Therapeutic lipid processing compositions and methods for treating age-related macular degeneration

Abstract

Compositions and methods for treating age-related macular degeneration (AMD) are described herein. In particular, polypeptides comprising a helix structure having ATP-binding cassette transporter membrane stabilization and agonist activity, transporter protein binding activity, and / or capable of effecting cholesterol efflux, and methods of using these peptides for treating AMD are described herein.

Description

Technical Field

[0001] Cross-reference to related applications

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 341,990, filed May 13, 2022, entitled "THERAPEUTIC LIPID PROCESSING COMPOSITIONS AND METHODS FOR TREATING AGE-RELATED MACULAR DEGENERATION", the entire disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporation by reference

[0002] All publications and patent applications mentioned herein are incorporated herein by reference in their entirety, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Background Art

[0003]

[0003] Age-related macular degeneration (AMD) is a chronic metabolic and inflammatory disease of the eye. AMD is the leading cause of blindness in people over 55 years old and has a high prevalence in the United States (e.g., 8.7%) and worldwide. Furthermore, this problem is expected to increase with the aging of the world's population. AMD is categorized into various types (e.g., early, intermediate, exudative, and atrophic), but the majority of AMD cases are considered "atrophic" AMD, for which there is no approved treatment.

[0004]

[0004] There are many associated factors that are thought to contribute to AMD. Dysregulation of lipid transport and processing is thought to be the cause of the initiation and progression of AMD. Dysregulation of lipid homeostasis can lead to the accumulation of lipid deposits (drusen) throughout the extracellular matrix, such as Bruch's membrane. Drusen are the first pathological signs of AMD that disrupt and compress retinal pigment epithelium (RPE) cells, and the loss of RPE cells leads to severe late-stage disease, including photoreceptor degeneration and geographic atrophy (GA), and can further cause neovascular AMD (nvAMD).

[0005]

[0005] Currently, there is no treatment for AMD, and the treatment is generally ineffective. Statins or other potential treatments often consider disrupting cholesterol synthesis. However, efforts regarding the underlying defects involved in the etiology of AMD in the current state of the art have failed. There is a need for a treatment that can treat both atrophic and exudative AMD.

Summary of the Invention

[0006]

[0006] Compositions for the treatment of AMD and methods of using them are described herein. Certain compositions and methods consider cellular lipid efflux, the cholesterol reverse transport (RCT) mechanism, and the transport of lipids from within the cell. For example, compositions containing one or more small peptides (e.g., peptides of 80 amino acids or less, 75 amino acids or less, 70 amino acids or less, 65 amino acids or less, 60 amino acids or less, 55 amino acids or less, 50 amino acids or less, 45 amino acids or less, 40 amino acids or less, etc.) that mimic the ability of apolipoproteins to regulate lipid transport and promote cellular lipid efflux via the RCT mechanism, such as ATP-binding cassette (ABC) transporters, high-density lipoprotein (HDL), or scavenger receptor class B type 1 (SR-B1), are described herein. These peptides may be referred to herein as apolipoprotein peptidomimetics, therapeutic apolipoprotein peptidomimetics, test peptides, candidate peptides, or simply therapeutic peptides, and may include sequences engineered for an improved or comparable amphipathic helix structure and function related to one or more endogenous apolipoproteins (e.g., apoE, apoA, apoJ). For example, in some of the variants described herein, the peptide may include a modified or partial polypeptide sequence corresponding to a sequence related to the lipid-binding or lipid-receptor structure of an apolipoprotein.

[0007]

[0007] Generally, these therapeutic peptides can initiate or mediate the transport of lipids from cells via one or more transporter proteins involved in the cholesterol efflux mechanism (as shown herein by promoting cholesterol efflux from human microglial cell lines and human retinal pigment epithelial cell lines). For example, cholesterol efflux regulatory protein, ATP-binding cassette transporter proteins (e.g., ABCA1 or ABCG1, e.g., "ATP-binding cassette subfamily A member 1"), or scavenger proteins can be involved in lipid efflux for cholesterol removal via the RCT mechanism. The therapeutic peptides described herein can have ABCA1-dependent lipid efflux activity (as measured, for example, by a reduction in lipid efflux by the addition of siRNA targeting ABCA1). These therapeutic peptides can treat, prevent, or ameliorate cholesterol homeostasis dysregulation associated with deficiencies or mutations in one or more endogenous apolipoproteins, apolipoprotein receptors, or lipoprotein particle maturation factors.

[0008] [

[0008] ]Also, compositions for the treatment of AMD and methods of using them are described herein. Certain compositions and methods contemplate lipid influx, or the transport of extracellular lipids via lipid influx mechanisms. For example, one or more small peptides (e.g., peptides of 80 amino acids or less, 75 amino acids or less, 70 amino acids or less, 65 amino acids or less, 60 amino acids or less, 55 amino acids or less, 50 amino acids or less, 45 amino acids or less, 40 amino acids or less, etc.) that mimic the ability of apolipoproteins to solubilize lipids and transport them into cells via cellular lipid uptake mechanisms, such as the low density lipoprotein receptor (LDLR), scavenger receptor class B type 1 (SR-B1), or glycosaminoglycan (GAG)-dependent mechanisms, are described herein. These peptides may be referred to herein as apolipoprotein peptide mimetics, therapeutic apolipoprotein peptide mimetics, test peptides, or simply therapeutic peptides, and may include sequences engineered for improved amphipathic helix properties that differ from the native sequence properties of apolipoproteins (apoE, apoA, apoJ). For example, in some of the variants described herein, the peptide may include a modified or partial polypeptide sequence corresponding to helix 4 of apoE (e.g., amino acids 140 - 150, see e.g., SEQ ID NO: 8).

[0009]

[0009] Thus, generally, these therapeutic peptides can bind to the LDLR in a lipid-dependent manner and transport lipids into cells via the LDLR. These therapeutic peptides can overcome lipid transport defects present in the apoE2 carrier (e.g., the apoE2 variant has a defect in LDLR binding activity). The therapeutic peptides described herein can have lipid-dependent LDLR binding activity in surprisingly small polypeptides (e.g., 80 amino acids or less, 75 amino acids or less, 70 amino acids or less, 65 amino acids or less, 60 amino acids or less, 55 amino acids or less, 50 amino acids or less, 49 amino acids or less, 48 amino acids or less, 47 amino acids or less, 46 amino acids or less, 45 amino acids or less, 44 amino acids or less, 43 amino acids or less, 42 amino acids or less, 41 amino acids or less, 40 amino acids or less, etc.). These therapeutic peptides can preserve lipid-dependent LDLR binding and lipid transport activity in even smaller peptides.

[0010] In addition, these therapeutic peptides can solubilize lipids (e.g., as shown herein by reducing the turbidity of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposome solution), and can preferentially bind to oxidized lipids over non-oxidized lipids (measured by surface plasmon resonance (SPR)). The therapeutic peptides described herein can transfer lipids into cells (as shown by the transfer of labeled cholesterol into ARPE-19 and HepG2 cells), and this lipid transfer can be dependent on the GAG-binding domain of the peptide (GAG-dependent lipid transfer activity, measured by reducing the lipid transfer into cells by the addition of exogenous heparin). The therapeutic peptides described herein can have LDLR-dependent lipid transfer activity (e.g., measured by reducing the lipid transfer into cells by the addition of siRNA targeting LDLR). Some of the exemplary therapeutic peptides described herein can have SR-B1-dependent lipid transfer activity (measured by reducing the lipid transfer into cells by the addition of siRNA targeting SR-B1). Generally, the cytotoxicity of the therapeutic polypeptides described herein is minimal (measured by membrane-permeable dye staining of ARPE-19 cells and hemolysis of human RBCs).

[0011]

[0011] Thus, the therapeutic peptides described herein can function as lipid transport peptides that mimic the behavior of apolipoproteins, lipoprotein particles, lipid exporters, and lipid importers. Generally, the therapeutic peptides described herein can bind to LDLR, + increase the transfer of lipids into cells, and improve or prevent lipid transport dysregulation, which is characteristic of drusen formation and AMD disease progression. Also, the therapeutic peptides described herein can bind to ABCA1, +It can increase the excretion of lipids from cells and improve or prevent dysregulation of lipid transport. The methods described herein may include replacing a defective lipid transport function by intravitreal or systemic injection of a therapeutic peptidomimetic of apolipoprotein function. Therefore, these treatment methods can address the LDLR, GAG, and / or SR-B1-dependent mechanisms of lipid uptake into cells. Therefore, these treatment methods can address the ABCA1-dependent or ABCA1-independent mechanisms of lipid excretion from cells. These methods and compositions (e.g., therapeutic peptides) can modulate a patient's cholesterol homeostasis. Therapeutic peptides are generally small (e.g., less than 50 amino acids, less than 49 amino acids, less than 48 amino acids, etc.) and may be amphiphilic and capable of packaging lipids. Therapeutic peptides are engineered to bind to lipids and sequester them, and may deposit lipids intracellularly via uptake receptors or enable the excretion of lipids from cells via excretion receptors. For example, these therapeutic peptides can improve lipid clearance from drusen through increased interaction with the LDLR and other lipid uptake pathways. As an additional example, these therapeutic peptides can reduce the drusen burden through increased interaction with ABCA1 and other lipid excretion pathways, restoring natural lipid transport homeostasis and clearance mechanisms. Since drusen is a major risk factor for AMD disease progression, therapeutic peptides with the potential to reduce drusen formation appear to improve patient outcomes. These peptides can be synthesized synthetically. In some examples, these compositions (e.g., therapeutic peptides) can be formulated with one or more pharmaceutically acceptable carriers.

[0012]

[0012] Also described herein is a method of treating a subject having a disorder associated with an undesirable activity of a lipid regulatory pathway, the method comprising administering to the subject any of the compositions disclosed herein.

[0013] In some examples, the present disclosure provides a method of treating a subject having age-related macular degeneration (AMD), the method comprising administering to the subject any of the compositions disclosed herein. In some examples, the composition is administered intravitreally. In some examples, the subject is human. In some examples, the human is at least 40 years old. In some examples, the human is at least 50 years old. In some examples, the human is at least 65 years old. In some examples, the composition is administered topically. In some examples, the composition is administered systemically. In some examples, the composition has an amino acid sequence of any one of SEQ ID NOs: 35-39, 87, 101, or 114. For example, a polypeptide for use in the treatment of age-related macular degeneration (AMD) having a peptide sequence that is less than 80 amino acids in length and has 65% or more (e.g., 70% or more, 80% or more, 85% or more, 90% or more, etc.) homology with one of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, or 178 is described herein.In particular, a polypeptide having a peptide sequence less than 80 amino acids in length and having at least 65% (e.g., 70% or more, 80% or more, 85% or more, 90% or more, etc.) homology with one of SEQ ID NOs: 87, 101, or 114 is described herein for use in the treatment of age-related macular degeneration (AMD).

[0014]

[0014] In some examples, a polypeptide for use in the treatment of age-related macular degeneration (AMD) may have a peptide sequence that is at least 85% identical to one of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116.

[0015]

[0015] In some examples, the polypeptide is more than 90% identical to one of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116.

[0016] A polypeptide for use in the treatment of age-related macular degeneration (AMD) may have a peptide sequence that is at least 85% identical to SEQ ID NOs: 35, 36, 37, 38, or 39.

[0016]

[0017] Examples of one or more polypeptides for use in the treatment of age-related macular degeneration (AMD) having a peptide sequence of one of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116 are described herein.

[0017]

[0018] Examples of one or more polypeptides for use in the treatment of age-related macular degeneration (AMD) having a peptide sequence of one of SEQ ID NOs: 35, 36, 37, 38, or 39 are described herein.

[0018]

[0019] Peptide sequences related to apolipoprotein mimetics having ATP-binding cassette transporter-binding activity and comprising a sequence that is at least 65% identical to one or more of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116 for use in the treatment of age-related macular degeneration (AMD) are described herein.

[0019]

[0020] One or more polypeptides having cholesterol efflux regulatory protein (CERP) binding activity and comprising a sequence that is at least 65% homologous to one or more of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116 are described herein for use in the treatment of age-related macular degeneration (AMD).

[0020]

[0021] One or more polypeptides having transporter protein binding activity and comprising a sequence that is at least 65% homologous to one or more of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116 are described herein for use in the treatment of age-related macular degeneration (AMD).

[0021]

[0022] One or more polypeptides having ATP-binding cassette transporter binding activity and comprising a sequence that is at least 65% homologous to one or more of SEQ ID NOs: 84, 86, 87, 101, 112, 114, or 116 are described herein for use in the treatment of age-related macular degeneration (AMD).

[0022]

[0023] Polypeptides for use in the treatment of age-related macular degeneration (AMD) may have a peptide sequence that is at least 85% homologous to one of SEQ ID NOs: 25, 27, or 29. In some examples, the polypeptide is more than 90% homologous to one of SEQ ID NOs: 25, 27, or 29.

[0023]

[0024] Polypeptides for use in the treatment of age-related macular degeneration (AMD) may have a peptide sequence of one of SEQ ID NOs: 25, 27, or 29. For example, polypeptides for use in the treatment of age-related macular degeneration (AMD) may have 80 or fewer amino acids, wherein the N-terminus of the polypeptide has 65% or more (e.g., 70% or more, 80% or more, 85% or more, 90% or more, etc.) homology to SEQ ID NO: 8.

