E141-150-amL-mediated LDL uptake in wild-type HepG2 cells ( Fig. 18C). This suggests that HSPG plays a role in peptide-mediated LDL uptake at low peptide concentrations, but its role is lower at high peptide concentrations. The same experiment with LDLR-KO HepG2 cells showed similar results ( Fig.18B ), suggesting that at higher peptide doses, some other receptors, such as LRP1, may be involved in peptide-mediated uptake, or that some nonspecific peptide-mediated uptake exists.

[0168] Fluorescently labeled LDL (Alexa Fluor 568-NHS ester) at a final protein concentration of 50 μg / ml was preincubated with or without 10 μM MyrN-E141-150-amL peptide for 30 min at 37°C (solutions prepared in DMEM (phenol red-free, serum-free) containing 0.1% BSA). Heparin was added to some LDL and some LDL / peptide samples (final concentration of 10 USP / ml; heparin, sodium injection from porcine intestinal mucosa (Fresenius Kabi USA, Lake Zurich, IL)). The mixture was preincubated for an additional 5 min at 37°C. Next, wild-type HepG2 cells were incubated with the LDL / peptide / heparin mixture at 37°C for 1 h. Lipoprotein uptake was assessed by flow cytometry. Heparin reduced the effect of MyrN-E141-150-amL peptide on LDL uptake in wild-type HepG2 cells ( Fig.19 ).

[0169] Example 6 Additional in vivo characterization of ApoE mimetic peptides

[0170] MyrN-E139-150 and OctaN-E139-150 peptides were tested in a single-dose ApoE KO mouse study. ApoE KO mice (n=9) were divided into 3 groups: control group (PBS), n=3; MyrN-E139-150 (2.5 μmol / kg; 4.6 mg / kg), n=3; OctaN-E139-150 (2.5 μmol / kg; 4.4 mg / kg), n=3. Mice were maintained on a normal diet throughout the study. The peptides were injected intravenously, and blood samples were collected before and 1 hour, 3 hours, and 6 hours after the peptide injection. Plasma total cholesterol and triglycerides were measured as described in Example 2. The peptides lowered plasma total cholesterol ( Fig. 20A ; 37% at 3 hours; 29% at 6 hours) and triglycerides ( Fig. 20B ; 30-50% at 1 hour).

[0171] MyrN-E141-150-amL and OctaN-E141-150 were tested in a single-dose LDLR-KO mouse study. LDLR-KO mice (n=9) were divided into 3 groups: control group (PBS), n=3; MyrN-E141-150-amL (2.5 μmol / kg; 4.6 mg / kg), n=3; OctaN-E141-150 (2.5 μmol / kg; 4.4 mg / kg), n=3. Mice were kept on a normal diet throughout the study. The peptide was injected intravenously, and blood samples were collected before and 1 hour, 3 hours, 6 hours and 24 hours after the injection of the peptide. Plasma total cholesterol was determined as described in Example 2. MyrN-E141-150-amL reduced plasma total cholesterol by approximately 15-30% (3 hours and 6 hours after injection). Fig.21A ).

[0172] Obviously, the precise details of the methods or compositions described may be changed or modified without departing from the spirit of the described aspects of the present disclosure. We claim all such modifications and variations to be within the scope and spirit of the following claims.

Claims

1. An apolipoprotein E (ApoE) mimetic peptide comprising the receptor binding region of ApoE or a portion thereof of 8-17 amino acids in length, comprising one or more covalent linkages linking at least two non-contiguous amino acids of the peptide.

2. The ApoE mimetic peptide according to claim 1, wherein the peptide comprises an amphipathic helical domain, and the covalent linkage connecting at least two discontinuous amino acids is located between two amino acids on the hydrophobic side of the amphipathic helical domain.

3. The ApoE mimetic peptide according to claim 1 or 2, wherein the one or more covalent linkages are hydrocarbon staples, hydrocarbon stitches, lactam bridges or disulfide bonds.

4. An ApoE mimetic peptide according to claim 3, wherein the hydrocarbon staple or hydrocarbon stitch comprises a bond comprising one or more of (S)-α-methyl α-pentenylglycine (S5), (S)-α-methyl α-octenylglycine (S8), dipentenylglycine (B5), (R)-α-methyl α-pentenylglycine (R5) and (R)-α-methyl α-octenylglycine (R8).

5. The ApoE mimetic peptide according to any one of claims 1 to 4, wherein the peptide comprises one or more additional modifications.

6. An ApoE mimetic peptide according to claim 5, wherein the modification comprises one or more amino acid substitutions, additions or deletions; C-terminal amidation; N-terminal acylation; one or more D-isomer amino acids; modified amino acids; N-terminal fatty acids; C-terminal fatty acids; or a combination of two or more thereof. The ApoE mimetic peptide according to claim 6 , wherein the fatty acid is caprylic acid or myristic acid.

8. The ApoE mimetic peptide according to any one of claims 1 to 7, wherein the peptide comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 31, 2-4, 7-12, 14-21, 30, 32 and 33.