[0024]

[0025] In some examples, a polypeptide for use in the treatment of age-related macular degeneration (AMD) has an N-terminus of a polypeptide having at least 65% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, etc.) homology with SEQ ID NO: 29, wherein the first 11 amino acids of the polypeptide have 4 or fewer (e.g., 3 or fewer, 2 or fewer, or 1) substitutions compared to SEQ ID NO: 29.

[0025]

[0026] For example, a polypeptide for use in the treatment of age-related macular degeneration (AMD) can include a sequence having lipid-dependent low density lipoprotein receptor (LDLR) binding activity and being at least 65% homologous to one or more of SEQ ID NO: 25, 27, or 29. In some examples, a polypeptide for use in the treatment of age-related macular degeneration (AMD) includes a sequence having lipid-dependent low density lipoprotein receptor (LDLR) binding activity and being at least 65% homologous to one or more of SEQ ID NO: 5, 7, 9, 13, 18, and 29.

[0026]

[0027] A polypeptide for use in the treatment of age-related macular degeneration (AMD) has 80 or fewer amino acids, wherein the N-terminus of the polypeptide has at least 65% homology with the L-confirmation shown in SEQ ID NO: 8, or the C-terminus of the polypeptide has at least 65% homology with the D-confirmation of SEQ ID NO: 8.

[0027]

[0028] A polypeptide for use in the treatment of age-related macular degeneration (AMD) may have 80 or fewer amino acids, wherein the N-terminus of the polypeptide is homologous to the L-confirmation shown in SEQ ID NO: 8 with 2 or fewer amino acid substitutions or deletions, or the C-terminus of the polypeptide is homologous to the D-confirmation of SEQ ID NO: 8 with 2 or fewer amino acid substitutions or deletions.

[0028]

[0029] A polypeptide for use in treating age-related macular degeneration (AMD) may have 80 or fewer amino acids, while the N-terminus of the polypeptide has at least 65% homology with SEQ ID NO: 8.

[0029]

[0030] A polypeptide for use in treating age-related macular degeneration (AMD) may have the N-terminus of a polypeptide having at least 65% homology with SEQ ID NO: 29, wherein the first 11 amino acids of the polypeptide have two or fewer substitutions compared to SEQ ID NO: 29.

[0030]

[0031] A polypeptide for use in treating age-related macular degeneration (AMD) may have 80 or fewer amino acids, wherein the N-terminus of the polypeptide is at least 65% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, etc.) homologous to SEQ ID NO: 27.

[0031]

[0032] Any of the polypeptides described herein may have a lipid-dependent low-density lipoprotein receptor (LDLR) binding activity such that the polypeptide binds to LDLR at least 2-fold (e.g., at least 2.5-fold, at least 3-fold, at least 5-fold, at least 10-fold, etc.) more strongly in the presence of lipids than in the absence of lipids.

[0032]

[0033] Any of the therapeutic peptides described herein may increase the efflux of lipids from cells (e.g., microglial cells or retinal pigment epithelial cells) via the ATP-binding cassette transporter ABCA1 and increase the presence of ABCA1 on the cell membrane after a pro-inflammatory stimulus.

[0033]

[0034] Any of the therapeutic peptides described herein may include N-terminal acetylation and C-terminal amidation. Also, the therapeutic peptides described herein are intended to include both the L- and D-forms of the peptides described herein.

[0034]

[0035] Also described herein is a pharmaceutical composition for use in preventing or treating age-related macular degeneration (AMD) in a patient, the composition comprising any of the polypeptides described herein and a pharmaceutically acceptable excipient. The composition can be for administration by intravitreal injection and / or intravenous (IV) injection and / or subcutaneous (SC) injection. The pharmaceutical composition can comprise two or more of the polypeptides described above.

[0035]

[0036] For example, a method of treating or preventing age-related macular degeneration (AMD) in a patient using the described polypeptide or pharmaceutical composition can be used when the patient exhibits signs or symptoms of AMD. These methods of treating or preventing age-related macular degeneration (AMD) in a patient using the polypeptide or pharmaceutical composition described herein can be for the treatment of early-stage AMD. Any of these methods of treating a patient for age-related macular degeneration (AMD) can include, for example, delivering the polypeptide or composition to the patient's eye by intravitreal injection (and / or by intravenous injection and / or subcutaneous injection, etc.). As mentioned, the method can include delivering two or more of the polypeptides or compositions described herein.

[0036]

[0037] In particular, described herein is a peptide for treating or preventing age-related macular degeneration (AMD) in a patient, comprising or derived from a peptide having the sequence shown below.

[0037]

[0038] DAWERFRALFKELADYFR (SEQ ID NO: 87)

[0039] It is shown herein that these polypeptides have surprising beneficial properties for treating AMD. As illustrated herein, polypeptides having a sequence that is at least 65% homologous (e.g., including six or fewer conservative and / or conservative hydrophobic substitutions), a hydrophobic moment (μH) of 0.65 or greater, ATP-binding cassette transporter membrane stabilization and agonist activity, transporter protein binding activity, and / or one or more demonstrable properties such as cholesterol efflux of 17.5% or greater can be therapeutically effective in treating AMD. Non-limiting examples of polypeptides that are homologous to T-087 and demonstrate either maintenance or improvement of activity in important assays include T-152, T-160, T-161, T-163, and T-172.

[0038]

[0040] Accordingly, polypeptides having a peptide sequence that is at least 65% homologous to SEQ ID NO: 87 and having a helical coil with a hydrophobic moment of 0.65 μH or greater for use in the treatment of age-related macular degeneration (AMD) are described herein. As mentioned, in the framework of the T-087 polypeptide, the hydrophobic moment typically results in an increase in cholesterol efflux of greater than 15% (e.g., 16% or greater, 17% or greater, 17.5% or greater, 18% or greater, etc.), and the hydrophobic moment correlates with the percentage of cholesterol efflux.

[0039]

[0041] For example, polypeptides having a peptide sequence that is at least 65% homologous to SEQ ID NO: 87 and having ATP-binding cassette transporter membrane stabilization and agonist activity for use in the treatment of age-related macular degeneration (AMD) are also described herein.

[0040]

[0042] Also described herein are polypeptides having a sequence that is at least 65% homologous to SEQ ID NO: 87 for use in the treatment of age-related macular degeneration (AMD) and having transporter protein binding activity.

[0041]

[0043] Any of these polypeptides can have a hydrophobic moment of 0.65 μH or more and can result in cholesterol efflux of 17.5% or more. Any of these polypeptides can have ATP-binding cassette transporter-binding activity.

[0042]

[0044] Generally, for any of these polypeptides based on T-087, any peptide residue different from the sequence of SEQ ID NO: 87 at positions 2 to 7, 9, and 10 to 17 is a conservative substitution or conservative hydrophobic substitution having a hydrophobic value within 0.25 of the hydrophobic value of the peptide residue at the corresponding position of SEQ ID NO: 87 calculated using the method of Fauchere and Pliska, where any different peptide residue at positions 1, 8, 10, and 18 can be any amino acid. For example, the polypeptide sequence can be one of SEQ ID NO: 87, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, or 178. In particular, the polypeptide sequence can be one of SEQ ID NO: 87, 152, 160, 161, 163, or 172.

[0043]

[0045] In any of these examples, the polypeptide sequence can be 70% or more (e.g., 80% or more, 85% or more, 90% or more, etc.) homologous to SEQ ID NO: 87. As mentioned, in any of these polypeptide sequences based on T-087, the polypeptide sequence can be as shown in SEQ ID NO: 35 or 36, where X can be any amino acid.

[0044]

[0046] Also described herein are peptides for treating or preventing age-related macular degeneration (AMD) in a patient, comprising or derived from a peptide having the sequence shown below.

[0045]

[0047] RSGADALESALKELKRFIREWT (SEQ ID NO: 101)

[0048] In addition, it is shown herein that these polypeptides have surprising beneficial properties for treating AMD. Polypeptides having a sequence that is at least 65% identical (e.g., including eight or fewer conservative substitutions and / or conservative hydrophobic substitutions), a hydrophobic moment (μH) of 0.6 or greater (which can confer greater ABCA1 stability), ATP-binding cassette transporter membrane stabilization and agonist activity, transporter protein binding activity, and / or an overall hydrophobicity of 0.198 or greater (which can confer greater cholesterol efflux). Non-limiting examples of polypeptides that are identical to T-087 and demonstrate either maintenance or improvement of activity in important assays include T-122, T-123, T-129, T-136, and T-139.

[0046]

[0049] For example, polypeptides for use in the treatment of age-related macular degeneration (AMD) having a peptide sequence that is at least 65% identical to SEQ ID NO: 101 and comprising a helix-coil having a hydrophobic moment of 0.6 μH or greater are described herein. For example, polypeptides for use in the treatment of age-related macular degeneration (AMD) having a peptide sequence with at least 65% homology to SEQ ID NO: 101 and having ATP-binding cassette transporter membrane stabilization and agonist activity are described herein. Also, polypeptides for use in the treatment of age-related macular degeneration (AMD) having a sequence that is at least 65% identical to SEQ ID NO: 101 and having transporter protein binding activity are described herein. In some examples, the polypeptides for use in the treatment of age-related macular degeneration (AMD) have an overall hydrophobicity of 0.198 or greater and can result in an improvement in cholesterol efflux. In any of these examples, the polypeptide has a hydrophobic moment of 0.6 μH or greater and can result in an ABCA1 stability that is greater than 40% of the ABCA1 stability of the polypeptide of SEQ ID NO: 101. The polypeptide can have ATP-binding cassette transporter binding activity.

[0047]

[0050] In any polypeptide that is at least 65% identical, it has a conservative substitution or conservative hydrophobic substitution with a hydrophobicity value within 0.25 of the hydrophobicity value of the peptide residue at the corresponding position of SEQ ID NO: 101, calculated using the method of Fauchere and Pliska, substituting any peptide residue that differs from the main sequence (e.g., the sequence of SEQ ID NO: 101) at, for example, positions 1, 4 - 8, 10 - 15, 17 - 18, or 20 - 22, where any different peptide residues at positions 2, 3, 9, 16, and 19 can be any amino acid. For example, the polypeptide sequence can be one of SEQ ID NO: 101, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or 151. In particular, the polypeptide sequence can be one of SEQ ID NO: 101, 122, 123, 129, 136, or 139. The polypeptide sequence can be at least 70% identical to SEQ ID NO: 101 (e.g., at least 75% identical, at least 80%, at least 85%, at least 90%, etc.).

[0048]

[0051] Also, polypeptides based on the framework of the T - 114 polypeptide are described herein. For example, a polypeptide having a peptide sequence that is at least 65% identical to SEQ ID NO: 114 and used for the treatment of age - related macular degeneration (AMD), the polypeptide having strong lipid - dependent binding to the low - density lipoprotein receptor, is described herein. The polypeptide for use in the treatment of age - related macular degeneration (AMD) may have a sequence that is at least 65% identical to SEQ ID NO: 114, where the polypeptide has transporter protein - binding activity. In any of these polypeptides, the polypeptide may also include ATP - binding cassette transporter - binding activity.

[0049]

[0052] As mentioned above, and generally, pharmaceutical compositions for use in the prevention or treatment of age-related macular degeneration (AMD) in a patient, comprising a polypeptide as described in any of the above examples and a pharmaceutically acceptable excipient, are described herein. The compositions can be for administration by intravitreal injection, intravenous (IV) injection, and / or subcutaneous (SC) injection. Any of these pharmaceutical compositions can comprise two or more of the various polypeptides described herein.

[0050]

[0053] Also described herein is a method of treating or preventing age-related macular degeneration (AMD) in a patient using any of these polypeptides or pharmaceutical compositions, where the prevention or treatment is for the prevention of AMD and the patient is diagnosed as having a tendency to develop AMD. For example, a method of treating or preventing age-related macular degeneration (AMD) in a patient can comprise using any of the polypeptides described herein, where the prevention or treatment is for treatment and the patient exhibits signs or symptoms of AMD. A method of treating or preventing age-related macular degeneration (AMD) in a patient using one or more of the engineered polypeptides or pharmaceutical compositions described herein can include treating early-stage AMD. A method of treating a patient for age-related macular degeneration (AMD) can include, for example, delivering a polypeptide or composition to the patient's eye by intravitreal injection, intravenous (IV) injection, and / or subcutaneous (SC) injection. Delivering can include delivering two or more of the polypeptides or compositions described herein. As mentioned, in some examples, the patient can be, for example, 40 years of age or older.

[0051]

[0054] All of the methods and devices described herein are contemplated in any combination and can be used to achieve the benefits described herein.

[0052]

[0055] This patent or application file contains at least one colored drawing. Copies of this patent or patent application publication containing colored drawings are provided by the Office upon payment of the claims and the necessary fees.

[0053]

[0056] A better understanding of the features and advantages of the methods and apparatuses described herein can be obtained by reference to the following detailed description, which illustrates exemplary examples, and the accompanying drawings below.

Brief Description of the Drawings

[0054]

Figure 1A

[0057] FIG. 1A is a list of peptides including the control peptides (T-001 to T-004, T-031 to T-034, and T-081 to T-083) described herein, and also shows the hydrophobic moment. The hydrophobic moment is defined in Eisenberg (Eisenberg et al., 1982. The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature).

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 1H

Figure 1I

Figure 2A-2B

[0058] Figures 2A - 2D show the results of a DMPC solubility assay measuring the solubilization of 0.5 mM 1,2 - dimyristoyl - sn - glycero - 3 - phosphocholine (DMPC) by 100 μM peptide concentration for the selected polypeptides shown in Figures 1A - 1I.

Figure 2C-2D

Figure 3A

[0059] Figure 3A is a graph showing the results of the solubilization of 0.5 mM DMPC over a range of peptide concentrations for the controls (T - 001, T - 002, T - 004, and T - 031) and the therapeutic polypeptides (T - 025, T - 027) described herein.

Figure 3B

Figure 3C

Figure 4

[0060] Figure 4A graphically shows the solubilization of 1 mM DMPC over a range of peptide concentrations for the selected peptides shown in FIGS. 1A-1I described herein. Figure 4B graphically shows the solubilization of 1 mM DMPC over a range of peptide concentrations for the selected peptides shown in FIGS. 1A-1I described herein.

Figure 5

[0061] Figure 5A is a graph showing the results of an ARPE-19 cell lysis assay at a single concentration (10 μM) for the selection controls (T-031, T-032, T-033, and T-034) and therapeutic polypeptides (T-001, T-002, T-004, T-031, T-032, T-033, and T-034) described herein. Figure 5B is a graph showing the results of an ARPE-19 cell lysis assay at a single concentration (10 μM) for the therapeutic polypeptides (T-006, T-007, T-009, T-010, T-011, T-012, T-013, and T-014) described herein. Figure 5C is a graph showing the results of an ARPE-19 cell lysis assay at a single concentration (10 μM) for the therapeutic polypeptides (T-018, T-019, T-021, T-025, T-027, T-028, and T-029) described herein. Figure 5D is a graph showing the results of an ARPE-19 cell lysis assay at a single concentration (10 μM) for the therapeutic polypeptides (T-024, T-026, and T-030) described herein.

Figure 6

[0062] Figure 6 is a graph showing an ARPE-19 cell lysis titration curve over a range of peptide concentrations for the controls (T-001, T-002, T-004, and T-034) and selected therapeutic polypeptides (T-025 and T-027) described herein.

Figure 7A-7B

[0063] Figures 7A-7B illustrate the results of an ARPE-19 cell viability assay at a single concentration (100 μM) for the selected peptides shown in Figures 1A-1I.

Figure 7C-7D

Figure 8

[0064] Figure 8A shows a graph summarizing the results of a human red blood cell (hRBC) lysis assay at a single concentration (10 μM) using the control peptides (T-001, T-002, T-004, T-031, T-032, T-033, and T-034) described herein. Figure 8B shows a graph summarizing the results of a human red blood cell (hRBC) lysis assay at a single concentration (10 μM) using the therapeutic polypeptides (T-006, T-007, T-009, T-010, T-011, T-012, T-013, and T-014) described herein. Figure 8C shows a graph summarizing the results of a human red blood cell (hRBC) lysis assay at a single concentration (10 μM) using the therapeutic polypeptides (T-024, T-026, and T-030) described herein. Figure 8D shows a graph summarizing the results of a human red blood cell (hRBC) lysis assay at a single concentration (10 μM) using the therapeutic polypeptides (T-018, T-019, T-021, T-025, T-027, T-028, and T-029) described herein.

Figure 9

[0065] Figure 9A illustrates the results of an hRBC lysis assay at a single concentration (10 μM) for the selected peptides shown in FIGS. 1A-1I. Figure 9B illustrates the results of an hRBC lysis assay at a single concentration (10 μM) for the selected peptides shown in FIGS. 1A-1I.

Figure 10

[0066] Figure 10 is a graph showing an hRBC lysis titration curve over a range of concentrations for the selected peptides shown (controls: T-001, T-002, T-004, and T-034, and therapeutic polypeptides: T-025 and T-027).

Figure 11

[0067] Figure 11 is a graph summarizing the results of an hRBC cell lysis titration curve over a range of peptide concentrations using the control peptides (T-001 and T-034) and therapeutic polypeptides (T-025, T-086, T-087, T-101, T-112, T-114, and T-116) described herein.

Figure 12

[0068] Figure 12 is a graph of ARPE-19 cholesterol uptake at a single concentration (10 μM) for the selected peptides shown in FIGS. 1A-1I.

Figure 13

[0069] Figure 13 is a graph summarizing the results of an ARPE-19 GAG-dependent cholesterol uptake assay at a single concentration (10 μM) using the control peptides (T-002) and therapeutic polypeptides (T-013, T-021, T-025, T-027, T-028, T-029, and T-030) described herein.

Figure 14

[0070] Figure 14A is a graph summarizing an ARPE-1 cholesterol uptake assay at a single concentration (10 μM) for the selected peptides shown in FIGS. 1A-1I. Figure 14B is a graph summarizing an ARPE-1 cholesterol uptake assay at a single concentration (10 μM) for the selected peptides shown in FIGS. 1A-1I.

Figure 15

[0071] Figure 15 is a graph showing an ARPE-19 cholesterol uptake titration curve over a range of peptide concentrations for the control peptides (T-001, T-002, T-004, and T-032) and selected therapeutic polypeptides (T-025 and T-027).

Figure 16

[0072] Figure 16 is a graph showing an ARPE-19 cholesterol uptake titration curve over a range of peptide concentrations for the control peptides (T-001, T-002) and selected therapeutic polypeptides (T-086, T-087, T-101, T-112, T-114, and T-116) described herein.

Figure 17

[0073] Figure 17 is a graph showing HepG2 cholesterol uptake screening data at a single concentration (10 μM) for the selected peptides shown in FIGS. 1A-1I.

Figure 18

[0074] Figure 18 is a graph showing HepG2 cholesterol uptake screening data at a single concentration (10 μM) for the selected peptides shown in Figures 1A - 1I.

Figure 19

[0075] Figure 19 is a graph showing the screening results in the HepG2 GAG - dependent cholesterol uptake assay at a single concentration (10 μM) for the selected peptides shown in Figures 1A - 1I.

Figure 20

[0076] Figure 20 shows examples of HepG2 cholesterol uptake titration curves for controls (T - 001, T - 002, T - 004, and T - 032) and therapeutic peptides (T - 025 and T - 027).

Figure 21

[0077] Figure 21 shows examples of HepG2 cholesterol uptake titration curves for controls (T - 001, T - 002) and therapeutic peptides (T - 086, T - 087, T - 101, T - 112, T - 114, and T - 116).

Figure 22

[0078] Figure 22 graphically summarizes the results of a lipid - dependent LDLR binding (SPR) assay at a single concentration (1 μM) for recombinant apoE2 and apoE4, control peptides (T - 001, T - 002, T - 031, T - 032, T - 033, and T - 034), and the selected test peptides shown in Figures 1A - 1I.

Figure 23

[0079] Figure 23 is a table showing the results of the LDLR binding assay (SPR) and the binding kinetics of high - surfactant preparations for the selected peptides shown in Figures 1A - 1I as described herein.

Figure 24A

[0080] Figure 24A is a table showing the results of the LDLR binding assay (SPR) and the binding kinetics of lipid - dependent binding with low - surfactant preparations for the selected peptides shown in Figures 1A - 1I as described herein.

Figure 24B

Figure 25A-25D

[0081] Figures 25A-25D are graphs showing the activities of non-oxidized and oxidized lipids that bind to various immobilized peptides in the list of FIGS. 1A-1I (SPR).

Figure 25E-25H

Figure 25I-25L

Figure 25M-25O

Figure 26

[0082] Figure 26 summarizes the results of the LDL oxidation assay. Figure 26A is a graph showing the change over time in LDL oxidation measured by UV spectrophotometry at a single concentration (25 μM) for the selected peptides shown in FIGS. 1A-1I. Figure 26B is a bar graph showing the LDL oxidation rate calculated as the maximum absorbance divided by the length of the lag period at a single concentration (25 μM) for the selected peptides shown in FIGS. 1A-1I.

Figure 27

[0083] Figure 27A is a graph showing the results of the HMC3 cholesterol efflux assay at a single concentration (20 μM) for the selected peptides shown in FIGS. 1A-1I. Figure 27B is a graph showing the results of the HMC3 cholesterol efflux assay at a single concentration (20 μM) for the selected peptides shown in FIGS. 1A-1I.

Figure 28

[0084] Figure 28 summarizes the results of the HMC3 cholesterol efflux assay (compared to the control) at a single concentration (20 μM) for variants of peptide T-101 (peptides T-121 to T-151) described herein.

Figure 29

[0085] Figure 29 summarizes the results of the ARPE-19 cholesterol efflux assay (compared to the control) at a single concentration (20 μM) for variants of peptide T-087 (peptides T-152 to T-178) described herein.

Figure 30A-30F

[0086] Figures 30A - 30F are graphs of the HMC3 cholesterol efflux titration curves for a range of peptide concentrations for the selected peptides shown in Figures 1A - 1I.

Figure 30G-30J

Figure 31

[0087] Figure 31A illustrates the results of the cholesterol efflux assay in ARPE-19 cells after siRNA knockdown of various ATP-binding cassette family members and SR-B1 at a single concentration (20 μM) for peptide T-087. Figure 31B illustrates the results of the cholesterol efflux assay in ARPE-19 cells after siRNA knockdown of various ATP-binding cassette family members and SR-B1 at a single concentration (20 μM) for peptide T-101.

Figure 32

[0088] Figure 32 is a graph of cholesterol efflux in iPS-RPE cells at a single concentration (10 μM) for peptides T-087 and T-101 described herein.

Figure 33

[0089] Figure 33 illustrates the results of an ABCA1 stability assay using J774 cells. Figure 33A shows a representative Western blot of ABCA1 membrane levels after washing out 8-Br-cAMP and treatment with buffer control or peptide T-101 or T-087 (10 μM), or the positive control protein apoA1 (350 nM). Figure 33B is a graph showing the quantification of band density measurements for membrane ABCA1 after treatment with either buffer control or peptide T-101 or T-087 (10 μM). Figure 33C is a graph showing the change in percentage of membrane ABCA1 in various concentrations of peptide T-101 or T-087 described herein.

Figure 34

[0090] Figure 34 illustrates the results of an ABCA1 stability assay using ARPE-19 cells treated with mCRP and peptides T-101 and T-087. Figure 34A shows Western blot data from cells treated with 10 μg / mL of mCRP and 20 μM of peptides T-101 and T-087. Figure 34B is a graph showing the concentration-response of peptides T-087 and T-101 in this assay.

Figure 35

[0091] Figure 35 is a graph showing the results of an ABCA1 stability assay using ARPE-19 cells at a single concentration (20 μM) for peptide T-087 and modifications of peptide T-087 described herein.

Figure 36

[0092] Figure 36 is a graph showing the results of an ABCA1 stability assay using ARPE-19 cells at a single concentration (20 μM) for peptide T-101 and modifications of peptide T-101 described herein.

Figure 37

[0093] Figure 37 is a graph showing a positive correlation between cholesterol efflux (shown in Figure 29) and hydrophobic moment from ARPE-19 cells for variants of peptide T-087 (T-152 to T178). Linear regression was used to determine the slope deviation from zero.

Figure 38

[0094] Figure 38A graphically shows the positive correlation between cholesterol efflux (shown in Figure 28) and hydrophobicity from HMC3 cells for peptides T-121 to T-151 (variants of peptide T-101). Hydrophobicity is defined by Fauchere and Pliska (Fauchere and Pliska, 1983. Hydrophobic Parameters II of Amino-Acid Side Chains from the Partitioning of N-Acetyl-Amino-Acid Amides. Eur J Med Chem). Linear regression was used to determine the slope deviation from zero.

[0055]

[0095] Figure 38B illustrates the positive correlation between ABCA1 stability (shown in Figure 36) and hydrophobic moment in ARPE-19 cells for peptides T-121 to T-151. Linear regression was used to determine the slope deviation from zero.

Figure 39

[0096] Figures 39A - 39E show representative H&E stained sections of the eyes of adult C57BL / 6 mice treated with the peptides (T-087, T-101, T-112, and T-114) described herein, demonstrating in vivo tolerance 7 days after intravitreal injection of 1 μL of 520 μM peptide.

Figure 40

[0097] Figures 40A - 40B illustrate the evaluation of peptide-mediated reduction of sub-RPE BODIPY+ lipid deposits in apoE- / - mice for peptides T-087 and T-101. Peptides (1 μL at 520 μM or 260 μM) were delivered by intravitreal injection, and sub-RPE lipid deposits were measured on day 14 after injection by BODIPY staining of retinal sections.