9. The ApoE mimetic peptide according to claim 8, wherein the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 31, 2-4, 7-12, 14-21, 30, 32 and 33.

10. The ApoE mimetic peptide according to claim 9, wherein the peptide consists of the amino acid sequence of any one of SEQ ID NOs: 31, 2-4, 7-12, 14-21, 30, 32 and 33.

11. The ApoE mimetic peptide according to any one of claims 1 to 10, wherein the peptide is 8 amino acids in length.

12. The ApoE mimetic peptide according to any one of claims 1 to 11, wherein the peptide binds to lipoprotein.

13. The ApoE mimetic peptide according to claim 12, wherein the peptide binds to one or more of low-density lipoprotein (LDL), high-density lipoprotein (HDL), intermediate-density lipoprotein (IDL), very low-density lipoprotein (VLDL), proteoglycan, LDL receptor (LDLR) and LDLR-related protein 1 (LRP1).

14. A fusion protein comprising the ApoE mimetic peptide of any one of claims 1 to 13 linked to a human neonatal Fc receptor (FcRn) IgG binding site or an FcRn-albumin binding site.

15. The fusion protein of claim 14, wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 22-25. The fusion protein according to claim 15 , wherein the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 22-25. The fusion protein according to claim 16 , wherein the fusion protein consists of the amino acid sequence of any one of SEQ ID NOs: 22-25.

18. A pharmaceutical composition comprising the ApoE mimetic peptide according to any one of claims 1 to 13 or the fusion protein according to any one of claims 14 to 17 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18, further comprising a phospholipid.

20. The pharmaceutical composition of claim 19, wherein the phospholipid comprises 1,2-dimyristoyl-sn-glycero-3-phosphocholine.

21. The pharmaceutical composition according to any one of claims 18 to 20, wherein the composition is formulated for intravenous administration, subcutaneous administration or oral administration.

22. A method for treating dyslipidemia in a subject, comprising administering to the subject an effective amount of the ApoE mimetic peptide of any one of claims 1 to 13, the fusion protein of any one of claims 14 to 17, or the composition of any one of claims 18 to 21.

23. The method of claim 22, wherein the method reduces triglyceride levels in the subject.

24. The method of claim 23, wherein the subject suffers from hypertriglyceridemia.

25. The method of any one of claims 22 to 24, wherein the subject has a pre-treatment serum triglyceride level of 150 mg / dL or greater.

26. The method of claim 22, wherein the method reduces the subject's total cholesterol level.

27. The method of claim 26, wherein the subject suffers from hypercholesterolemia.

28. The method of any one of claims 22, 26 or 27, wherein the subject has a pre-treatment total cholesterol level of 200 mg / dL or greater.

29. A method for treating a viral infection in a subject, comprising administering to the subject an effective amount of the ApoE mimetic peptide of any one of claims 1 to 13, the fusion protein of any one of claims 14 to 17, or the composition of any one of claims 18 to 21, thereby treating the viral infection.

30. The method of claim 29, wherein the viral infection is an infection with an enveloped virus.

31. The method of claim 30, wherein the enveloped virus is a betacoronavirus.

32. The method of claim 31, wherein the betacoronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

33. The method of any one of claims 29 to 32, wherein treating the viral infection comprises inhibiting replication of the virus in the subject.

34. The method of any one of claims 22 to 33, wherein the administering comprises intravenous administration, subcutaneous injection, oral administration, nasal administration, or aerosol administration.

35. The method of any one of claims 22 to 34, wherein the subject is a human.

36. A method for preparing the ApoE mimetic peptide of any one of claims 1 to 13 or the fusion protein of any one of claims 14 to 17, comprising recombinantly producing the peptide or fusion protein.

37. The method of claim 36, wherein the ApoE mimetic peptide or the fusion protein is produced by chemical synthesis.

38. A method for reducing lipoprotein in a sample, comprising: contacting a sample containing lipoproteins with one or more ApoE mimetic peptides of any one of claims 1 to 13 under conditions sufficient to allow the lipoproteins to bind to the one or more peptides, thereby forming a peptide / lipoprotein complex; contacting the peptide / lipoprotein complex and the glycosaminoglycan under conditions sufficient to allow the peptide / lipoprotein complex to bind to the glycosaminoglycan, thereby forming a peptide / lipoprotein / glycosaminoglycan complex; and Remove peptide / lipoprotein / glycosaminoglycan complexes from samples.

39. The method of claim 38, wherein the sample comprises plasma.

40. The method of claim 38 or 39, wherein the sample is from a subject with familial hypercholesterolemia.

41. The method of any one of claims 38 to 40, wherein the glycosaminoglycan is immobilized on a solid support.

42. The method according to any one of claims 38 to 41, wherein the method is performed by an apheresis system.

43. The method of any one of claims 38 to 42, wherein the glycosaminoglycan is dextran sulfate, heparin, or heparan sulfate.

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