Figure 41

[0098] Figure 41A shows the residues of peptide T-087 (shown in circles) that are the same as or can be substituted for conservative amino acid substitutions for maintaining the desired therapeutic activity of the peptide, including activities regarding safety, cholesterol efflux ability, and ABCA1 stabilization ability. The residues shown without circles indicate positions where any amino acid can be substituted to maintain the desired therapeutic activity of the peptide, including activities regarding safety, cholesterol efflux ability, and ABCA1 stabilization ability. Figure 41B shows the residues of peptide T-101 (shown in circles) that are the same as or can be substituted for conservative amino acid substitutions for maintaining the desired therapeutic activity of the peptide, including activities regarding safety, cholesterol efflux ability, and ABCA1 stabilization ability. The residues shown without circles indicate positions where any amino acid can be substituted to maintain the desired therapeutic activity of the peptide, including activities regarding safety, cholesterol efflux ability, and ABCA1 stabilization ability.

Mode for Carrying Out the Invention

[0056]

[0099] The compositions and methods described herein can be used to treat AMD, particularly AMD patients in whom the disease is driven primarily by dysfunction of lipid transport and processing. For example, the synthetically engineered mimetic peptides described herein provide therapeutic opportunities for safely and effectively addressing pathophysiology, such as dysregulated lipid homeostasis.

[0057] [000100] In particular, apolipoproteins are endogenously synthesized in response to intracellular and extracellular lipid transport and processing requirements. Generally, apolipoproteins are a family of amphipathic molecules that, as part of the lipid homeostasis mechanism, have the ability to bind to lipids and transport lipids into and out of cells and peripheral tissues. For example, apolipoproteins are components of HDL / LDL molecules. Apolipoprotein E (apoE) is generally associated with LDL, while apolipoprotein A1 (apoA1) is generally associated with HDL.

[0058] [000101]The cellular uptake of lipids such as cholesterol can occur due to the metabolic requirements of the cell or in response to high extracellular cholesterol concentrations. Apolipoprotein E (e.g., apoE) is generally associated with mechanisms related to cholesterol uptake. The low-density lipoprotein receptor (LDLR) is a surface receptor that has a high affinity for apoE. Retinal pigment epithelial (RPE) cells have been shown to express LDLR, and LDLR enables a pathway for the uptake of cholesterol from cholesterol-rich LDL / apoE complexes.

[0059] [000102]Alternatively, cholesterol efflux is the mechanism by which cholesterol is excreted from within the cell to the extracellular environment through one or more transporter proteins (e.g., ATP-binding cassette transporters or SR-B1). The transporter effluxes cholesterol, and that cholesterol then binds to lipid-poor apoA1 as a component in the formation of HDL. As apoA1 accumulates cholesterol from the transporter protein, HDL becomes saturated and continues RCT as part of the lipid transport and processing mechanisms for cholesterol homeostasis and lipid regulation.

[0060] [000103]Genotypic variations, environmental factors, and aging can contribute to dysregulation of lipid homeostasis associated with defective apolipoprotein activity. Synthetically engineered mimetic peptides having lipid transport and processing activities, as detailed below, are described herein. These synthetic peptides may account for a family of peptides that exhibit significant therapeutic efficacy (including conservative substitutions of the peptides described herein).

[0061] [000104]In some examples, dysfunction of lipid transport and processing may be related to impaired lipid efflux from one or more cells or tissues associated with the etiology of AMD. For example, RPE cell-specific deletion of the ABCA1 gene in a mouse model causes abnormal lipid accumulation, retinal inflammation, and RPE photoreceptor degeneration (PMID 30864945). Apolipoproteins, and particularly apolipoprotein A1 (apoA1), act as regulators of lipid processing and transport of lipid molecules to maintain cholesterol homeostasis. ApoA1 can interact with one or more cholesterol efflux regulatory proteins, such as ABCA1, ABCG1, or SR-B1. Cholesterol efflux regulatory proteins transport cholesterol to apoA1, and apoA1 binds to cholesterol in the formation of nascent HDL. As cholesterol accumulates, lipid-rich HDL circulates and is sent to the liver where lipid deposition and metabolism occur.

[0062] [000105]In some examples where a major dysfunction in lipid processing is related to lipid accumulation due to inappropriate exocytosis of lipids by one or more cholesterol regulatory proteins, patients with AMD may benefit from administration of one or more of the therapeutic peptides described herein (e.g., those included in SEQ ID NOs: 5-30; or 35-178). For example, in the presence of an excessive rate of cholesterol influx and corresponding intracellular lipid concentrations, administration of one or more of the therapeutic peptides described herein (e.g., those included in SEQ ID NOs: 5-30; or 35-178) establishes or restores an effective lipid transport and processing mechanism mediated by one or more apolipoproteins. As described in more detail herein, some of these peptides may be more effective than others, but generally, they may provide therapeutic use.

[0063] [000106]In some examples, including but not limited to, examples where the main contributing factor is one or more dysfunctions related to lipid transport and processing pathways, a patient can be treated with one or more of the therapeutic peptides described herein or modified versions of these peptides (e.g., those included in SEQ ID NOs: 5 - 30; or 35 - 178). In particular, these one or more therapeutic peptides are peptides that reduce lipid accumulation in Bruch's membrane, the subretinal space, or reticular pseudodrusen, preventing or reducing AMD-related effects. For example, the methods described herein can replace lipid regulatory defects in Bruch's membrane by intravitreal injection of one or more of the therapeutic peptides described herein.

[0064] [000107]The present disclosure herein provides compositions and methods for treating, preventing, or inhibiting eye diseases. For example, the present disclosure herein provides engineered therapeutic peptides (therapeutic polypeptides) that selectively bind to lipids, solubilize lipids, activate lipid efflux mechanisms via ABCA1 and related transporters, improve cholesterol homeostasis, and reduce drusen formation. The present disclosure provides methods for treating, preventing, or inhibiting eye diseases by administering an effective amount of these compositions of the present disclosure intravitreally (e.g., intravitreally) to the eye using the methods provided herein. Eye diseases that can be treated or prevented using these methods include, but are not limited to, glaucoma, macular degeneration (e.g., age-related macular degeneration, AMD), diabetic retinopathy, hereditary retinal degenerations such as retinitis pigmentosa, retinal detachment or injury, and retinopathy (whether hereditary; surgical; traumatic; underlying etiology, e.g., severe anemia, SLE, hypertension, blood disorders, diabetes, systemic infections, or underlying carotid artery disease; induced by toxic compounds or drugs; or induced by light, etc.).

[0065] [000108]The present disclosure herein provides compositions and methods for treating, preventing, or inhibiting eye diseases. For example, the present disclosure herein provides engineered therapeutic peptides (therapeutic polypeptides) that selectively bind to lipids, solubilize lipids, increase LDLR binding activity, increase lipid transport activity via LDLR and related receptors, improve cholesterol homeostasis, and reduce drusen formation. In addition, the present disclosure herein provides engineered therapeutic peptides that selectively activate ABCA1, promote cholesterol efflux from cells through ABCA1-specific mechanisms, improve cholesterol homeostasis, and reduce drusen formation and progression. The present disclosure provides methods for treating, preventing, or inhibiting eye diseases by administering an effective amount of these compositions of the present disclosure intravitreally (e.g., intravitreally) to the eye using the methods provided herein. Eye diseases that can be treated or prevented using these methods include glaucoma, macular degeneration (e.g., age-related macular degeneration, AMD), diabetic retinopathy, hereditary retinal degenerations such as retinitis pigmentosa, retinal detachment or injury, and retinopathy (whether hereditary; surgical; traumatic; underlying etiology, e.g., severe anemia, SLE, hypertension, blood disorders, diabetes, systemic infections, or underlying carotid artery disease; induced by toxic compounds or drugs; or induced by light, etc.), but are not limited thereto.

[0066] [000109]Unless otherwise defined herein, scientific and technical terms used in this application have the meanings commonly understood by one of ordinary skill in the art. [000110]Generally, the nomenclature and techniques used in connection with pharmacology, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, genetics, and protein and nucleic acid chemistry as described herein are those well known and commonly used in the art. In case of conflict, this specification, including definitions, will control.

[0067] [000111]The practice of the present disclosure uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art unless otherwise indicated. Such techniques are described in Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait (ed.), 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis (ed.), 1998) Academic Press; Animal Cell Culture (R.I. Freshney (ed.), 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell (eds.), 1993-1998) J.Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos (eds.), 1987); Current Protocols in Molecular Biology (F.M.Fully described in the literature such as Ausubel et al. (eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al. (eds., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0068] [000112]Enzyme reactions and purification techniques are carried out according to the manufacturer's instructions, as generally achieved in the art or as described herein. The nomenclature used in connection with analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as their laboratory procedures and techniques, are well known and commonly used nomenclature, procedures, and techniques in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0069] [000113]When aspects or examples of the present disclosure are described with respect to a Markush group or other group of alternatives, the present disclosure encompasses not only the entire recited group as a whole, but also each individual member of the group and all possible subgroups of the main group, as well as the main group with one or more of the members of the group being absent. The present disclosure also contemplates any one or more explicit exclusions of members of the group in the disclosed examples. Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present disclosure. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0070] Definitions [000114]The following terms are understood to have the following meanings unless otherwise indicated. [000115]As used herein, "residue" refers to a position in a protein and its attendant amino acid identity. As is known in the art, "polynucleotide" or "nucleic acid" are used interchangeably herein and refer to a chain of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substitutions that can be incorporated into the chain by DNA polymerase or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. Where present, modifications to the nucleotide structure can be imparted before or after assembly of the chain. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "cap" substitution of one or more of the naturally occurring nucleotides with an analog; internucleotide modifications, such as modifications by uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.); modifications containing pendant moieties, such as proteins (e.g., nuclease, toxin, antibody, signal peptide, poly-L-lysine, etc.); modifications by intercalators (e.g., acridine, psoralen, etc.); modifications containing chelators (e.g., metal, radioactive metal, boron, metal oxide, etc.); modifications containing alkylating agents; modifications having modified linkages (e.g., α-anomer nucleic acid, etc.); and polynucleotides in unmodified form. Further, any of the hydroxyl groups normally present on the sugar can be replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to provide additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OHs can be phosphorylated or replaced by an amine or an organic capping group moiety of 1 to 20 carbon atoms. Also, other hydroxyls can be derivatized with standard protecting groups.In addition, the polynucleotide may contain analogs of ribose or deoxyribose sugars commonly known in the art, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups are such that the phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NRi (“amidate”), P(O)R, P(O)OR’, CO or CH2 (“formacetal”), where each R or R’ is independently H, or substituted or unsubstituted alkyl (C. 1~20 ) and includes, but is not limited to, examples containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in the polynucleotide need to be the same. The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0071] [000116] The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a chain of amino acids of any length. The chain may be linear or branched and may contain modified amino acids and / or be interrupted by non-amino acids. The term also encompasses amino acid chains that are naturally or artificially modified, such as by disulfide bond formation, glycosylation, lipid addition, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included in this definition are polypeptides containing, for example, one or more analogs of amino acids (including, for example, non-natural amino acids) and other modifications known in the art. It is understood that the polypeptide may occur as a single chain or as associated chains.

[0072] [000117] "Identical" refers to the relationship between two proteins that possess a common sequence, including protein sequences from the same superfamily within the same biological species and homologous proteins from different biological species, in all their grammatical forms and spelling variations. Such proteins (and their coding nucleic acids) have sequence homology reflected by their percent identity or their sequence similarity based on the presence and conserved positions of specific residues or motifs. However, in general use and in this application, the term "identical" may refer to sequence similarity, particularly (but not exclusively) when modified by a percentage, and may or may not be related to common evolutionary origin.

[0073] [000118] The term "sequence similarity" refers to the degree of identity or correspondence between nucleic acid or amino acid sequences, in all their grammatical forms, which may or may not share a common evolutionary origin. "Percent sequence identity" or "identical to %" for a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to the amino acid residues (or nucleic acids) in the reference polypeptide (nucleotide) sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, using, for example, publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithm required to achieve the maximum alignment over the entire length of the sequences being compared.

[0074] [000119] The term "conservative substitution" refers to the substitution of an amino acid in a polypeptide with a natural or non-natural amino acid that is functionally, structurally, or chemically similar. In certain embodiments, each of the following groups contains natural amino acids that are conservative substitutions for one another. 1) Glycine (G), Alanine (A); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K), Histidine (H); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), Alanine (A); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); and 7) Serine (S), Threonine (T), Cysteine (C).

[0075] [000120]The peptides described herein may include substitutions that do not disrupt the helical structure of the peptide in a manner that disrupts the functions described below. In particular, a peptide (e.g., T-087, T-101, etc.) may include one or more substitutions (referred to herein as conservative hydrophobic substitutions) such that the hydrophobicity of the residue being substituted is within 0.25 of the hydrophobicity value of the original amino acid. For example, (1) alanine (A) may be substituted by threonine (T) or histidine (H); (2) glutamic acid (E) may be substituted by asparagine (N) or aspartic acid (D); (3) arginine (R) may be substituted by aspartic acid (D) or lysine (K); (4) phenylalanine (F) may be substituted by cysteine (C), leucine (L), or isoleucine (I); (5) leucine (L) may be substituted by isoleucine (I), phenylalanine (F), or cysteine (C); (6) lysine (K) may be substituted by aspartic acid (D) or arginine (R); (7) aspartic acid (D) may be substituted by asparagine (N), glutamic acid (E), lysine (K), or arginine (R); (8) tyrosine (Y) may be substituted by proline (P); (9) serine (S) may be substituted by glutamine (Q), glycine (G), or histidine (H); (10) isoleucine (I) may be substituted by leucine (L) or phenylalanine (F); (11) threonine (T) may be substituted by alanine (A) or histidine (H); (12) serine (S) may be substituted by histidine (H), serine (S), or glutamine (Q). Hydrophobicity is determined using the techniques of Fauchere and Pliska (e.g., Fauchere and Pliska, Eur J Med Chem, 1983).

[0076] [000121]Alternatively or in addition, the peptides described herein may include substitutions (referred to herein as conservative size substitutions) where the size of the residue to be substituted is approximately the same as the original amino acid and / or is the next closest in size. For example, aspartic acid (D) may be substituted by asparagine (N) or lysine (K); alanine (A) may be substituted by glycine (G) or serine (S); tryptophan (W) may be substituted by tyrosine (Y); glutamic acid (E) may be substituted by glutamine (Q) or methionine (M); arginine (R) may be substituted by phenylalanine (F) or tyrosine (Y); phenylalanine (F) may be substituted by histidine (H) or arginine (R); leucine (L) may be substituted by cysteine (C) or isoleucine (I); lysine (K) may be substituted by aspartic acid (D) or glutamine (Q); tyrosine (Y) may be substituted by arginine (R) or tryptophan (W); serine (S) may be substituted by alanine (A) or proline (P); glycine (G) may be substituted by alanine (A); isoleucine (I) may be substituted by leucine (L) or asparagine (N); threonine (T) may be substituted by valine (V) or cysteine (C).

[0077] [000122]As used herein, an "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide or fragment thereof) is one that is not associated with one or more of the naturally associated components that accompany it in its native state, (2) is substantially free of one or more other molecules from the same species, (3) is expressed by cells from a different species, or (4) is a molecule that does not occur naturally, due to its source of origin or derivation. The therapeutic polypeptides described herein can be isolated.

[0078] [000123]As used herein, "purify" and its grammatical variations refer to the removal of at least one impurity from a mixture containing a polypeptide and one or more impurities, whether complete or partial, and the removal thereby improves the level of purity of the polypeptide in the composition (i.e., by reducing the amount (ppm) of impurities in the composition). The therapeutic polypeptides described herein may be referred to as purified.

[0079] [000124]As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure. The therapeutic polypeptides described herein may be substantially pure.

[0080] [000125]The terms "patient", "subject", or "individual" are used interchangeably herein and refer to either a human or a non-human animal. These terms include mammals such as humans, non-human primates, laboratory animals, domestic animals (including cows, pigs, camels, etc.), companion animals (e.g., dogs, cats, other pet animals, etc.), and rodents (e.g., mice and rats). In some examples, the subject is a human who is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old.

[0081] [000126]In one example, the subject has or is at risk of developing eye diseases. Eye diseases include, but are not limited to, retinitis pigmentosa, rod-cone dystrophy, Leber congenital amaurosis, Usher syndrome, Bardet-Biedl syndrome, Best disease, retinal detachment, Stargardt disease (autosomal dominant or autosomal recessive), untreated retinal detachment, pattern dystrophy, cone-rod dystrophy, color vision abnormalities, albinism, S cone opsin excess syndrome, diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, sickle cell retinopathy, congenital stationary night blindness, glaucoma, or retinal vein occlusion. In another example, the subject has or is at risk of developing glaucoma, Leber hereditary optic neuropathy, lysosomal storage disease, or peroxisomal disease. In some examples, the subject exhibits clinical signs of an eye disease.

[0082] [000127]In some examples, the subject has or is at risk of developing a kidney disease or complication. In some examples, the kidney disease or complication is associated with AMD or atypical hemolytic uremic syndrome (aHUS). In some examples, the subject has or is at risk of developing AMD or aHUS.

[0083] [000128]Clinical signs of an eye disease include, but are not limited to, a reduction in peripheral vision, a reduction in central (reading) vision, a reduction in night vision, loss of color vision, reduced visual acuity, reduced photoreceptor function, and pigmentary changes. In one example, the subject exhibits degeneration of the outer nuclear layer (ONL). In another example, the subject is diagnosed with an eye disease. In yet another example, the subject has not yet exhibited clinical signs of an eye disease.

[0084] [000129]As used herein, the terms "prevent," "preventing," and "prevention" refer to the prevention of recurrence or onset of a disease or condition (e.g., an eye disease) in a subject, or a reduction in one or more symptoms of the disease or condition, as a result of administration of a treatment (e.g., a prophylactic or therapeutic agent). For example, with respect to administration of a treatment to a subject, "prevent," "preventing," and "prevention" mean inhibition or reduction in the onset, initiation, or progression of a disease or condition (e.g., an eye disease) in the subject, or prevention of recurrence, initiation, or onset of one or more symptoms of the disease or condition (e.g., an eye disease), resulting from administration of a treatment (e.g., a prophylactic agent or therapeutic agent) or a combination of treatments (e.g., a combination of prophylactic agents or therapeutic agents).

[0085] [000130]"Treating" a condition or patient refers to taking steps to obtain a beneficial or desired result, including a clinical result. For a disease or condition (e.g., an eye disease), treatment refers to a reduction or improvement in the progression, severity, and / or duration of the condition (e.g., an eye disease or a symptom associated therewith), or an improvement in one or more symptoms resulting from administration of one or more treatments (including, without limitation, administration of one or more prophylactic or therapeutic agents).

[0086] [000131]"Administering" or "administration" of a substance, compound, or agent to a subject can be effected using one of a variety of methods known to those of skill in the art. For example, a compound or agent can be administered intravitreally, subretinally, or systemically. In certain examples, a compound or agent is administered intravitreally. In some examples, administration can be local. In other examples, administration can be systemic. Also, administration can be effected, for example, once, a plurality of times, and / or over one or more extended periods. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have another person administer a drug to the patient, and / or provides a prescription for the drug to the patient, is administering the drug to the patient.

[0087] [000132]Each example described in this specification can be used individually or in combination with any other example described in this specification. [000133]In some examples, the therapeutic peptides described in this specification are synthetically engineered mimetics related to the structure or function of one or more endogenous molecules. For example, endogenous apolipoproteins involved in lipid transport and processing mechanisms are responsible for the solubilization and transport of lipid molecules for cell uptake or efflux. In particular, apolipoproteins have a direct involvement in cholesterol homeostasis in the RPE related to the uptake or efflux of lipids such as cholesterol.

[0088] [000134]The therapeutic compositions described in this specification may include one or more therapeutic peptides having lipid solubilization and lipid efflux activity, and the cytotoxicity of these therapeutic compositions can be minimal.

[0089] [000135]As mentioned, the therapeutic peptides described in this specification can be short amphiphilic polypeptides (e.g., 80 amino acids or less, 75 amino acids or less, 70 amino acids or less, 65 amino acids or less, 60 amino acids or less, 55 amino acids or less, 50 amino acids or less, 45 amino acids or less, 40 amino acids or less, less than 35 amino acids, less than 30 amino acids, etc.) that bind to one or more cholesterol efflux transporter proteins, such as ABCA1 or other transporters, and can promote lipid efflux from cells. Thus, the therapeutic peptide mimetics described in this specification may have a structure homologous to endogenous apolipoprotein A (e.g., apoA1), and the function can provide a therapeutic benefit in conditions based on dysregulation of the lipid efflux mechanism for the prevention of lipid accumulation or increased drusen formation.

[0090] [000136]The therapeutic compositions described in this specification may include one or more therapeutic peptides having lipid solubilization and lipid influx activity, and the cytotoxicity of these therapeutic compositions can be minimal.

[0091] [000137]As mentioned, the therapeutic peptides described herein can be short amphipathic substances that bind to the LDLR in a lipid-dependent manner and transport lipids into cells via the LDLR receptor. These therapeutic peptides can overcome lipid transport defects present in apoE2 carriers, such as apoE2 variants having a defect in LDLR binding activity. These therapeutic peptides preserve lipid-dependent LDLR binding and lipid transport activity in smaller peptides. Thus, the therapeutic peptidomimetics described herein may have a structure homologous to endogenous apolipoprotein E (such as apoE), and the function can provide a therapeutic benefit in conditions based on dysregulation of the lipid translocation mechanism for the prevention of lipid accumulation or increased drusen formation.

[0092] [000138]Figures 1A - 1I illustrate peptides including controls (T - 001 - T - 004 and T - 031 - T - 034) and therapeutic peptides (T - 005 - T - 030 and T - 35 - T - 178, corresponding to SEQ ID NOs: 5 - 30 and 35 - 178, respectively). These peptides, or modified versions of these peptides, can be used as the therapeutic peptides described herein. Also, the tables shown in Figures 1A - 1I include the helical hydrophobic moment (μH), which is a measure of the amphipathicity of the helices of the peptides.

[0093] [000139]As mentioned, any of the peptides, T-005 to T-030, T-35 to T-080, and T-084 to T-178, and related peptides (e.g., peptides having 65% or more homology) can be used therapeutically as described herein. Peptides that are at least 65% (in some examples, at least 75% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous) to any of the peptides of T-005 to T-030, T-35 to T-080, and T-084 to T-178 can refer to homologous peptides where the different amino acid residues are conservative substitutions. In any of these examples, the homologous peptides can refer to peptides where the amino acids different from the peptides of T-005 to T-030, T-035 to T-080, and T-084 to T-178 are conservative hydrophobic substitutions and / or conservative size substitutions and / or conservative charge substitutions. In any of these examples, the conservative substitutions (and / or conservative hydrophobic substitutions and / or conservative size substitutions and / or conservative charge substitutions) maintain the helical structure described herein.

[0094] [000140]In particular, a peptide that is an example, the T-087 (SEQ ID NO: 87) peptide, or a peptide that is at least 65% identical to the T-087 peptide can be used therapeutically as described herein. For example, FIG. 41A shows the sequences of one family of homologous peptides based on the T-087 peptide. In FIG. 41A, the amino acids within the enclosure indicate residues that contribute to the desired therapeutic activity of the peptide, including safety, cholesterol efflux ability, and ABCA1 stabilization ability. These residues were determined by testing derivatives of T-087 generated through alanine scanning (single and triple) mutagenesis in a functional assay as described in FIGS. 7D, 29, and 35. Peptides T-152 to T-178 exemplify some of the peptides of the examples corresponding to variants of T-087. In FIG. 41A, the amino acids outside the enclosure can correspond to any amino acid.In some examples, the amino acids within the enclosure correspond to conservative substitutions (e.g., using glycine (G) at position 2, phenylalanine (F) at position 3, aspartic acid (D) at position 4, lysine (K) at position 5, leucine (L) at position 6, arginine (R) at position 7, isoleucine (I) at position 9, arginine (R) at position 11, aspartic acid (D) at position 12, valine (V) at position 13, glycine (G) at position 14, glutamic acid (E) at position 15, tryptophan (W) at position 16, and tyrosine (Y) at position 17), conservative hydrophobic substitutions (e.g., threonine (T) at position 2, aspartic acid (D) at position 4, aspartic acid (D) at position 5, isoleucine (I) at position 6, aspartic acid (D) at position 7, phenylalanine (F) at position 9, aspartic acid (D) at position 11, asparagine (N) at position 12, phenylalanine (F) at position 13, threonine (T) at position 14, lysine (K) at position 15, proline (P) at position 16, and leucine (L) at position 17), or conservative size substitutions (e.g., glycine (G) or serine (S) at position 2, tyrosine (Y) at position 3, glutamine (Q) or methionine (M) at position 4, phenylalanine (F) or tyrosine (Y) at position 5, histidine (H) or arginine (R) at position 6, phenylalanine (F) or tyrosine (Y) at position 7, cysteine (C) or isoleucine (I) at position 9, aspartic acid (D) or glutamine (Q) at position 11, glutamine (Q) or methionine (M) at position 12, cysteine (C) or isoleucine (I) at position 13, glycine (G) or serine (S) at position 14, asparagine (N) or lysine (K) at position 15, arginine (R) or tryptophan (W) at position 16, and histidine (H) or arginine (R) at position 17)).

[0095] [000141]In another example, the T-101 (SEQ ID NO: 101) peptide or a peptide that is at least 65% identical to the T-101 peptide can be used therapeutically as described herein. For example, FIG. 41B shows the sequences of one family of homologous peptides based on the T-101 peptide. In FIG. 41B, the amino acids within the box indicate the residues that contribute to the desired therapeutic activity of the peptide, including safety, cholesterol efflux ability, and ABCA1 stabilization ability. These residues were determined by testing derivatives of T-101 created through alanine scanning mutagenesis (single and triple) in a functional assay as described in FIGS. 7C, 28, and 36. Peptides T-121 to T-151 exemplify some of the peptides of the examples corresponding to variants of T-101. In FIG. 41B, the amino acids outside the box can correspond to any amino acid. In some examples, the amino acids within the box are conservative substitutions (e.g., using lysine (K) at position 1, glycine (G) at position 4, glutamic acid (E) at position 5, glycine (G) at position 6, valine (V) at position 7, aspartic acid (D) at position 8, glycine (G) at position 10, isoleucine (I) at position 11, arginine (R) at position 12, aspartic acid (D) at position 13, valine (V) at position 14, arginine (R) at position 15, tryptophan (W) at position 17, leucine (L) at position 18, aspartic acid (D) at position 20, phenylalanine (F) at position 21, and serine (S) at position 22), conservative hydrophobic substitutions (e.g., aspartic acid (D) at position 1, threonine (T) at position 4, lysine (K) or arginine (R) at position 5, histidine (H) at position 6, phenylalanine (F) at position 7, asparagine (N) at position 8, threonine (T) at position 10, isoleucine (I) at position 11, aspartic acid (D) at position 12, asparagine (N) at position 13, cysteine (C) at position 14, aspartic acid (D) at position 15, leucine (L) at position 17, phenylalanine (F) at position 18,Asparagine (N) at position 20 and histidine (H) at position 22), or conservative size substitutions (e.g., phenylalanine (F) or tyrosine (Y) at position 1, glycine (G) or serine (S) at position 4, asparagine (N) or lysine (K) at position 5, glycine (G) or serine (S) at position 6, cysteine (C) or isoleucine (I) at position 7, glutamine (Q) or methionine (M) at position 8, glycine (G) or serine (S) at position 10, cysteine (C) or isoleucine (I) at position 11, aspartic acid (D) or glutamine (Q) at position 12, glutamine (Q) or methionine (M) at position 13, cysteine (C) or isoleucine (I) at position 14, aspartic acid (D) or glutamine (Q) at position 15, histidine (H) or arginine (R) at position 17, leucine (L) or asparagine (N) at position 18, glutamine (Q) or methionine (M) at position 20, tyrosine (Y) at position 21, and valine (V) or cysteine (C) at position 22).

[0096] [000142]The safety and functional activity of the peptides described herein are exemplified in the data shown in the drawings and herein. For example, FIGS. 2A-2D illustrate the lipid solubilization activity measured by reducing the turbidity of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposome solution for each of the peptides shown. In this example, the peptide stock was diluted to 200 μM in H2O (2× solution), and 50 μL of the peptide solution was transferred to the 96-well plate in three sets each. A 1 mM solution of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in H2O was prepared, and 50 μL of the DMPC solution was rapidly added to the plate using a multi-channel pipette. The peptide and lipid solutions were incubated at room temperature for 15 minutes at a final concentration of 100 μM peptide and 0.5 mM DMPC. After 15 minutes, the absorbance at 405 nm was read in a plate reader. The H2O blank was used for background correction, and 100% lipid clearance was determined using the absorbance of DMPC incubated in 1% Triton-X-100. The data are shown as the average value + SD of two runs. Almost all of the peptides tested solubilized more than 50% of the lipid over the course of the assay. Eleven of the test peptides (T-025, T-027, T-051, T-052, T-054, T-059, T-062, T-063, T-065, T-069, and T-080) were examined in more detail as shown in FIGS. 3A-3C. In these experiments, DMPC solubility titration curves were generated. The peptide stock was diluted to 200 μM in H2O (2× solution), serially diluted over 4 concentrations in H2O, and then 50 μL of the peptide solution was dispensed into the 96-well plate in three sets each. A 1 mM solution of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in H2O was prepared, and 50 μL of the DMPC solution was rapidly added to the plate using a multi-channel pipette. The peptide and lipid solutions were incubated at room temperature for 15 minutes at a final concentration of 3.16-100 μM peptide and 0.5 mM DMPC. After 15 minutes, the absorbance at 405 nm was read in a plate reader.The H2O blank was used for background correction, and 100% lipid clearance was determined using the absorbance of DMPC incubated in 1% Triton-X-100. Data are shown as the mean value + SD of two runs. Non-linear regression was used to calculate the EC. 50 values. The test peptide EC 50 values in the assay were (T-025, 25.33 μM; T-027, 56.55 μM; T-051, 15.6 μM; T-052, 20.6 μM; T-054, 122.6 μM; T-062, 213.4 μM; T-063, 110.6 μM; T-065, 24.4 μM; T-069, 38.77 μM; T-080, 154.8 μM).

[0097] [000143] From these results shown in FIGS. 2A-2D and 3A-3C, the peptides appear to be particularly therapeutically useful when more than 50% are cleared at 100 μM in 15 minutes (and especially when active at 10 μM). Many of the non-control peptides had significant activity. See, for example, T-006, T-007, T-009, T-010, T-011, T-012, T-013, T-014, T-024, T-026, T-030, T-018, T-019, T-021, T-025, T-027, T-028, T-029, T-086, T-087, T-101, T-112, T-114, T-116. Some of these peptides (T-005, T-008, T-015, T-017, T-020, and T-099) had no significant solubilizing activity.

[0098] [000144]Figures 4A - 4B illustrate a screening assay based on DMPC solubility, measured by reducing the turbidity of a DMPC liposome solution, for a subset of the peptides in Figures 1B - 1I. Figures 4A - 4B illustrate DMPC solubility titration curves. The peptide stock was diluted to 200 μM in H2O (2× solution), serially diluted over 4 concentrations in H2O, and then 50 μL of the peptide solution was dispensed in triplicate into a 96 - well plate. A 2 mM solution of 1,2 - dimyristoyl - sn - glycero - 3 - phosphocholine (DMPC) in H2O was prepared, and using a multi - channel pipette, 50 μL of the DMPC solution was rapidly added to the plate. The peptide and lipid solutions were incubated for 15 minutes at room temperature at final concentrations of 3.16 - 100 μM peptide and 1 mM DMPC. After 15 minutes, the absorbance at 405 nm was read in a plate reader. An H2O blank was used for background correction, and 100% lipid clearance was determined using the absorbance of DMPC incubated in 1% Triton - X - 100. The data are shown as the mean value + SD of two runs. Non - linear regression was used to calculate the EC 50 value. The test peptide EC 50 values in the assay were (T - 084, 16.15 μM; T - 086, 15.81 μM; T - 087, 17.82 μM; T - 101, 10.50 μM; T - 112, 8.26 μM; T - 113, 8.35 μM; T - 114, 12.21 μM; T - 116, 7.54 μM).

[0099] [000145]The selection test peptides were further evaluated as shown in FIGS. 5A-5D. These drawings illustrate ARPE-19 cell lysis. ARPE-19 cells were grown to 75-85% confluence in 96-well plates. A 10 mM peptide was diluted in serum-free DMEM:F12 medium to a 20 μM working solution (2× solution). Sytox Green was prepared in serum-free DMEM:F12 at a 1 μM working solution (2× solution). Equal volumes of the peptide and Sytox Green solutions were added to an empty 96-well plate and gently mixed. The final peptide concentration was 10 μM. ARPE-19 cells (approximately 80% confluent in 96-well plates) were washed with DPBS and then incubated at 37° C. for 2 hours in 100 μL of the peptide / Sytox Green mixture. The cells were fixed in a 4% paraformaldehyde solution in DPBS and then incubated in a 10 μM Hoechst solution for 10 minutes, washed, and imaged on a Yokogawa CQ1 high-content imager. Live / dead cells were counted at 20× magnification in 4 fields using CellPathfinder software. The % lysis was calculated by dividing the number of Sytox Green+ nuclei by the total number of Hoechst+ nuclei. In this assay, the test peptides did not induce cell lysis except for T-021, T-028, and T-034.

[0100] [000146]Figure 6 shows the ARPE-19 cell lysis titration curves for a subset of the peptides of FIGS. 1B - 1I. ARPE-19 cells were grown to 75 - 85% confluence in 96-well plates. A 10 mM peptide was diluted in serum-free DMEM:F12 medium to a 6.32 - 200 μM solution. Sytox Green was prepared as a 1 μM working solution in serum-free DMEM:F12 medium. Equal volumes of the peptide and Sytox Green solutions were added to an empty 96-well plate and gently mixed. The final peptide concentration was 3.16 - 100 μM. ARPE-19 cells (approximately 80% confluent in 96-well plates) were washed with DPBS and then incubated at 37 °C for 2 hours in 100 μL of the peptide / Sytox Green mixture. The cells were fixed in a 4% paraformaldehyde solution in DPBS and then incubated for 10 minutes in a 10 μM Hoechst solution, washed, and imaged on a Yokogawa CQ1 high-content imager. Live / dead cells were counted at 20x magnification in 4 fields using CellPathfinder software. The % lysis was calculated by dividing the number of Sytox Green+ nuclei by the total number of Hoechst+ nuclei. Non-linear regression was used to calculate the EC 50 value. The test peptide EC 50 values in the assay were (T-025, 144.1 μM; T-027, 40.14 μM).

[0101] [000147]Figures 7A - 7D illustrate the results of the ARPE - 19 cell viability assay for the peptides described herein. ARPE - 19 cells were seeded at 25,000 cells / well in a 96 - well plate and allowed to adhere to the plate surface for at least 24 hours. The peptides were diluted to a final concentration of 100 μM in serum - free DMEM:F12. The cells were washed and then incubated at 37°C for 1.5 hours in the 100 μM peptide solution. The plates were returned to room temperature for 30 minutes and then washed with DPBS. The cells were incubated at room temperature for 10 minutes in a 1:1 mixture of DPBS and Cell Titer Glo 2.0 reagent, and then the cell luminescence was read on a plate reader. The viability values were background - corrected, and 0% viability was determined by the luminescence value of cells treated with 1% Triton - X - 100. The data are shown as the mean value + SD of two separate runs. Treatment with the test peptides did not result in a substantial change in viability over the course of the assay, except for test peptides T - 062, T - 065, T - 069, T - 077, T - 085, T - 109, T - 110, T - 111, and T - 115.

[0102] [000148]Similar results were seen in human red blood cells (RBC) in the lysis assay, as shown in FIGS. 8A - 8D and 9A - 9B. FIGS. 8A - 8D show the results of the human red blood cell (hRBC) lysis assay for a subset of the peptides. Human red blood cells (hRBC; washed and suspended at 25% cell dilution in Alsevers solution) were added to a 96 - well plate containing 50 μL of a 40 μM peptide solution in two sets of 50 μL aliquots. The plate was incubated at 37 °C for 2 hours, after which the hRBC were pelleted at 500×g for 5 minutes. 50 μL of the supernatant was collected and transferred to a new plate containing 50 μL of PBS per well, and the absorbance at 560 nm was read in a plate reader to determine lysis. 100% lysis reflects wells treated with 0.1% Triton - X - 100. The data are shown as the mean value + SD of two runs. FIGS. 9A - 9B are graphs summarizing the hRBC lysis assay for additional peptides. The test peptides did not induce more than 10% hRBC lysis in the assay.

[0103] [000149]FIGS. 10 and 11 illustrate the human red blood cell (hRBC) lysis titration curves. Human red blood cells (hRBC; washed and suspended at 25% cell dilution in Alsevers solution) were added to a 96 - well plate containing 50 μL of a peptide solution at 6.32 - 200 μM in two sets of 50 μL aliquots (the final peptide concentration was 3.16 - 100 μM). The plate was incubated at 37 °C for 2 hours, after which the hRBC were pelleted at 500×g for 5 minutes. 50 μL of the supernatant was collected and transferred to a new plate containing 50 μL of PBS per well, and the absorbance at 560 nm was read in a plate reader to determine lysis. 100% lysis reflects wells treated with 0.1% Triton - X - 100. The data are shown as the mean value + SD of two runs. Non - linear regression was used to calculate the EC 50 values. The test peptide EC 50The values were (T-025, >100 μM; T-027, >100 μM; T-086, >100 μM; T-087, >100 μM; T-101, >100 μM; T-112, >100 μM; T-114, >100 μM; T-116, >100 μM).

[0104] [000150] Figures 12 and 14A - 14B illustrate the results of the ARPE - 19 cholesterol uptake assay. ARPE - 19 cells were seeded in a 96 - well plate at 25,000 cells per well and grown to confluence. A 2× solution of 500 μM DMPC and 50 μM BODIPY - cholesterol was prepared in serum - free DMEM:F12. Peptides were diluted to 20 μM in serum - free DMEM:F12 (2× solution). Peptides were added to the lipid solution in equal portions (60 μL each) and incubated at 37°C for 1 hour. ARPE - 19 cells were serum - starved in serum - free DMEM:F12 at 37°C for 1 hour. The peptide:lipid complex (total volume of 100 μL per well) was transferred to ARPE - 19 cells and left at 37°C for 2 hours. Cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation and 500 - 550 emission. Fluorescence values were background - corrected from untreated conditions. Data are shown as mean ± SD for two runs. The results illustrated in Figures 12 and 14A - 14B show that some of the peptides, e.g., T - 013, T - 018, T - 021, T - 025, T - 027, T - 028, T - 030, T - 042, T - 047, T - 065, T - 066, T - 067, T - 068, T - 069, T - 070, T - 073, T - 101, T - 109, T - 111, T - 112, T - 114, T - 115, T - 116, T - 117, T - 118, and T - 119 have cholesterol uptake activity (corresponding to fluorescence values at least 10 RFU higher than the baseline).

[0105] [000151]Figure 13 shows the results of the ARPE-19 GAG-dependent cholesterol uptake assay. ARPE-19 cells were seeded in a 96-well plate at 25,000 cells per well and grown to confluence. A 2× solution of 500 μM DMPC and 50 μM BODIPY-cholesterol was prepared in serum-free DMEM:F12 with or without 100 μg / mL heparin. Peptides were diluted to 20 μM in serum-free DMEM:F12 (2× solution). Peptides were added to the lipid solution in equal portions (60 μL each) and incubated at 37 °C for 1 h. ARPE-19 cells were serum-starved in serum-free DMEM:F12 at 37 °C for 1 h. The peptide:lipid complex (total volume of 100 μL per well) was transferred to the ARPE-19 cells and left at 37 °C for 2 h. Cells were washed, fixed, and intracellular fluorescence was read in a Promega GloMax at 475 nm excitation and 500–550 emission. Fluorescence values were background-corrected from untreated conditions. Data are shown as ±SD for two runs. Based on these data, the test peptides T-013, T-021, T-025, T-027, and T-028 rely on GAG binding for cholesterol uptake activity.

[0106] [000152]Figure 15 illustrates the ARPE-19 cholesterol uptake titration curve for one subset of peptides. ARPE-19 cells were seeded in a 96-well plate at 25,000 cells per well and grown to confluence. A 2× solution of 500 μM DMPC and 50 μM BODIPY-cholesterol was prepared in serum-free DMEM:F12. The peptides were diluted in serum-free DMEM:F12 to 0.74 - 60 μM (2× solution). The peptides were added to the lipid solution in equal portions (60 μL each) to create a final peptide concentration of 0.37 - 30 μM and incubated at 37 °C for 1 hour. ARPE-19 cells were serum-starved in serum-free DMEM:F12 at 37 °C for 1 hour. The peptide:lipid complex (total volume of 100 μL per well) was transferred to the ARPE-19 cells and left at 37 °C for 2 hours. The cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation and 500 - 550 emission. Fluorescence values were background corrected from the no-treatment condition. Data are shown as mean ± SD for three runs. Test peptides T-025 and T-027 demonstrated a concentration-dependent increase in cholesterol uptake.

[0107] [000153]Figure 16 shows the ARPE-19 cholesterol uptake titration curves for another subset of peptides. ARPE-19 cells were seeded in 96-well plates at 25,000 cells per well and grown to confluence. Peptide:lipid complexes were generated by preparing a solution of serum-free DMEM:F12 containing 30 μM peptide, 750 μM DMPC, and 75 μM BODIPY-cholesterol. The peptide:lipid complexes were serially diluted 1:3 to create five concentrations and incubated for 1 hour at 37 °C. ARPE-19 cells were serum-starved in serum-free DMEM:F12 at 37 °C for 1 hour. The peptide:lipid complexes (total volume of 100 μL per well) were transferred to the ARPE-19 cells and left at 37 °C for 2 hours. The cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation and 500–550 emission. Fluorescence values were background corrected from the no-treatment condition. Data are shown as mean ± SD for two runs. Test peptides T-101, T-112, T-114, and T-116 demonstrated a concentration-dependent increase in cholesterol uptake, while T-086 and T-087 were unable to increase cholesterol uptake over a range of concentrations.

[0108] [000154]Figures 17 and 18 summarize the HepG2 cholesterol uptake screening data for some of the peptides described herein. HepG2 cells were seeded in a 96-well plate at 10,000 cells per well and grown to confluence. A solution of 500 μM DMPC and 50 μM BODIPY-cholesterol was prepared in serum-free EMEM. Peptides were diluted to 20 μM in serum-free EMEM (2× solution). Peptides were added to the lipid solution in equal parts (60 μL each) to create a final 1× concentration and incubated at 37 °C for 1 hour. HepG2 cells were serum-starved in serum-free EMEM at 37 °C for 1 hour. The peptide:lipid complex (total volume of 100 μL per well) was transferred to the HepG2 cells and left at 37 °C for 2 hours. Cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation and 500 - 550 emission. Fluorescence values were background-corrected from the unprocessed conditions. Data are shown as mean ± SD for two runs. The results in Figures 17 and 18 show that many of the test peptides, including T-007, T-009, T-012, T-013, T-018, T-021, T-024, T-025, T-027, T-028, T-029, T-030, T-044, T-065, T-066, T-068, T-070, and T-080, have cholesterol uptake activity in HepG2 cells (corresponding to cell fluorescence values 20 RFU higher than baseline).

[0109] [000155]Figure 19 shows HepG2 GAG-dependent cholesterol uptake in one subset of peptides. HepG2 cells were seeded in a 96-well plate at 10,000 cells per well and grown to confluence. A 2× solution of 500 μM DMPC and 50 μM BODIPY-cholesterol was prepared in serum-free EMEM with or without 100 μg / mL heparin. Peptides were diluted to 20 μM in serum-free EMEM (2× solution). Peptides were added to the lipid solution in equal portions (60 μL each) and incubated at 37 °C for 1 hour. HepG2 cells were serum-starved in serum-free EMEM at 37 °C for 1 hour. The peptide:lipid complex (total volume of 100 μL per well) was transferred to HepG2 cells and left at 37 °C for 2 hours. Cells were washed, fixed, and intracellular fluorescence was read in a Promega GloMax at 475 nm excitation and 500–550 emission. Fluorescence values were background-corrected from the untreated condition. Data are shown as ±SD for two runs. Based on these data, the test peptides T-021, T-025, T-027, T-028, and T-029 rely on GAG binding for cholesterol uptake activity in HepG2 cells.

[0110] [000156]Figures 20 and 21 illustrate the HepG2 cholesterol uptake titration curves for several of the peptides shown (T-025, T-027, T-032, T-086, T-087, T-101, T-112, T-114, and T-116). HepG2 cells were seeded in 96-well plates at 10,000 cells per well and grown to confluence. Peptide:lipid complexes were generated by preparing a solution of serum-free EMEM containing 30 μM peptide, 750 μM DMPC, and 75 μM BODIPY-cholesterol. The peptide:lipid complexes were serially diluted 1:3 to create five concentration steps and incubated at 37 °C for 1 hour. HepG2 cells were serum-starved in serum-free EMEM at 37 °C for 1 hour. The peptide:lipid complexes (total volume of 100 μL per well) were transferred to the HepG2 cells and placed at 37 °C for 2 hours. The cells were washed, fixed, and intracellular fluorescence was read on a Promega GloMax at 475 nm excitation and 500–550 emission. Fluorescence values were background-corrected from the unprocessed conditions. Data are shown as mean ± SD for two runs. Test peptides T-025, T-027, T-101, T-112, T-114, and T-116 demonstrated concentration-dependent cholesterol uptake activity in HepG2 cells.

[0111] [000157]Figure 22 shows lipid-dependent LDLR binding (SPR) for one subset of peptides. Peptides were diluted to a final concentration of 1 μM in 1×HBS-N buffer containing 1 mM CaCl2 and incubated for 1 hour at room temperature with or without 25 μM DMPC to form lipid complexes. Each peptide was tested in duplicate. LDLR was diluted to 10 μg / mL in 10 mM acetate buffer pH 4.5 and immobilized (∼400 RU) on a CM5 chip by amine coupling using 1×HBS-P+ buffer containing 1 mM CaCl2. Test peptides (with or without DMPC) were exposed to the immobilized LDLR in HBS-N containing 1 mM CaCl2 at a flow rate of 30 μL / sec for 120 seconds. During the cycle, the surface was regenerated by three 30-second injections of 10 mM NaOH and 100 mM EDTA. Data were plotted as the average maximum RU value from three separate runs. Test peptides T-007, T-009, T-013, T-018, and T-029 demonstrated lipid-dependent LDLR binding activity, where the RU values for peptide binding to LDLR in the presence of lipid were higher than those for peptide alone without lipid.

[0112] [000158]The table shown in Figure 23 illustrates LDLR binding data (SPR) using high surface preparation. Recombinant LDLR was diluted in 10 mM acetate buffer pH 4.5 and amine-coupled to the CM5 chip at 1000 RU. Peptides were diluted to 5 μM in running buffer (HBS-P+, 1 mM CaCl2) and serially diluted 1:3 to create a total of five concentration steps. Peptides were exposed to the immobilized LDLR for 120 seconds at a rate of 30 μL / sec. During the cycle, the chip surface was regenerated by three injections of 50 mM NaOH / 100 mM EDTA. Multi-cycle kinetics were performed in Biacore Insight Evaluation software and the binding kinetics were analyzed by a 1:1 binding model. Several test peptides, including T-040, T-041, T-044, T-046, T-047, T-048, T-049, T-052, T-055, T-059, T-062, T-064, T-065, T-066, T-068, T-069, T-072, T-074, T-109, T-110, T-111, T-112, T-113, T-114, T-115, T-116, T-117, T-118, and T-120, had a binding KD of less than 10 μM to LDLR in this assay.

[0113] [000159]The tables of FIGS. 24A - 24B summarize the results of LDLR binding data (SPR) for lipid - dependent binding. Recombinant LDLR was diluted in 10 mM acetate buffer pH 4.5 and amine - coupled to the CM5 chip at 1000 RU. Peptides were diluted to 5 μM in running buffer (HBS - P, 1 mM CaCl2) and serially diluted 1:3 to create a total of five concentration steps. In separate preparations, peptides were diluted to 1 μM in running buffer containing 25 μM DMPC and serially diluted 1:3 in running buffer to create a total of five concentration steps. The peptide:DMPC complexes were incubated for 1 hour before use. Peptides or peptide:lipid complexes were exposed to the immobilized LDLR at a rate of 30 μL / sec for 120 seconds. During the cycle, the chip surface was regenerated by three injections of 50 mM NaOH / 100 mM EDTA. Multicycle kinetics were performed in Biacore Insight Evaluation software and the binding kinetics were analyzed by a 1:1 binding model. Several test peptides, including T - 065, T - 066, T - 068, T - 070, T - 109, T - 110, T - 111, T - 112, T - 113, T - 114, T - 115, T - 116, and T - 117, had more than a two - fold change in LDLR binding in the presence of DMPC lipid.

[0114] [000160]Figures 25A - 25o show the peptide binding activities to non - oxidized and oxidized lipids by SPR. The peptide was reconstituted in 10 mM acetate buffer pH 4.5 to 50 μg / mL and immobilized on the CM5 chip to approximately 200 RU by amine coupling. Non - oxidized lipids (DMPC and POPC) and oxidized lipids (POVPC and KOdiA - PC) were diluted to 100 μM in HBS - EP running buffer and exposed to the chip for binding (n = 3) for 120 s at a flow rate of 30 μL / s. The surface was regenerated with 20% EtOH. For each chip, T - 001 was used as a positive control and T - 031 as a negative control. For each peptide, the RU responses for all lipids were normalized to the DMPC RU value. If the peptide was unable to bind to DMPC, it was considered to be inert for immobilization. All of the test peptides T - 009, T - 013, T - 021, T - 025, T - 029, and T - 030 demonstrated binding to at least one oxidized lipid that was equivalent to or improved over binding to DMPC.

[0115] [000161]Figures 26A-26B show the results of the oxidized LDL test. In a 96-well microplate, LDL (stock concentration 10 mg / mL), copper(II) sulfate (stock concentration 1.5 mM), and the peptide (stock concentration 1 mM) were each mixed in six sets of wells to a final concentration of 100 μg / mL, 15 μM, and 25 μM, respectively. Control wells were filled with LDL (100 μg / mL) and copper(II) sulfate (15 μM), or LDL alone. The microplate was immediately read in a plate reader at OD234 nm every 5 minutes for 240 minutes. The increase in absorbance at 234 nm reflects the increase in conjugated diene formation and is used as a measure of oxidation. In Figure 26A, the data are shown as the mean ± SD over three technical replicates. In Figure 26B, the LDL oxidation rate was calculated as the maximum absorbance divided by the length of the lag period, and the data are shown as the mean + SD over three technical replicates. The test peptides T-087 and T-101 decreased the oxidation of LDL in the presence of copper(II) sulfate by 92% and 91.6%, respectively, in this assay.

[0116] [000162]Figures 27A - 27B and 28 illustrate HMC3 cholesterol efflux. A 96 - well plate of fully confluent HMC3 cells was treated for 1 hour in serum - free EMEM containing 10 mM methyl - β - cyclodextrin, 100 μM cholesterol, and 25 μM BODIPY cholesterol. Subsequently, the cells were washed and incubated overnight in serum - free EMEM containing 1 μM LXR agonist GW3965, 2 μg / mL ACAT inhibitor, and 0.2% BSA (LXR agonist treatment), or in serum - free EMEM containing 2 μg / mL ACAT inhibitor and 0.2% BSA (control). The cells were washed and incubated with 20 μM peptide diluted in serum - free Fluorobrite DMEM at 37°C for 4 hours. The medium was transferred to a new 96 - well plate and fresh serum - free Fluorobrite DMEM was added to the cells. The fluorescence of the medium and cells was read on a Promega GloMax at 475 nm excitation and 500 - 550 emission. The fluorescence values were background - corrected from the untreated condition, and the efflux percentage was calculated by the following formula. Efflux %=(medium fluorescence value) / (medium + cell fluorescence value). The data are shown as the mean value + SD over two runs for each peptide. Several test peptides, including T - 084, T - 085, T - 086, T - 087, T - 101, and the T - 101 - derived peptides T - 121, T - 122, T - 123, T - 124, T - 126, T - 128, T - 129, T - 134, T - 135, T - 136, T - 137, T - 138, T - 139, T - 140, T - 141, T - 142, T - 143, T - 144, T - 145, T - 146, T - 148, T - 149, T - 150, and T - 151, induced greater cholesterol efflux in HMC3 cells in the presence of an LXR agonist.

[0117] [000163]Figure 29 shows ARPE-19 cholesterol efflux for the peptides shown. A 96-well plate of confluent ARPE-19 cells was treated for 1 hour in serum-free DMEM containing 10 mM methyl-β-cyclodextrin, 100 μM cholesterol and 25 μM BODIPY cholesterol. The cells were then washed and incubated overnight in serum-free DMEM containing 1 μM GW3965, 2 μg / mL ACAT inhibitor and 0.2% BSA (LXR agonist treatment), or in serum-free DMEM containing 2 μg / mL ACAT inhibitor and 0.2% BSA (control). The cells were washed and incubated for 4 hours at 37 °C with 20 μM pe ptide diluted in serum-free Fluorobrite DMEM. The medium was transferred to a new 96-well plate and fresh serum-free Fluorobrite DMEM was added to the cells. Fluorescence of the medium and cells was read on a Promega GloMax at 475 nm excitation and 500 - 550 emission. Fluorescence values were background corrected from untreated conditions and the percent efflux was calculated by the following formula. Percent efflux = (medium fluorescence value) / (medium + cell fluorescence value). Data are shown as mean + SD over two runs per peptide. Several T-087-derived peptides, including T-152, T-153, T-154, T-155, T-156, T-157, T-158, T-159, T-160, T-161, T-162, T-163, T-164, T-166, T-167, T-168, T-170, T-171, T-172, T-174, T-175, T-176 AND T-178 demonstrated an increase in cholesterol efflux in ARPE-19 cells after LXR agonist treatment.

[0118] [000164]Figures 30A - 30J show the HMC3 cholesterol efflux titration curves for the peptides shown. A 96 - well plate of confluent HMC3 cells was treated for 1 hour in serum - free EMEM containing 10 mM methyl - β - cyclodextrin, 100 μM cholesterol, and 25 μM BODIPY cholesterol. The cells were then washed and incubated overnight in serum - free EMEM containing 1 μM GW3965, 2 μg / mL ACAT inhibitor, and 0.2% BSA (LXR agonist treatment), or in serum - free EMEM containing 2 μg / mL ACAT inhibitor and 0.2% BSA (control). The cells were washed and incubated at 37 °C for 4 hours with 0 - 100 μM peptide serially diluted in serum - free Fluorobrite DMEM. The medium was transferred to a new 96 - well plate and fresh serum - free Fluorobrite DMEM was added to the cells. The fluorescence of the medium and cells was read on a Promega GloMax at 475 nm excitation and 500 - 550 emission. The fluorescence values were background - corrected from the untreated condition, and the percent efflux was calculated by the following formula. Percent efflux=(medium fluorescence value) / (medium + cell fluorescence value). The data are shown as the mean ± SD over two runs per peptide. The peptides tested were (A) T - 001, positive control; (B) T - 082, positive control; (C) T - 083, positive control; (D) T - 084; (E) T - 086; (F) T - 087; (G) T - 101; (H) T - 112; (I) T - 114; and (J) T - 116. All test peptides demonstrated a concentration - dependent increase in cholesterol efflux in HMC3 cells in the presence of an LXR agonist.

[0119] [000165]Figures 31A - 31B show cholesterol efflux assays after knockdown of the ATP - binding cassette family. ARPE - 19 cells were seeded in 96 - well plates at 25,000 cells per well or in 12 - well plates at 100,000 cells per well and grown for 1 week until confluence. miR - 33a (which targets ABCA1) was diluted to up to 30 nM in Opti - MEM. siRNA for ABCA1, ABCA4, ABCA7, ABCG1, ABCG4, or SRB1 was diluted in Opti - MEM to a final concentration of 100 nM siRNA. RNAiMax Lipofectamine was added to the siRNA or miRNA preparation and incubated for 5 minutes, then transfection was performed at 37 °C for 24 hours in serum - free DMEM. To verify the siRNA or miRNA used in this experiment, qPCR was performed to confirm at least 85% knockdown of the target transcript and to confirm no effect on other transcripts being tested. After siRNA or miRNA treatment, the cells were treated for 1 hour in serum - free DMEM containing 10 mM methyl - β - cyclodextrin, 100 μM cholesterol, and 25 μM BODIPY cholesterol. Then the cells were washed and incubated overnight in serum - free DMEM containing 1 μM LXR agonist GW3965, 2 μg / mL ACAT inhibitor, and 0.2% BSA (LXR agonist treatment), or in serum - free EMEM containing 2 μg / mL ACAT inhibitor and 0.2% BSA (control). The cells were washed and incubated at 37 °C for 4 hours with 20 μM of peptide T - 087 or T - 101 diluted in serum - free Fluorobrite DMEM. The medium was transferred to a new 96 - well plate and fresh serum - free Fluorobrite DMEM was added to the cells. The fluorescence of the medium and cells was read in a Promega GloMax at 475 nm excitation and 500 - 550 emission. The fluorescence values were background - corrected from the untreated condition and the efflux percentage was calculated by the following formula. Efflux %=(medium fluorescence value) / (medium + cell fluorescence value).Data are shown as mean + SD from 2 runs (siABCA7, siABCG4, siSCARB1), 3 runs (miR-33), 4 runs (siABCA4, siABCG1), or 16 runs (siABCA1). Differences were tested by two-way ANOVA and Tukey's post hoc test. *, significant difference (P < 0.05). Peptides T-087 and T-101 demonstrated a reduction in cholesterol efflux after knockdown of miR-33 and siABCA1, but none of the other siRNAs tested did, indicating that these peptides direct cholesterol efflux activity mainly through ABCA1.

[0120] [000166]Figure 32 is a graph illustrating cholesterol efflux in iPS-RPE cells for two examples, T-087 and T-101. The iPS-derived RPE cells were obtained from Fujifilm (iCell retinal pigment epithelial cells) and grown for 28 days in a 96-well plate using vitronectin as the coating substrate. The cells were confirmed for their cobblestone morphology and pigmentation, and then the cells were treated for 1 hour in serum-free DMEM containing 10 mM methyl-β-cyclodextrin, 100 μM cholesterol, and 25 μM BODIPY cholesterol. The cells were then washed and incubated overnight in serum-free DMEM containing 1 μM LXR agonist GW3965, 2 μg / mL ACAT inhibitor, and 0.2% BSA (LXR agonist treatment), or in serum-free EMEM containing 2 μg / mL ACAT inhibitor and 0.2% BSA (control). The cells were washed and incubated for 4 hours at 37 °C with 20 μM of the peptide serially diluted in serum-free Fluorobrite DMEM. The medium was transferred to a new 96-well plate and fresh serum-free Fluorobrite DMEM was added to the cells. The fluorescence of the medium and the cells was read at 475 nm excitation and 500 - 550 emission in a Promega GloMax. The fluorescence values were background corrected from the untreated condition, and the efflux percentage was calculated by the following formula. Efflux % = (medium fluorescence value) / (medium + cell fluorescence value). The data are shown as the mean value + SD from two runs. Peptides T-087 and T-101 were able to increase cholesterol efflux in iPS-RPE cells after LXR agonist action.

[0121] [000167]Figures 33A to 33C illustrate the results of the membrane ABCA1 stability test in J774 cells. J774 mouse macrophage cells were seeded in a 12-well plate at 50,000 cells per well and grown to confluence. A medium solution of 0.1% BSA in serum-free DMEM was prepared for the control wells. An ABCA1 induction solution of 0.1% BSA containing 1 mM 8-Br-cAMP and 50 μg / mL AcLDL was prepared in serum-free DMEM and incubated with the cells at 37°C overnight to increase the ABCA1 protein level on the membrane. A solution of 10 μM peptide was prepared in serum-free DMEM and serially diluted 1:10 to create four concentrations. ApoAI was prepared at a concentration of 300 nM. The peptide or apoA1 was added to the cells and incubated at 37°C for 4 hours, after which the membrane proteins were isolated and the membrane ABCA1 level was measured by Western blot. The blot was quantified in ImageJ and the signal was normalized to the membrane Na + / K + pump level. The signal was further normalized to the 18-hour cAMP-treated group to determine the remaining membrane ABCA1%. In the absence of peptide or apoA1 treatment, the membrane ABCA1 level decreased after a 4-hour washout of 8-Br-cAMP, while treatment with either peptide T-087, T-101, or apoA1 preserved the membrane ABCA1 level. The EC 50 values for peptides T-087 and T-101 in this assay were 1.51 μM and 3.7 μM, respectively.

[0122] [000168]Figure 34 shows the results of the membrane ABCA1 stability test in ARPE-19 cells for peptides T-087 and T-101. ARPE-19 cells were seeded in a 12-well plate at 100,000 cells per well and grown to confluence. To prepare monomeric CRP (mCRP), recombinant CRP was prepared in a PBS solution containing 8 M urea and 10 mM EDTA, incubated at 37 °C for 2 hours, and then desalted using a Zeba spin column (7k MW cut-off). mCRP was prepared in a 2× solution at 20 μg / mL. A 2× solution of 40 μM peptide was prepared in serum-free DMEM and serially diluted 1:2 to create four concentrations. mCRP and the peptide were added to the cells at 1:1, incubated at 37 °C for 24 hours, and then the membrane proteins were isolated and the membrane ABCA1 level was measured by Western blot. The blot was quantified in ImageJ. The signal was further normalized for the LXR agonist-treated group to determine the remaining % of ABCA1. Treatment with the LXR agonist increased the membrane ABCA1 level in this assay, but the membrane ABCA1 level decreased after treatment with mCRP. In the presence of peptide T-087 or T-101 during the mCRP incubation period, the ABCA1 membrane level was preserved compared to without peptide treatment. The EC 50 values for peptides T-087 and T-101 in this assay were 4.6 μM and 3.5 μM, respectively.

[0123] [000169]Figures 35 and 36 show the membrane ABCA1 stability assay using ARPE-19 cells. ARPE-19 cells were seeded in 12-well plates at 100,000 cells per well and grown to confluence. To prepare monomeric CRP (mCRP), recombinant CRP was prepared in a PBS solution containing 8 M urea and 10 mM EDTA, incubated at 37 °C for 2 hours, and then desalted by a Zeba spin column (7k MW cut-off). mCRP was prepared in a 2× solution at 20 μg / mL. A 2× solution of 40 μM peptide was prepared in serum-free DMEM. CRP and the peptide were added to the cells at a 1:1 ratio, incubated at 37 °C for 24 hours, and then the membrane proteins were isolated and the membrane ABCA1 levels were measured by Western blot. The blots were quantified in ImageJ. In Figure 35, the signals were further normalized for the T-087 treatment group to determine the % ABCA1 levels compared to the peptide T-087-treated cells. In Figure 36, the signals were further normalized for the peptide T-101 treatment group to determine the % ABCA1 levels compared to the peptide T-101-treated cells. Some derivatives of peptide T-087, including peptides T-155, T-156, T-157, T-158, T-159, T-162, T-166, T-167, T-170, T-173, and T-177, resulted in a reduction in the membrane ABCA1 levels after mCRP treatment compared to the peptide T-087-treated cells, indicating that residues 3, 4, 5, 6, 7, 9, 13, 14, and 15 in peptide T-087 contribute to the activity of stabilizing membrane ABCA1 after mCRP treatment.In addition, several derivatives of peptide T-101, including peptides T-121, T-124, T-126, T-127, T-128, T-130, T-132, T-134, T-135, T-137, T-138, T-104, T-141, T-145, T-148, T-149, and T-150, result in a reduction of membrane ABCA1 levels after mCRP treatment compared to peptide T-101-treated cells, indicating that the group of residues at positions 1, 4, 6, 7, 8, 10, 12, 14, 15, 17, 18, 21, and residues between positions 6 - 19 are important in T-101 for the full activity of stabilizing ABCA1 after mCRP treatment.

[0124] [000170] Figure 37 shows the correlation between cholesterol efflux and hydrophobic moment for peptides T-152 - T-178 (SEQ ID NOs: 152 - 178). In this example, linear regression was used to determine the deviation of the slope from zero. The raw data for this figure are the data from Figure 28 and the calculated hydrophobic moments defined in Eisenberg (Eisenberg et al. 1982. The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature). The results of this analysis show that the hydrophobic moment of the test peptides correlates positively with their cholesterol efflux activity.

[0125] [000171]Figure 38A shows the correlation between cholesterol efflux and hydrophobicity for peptides T-121 to T-151 (SEQ ID NOs: 121 to 151). Figure 38B shows the correlation between ABCA1 membrane stability and hydrophobic moment for these peptides. Linear regression was used to determine the deviation of the slope from zero. The raw data for these figures are the data from Figures 29 and 36. Hydrophobicity is defined by Fauchere and Pliska (Fauchere and Plisk (1983). Hydrophobic Parameters II of Amino-Acid Side Chains from the Partitioning of N-Acetyl-Amino-Acid Amides. Eur J Med Chem). Hydrophobic moment is defined by Eisenberg (Eisenberg et al 1982. The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature). The results of the analysis shown in Figure 38A indicate that the hydrophobicity of the test peptides is positively correlated with their cholesterol efflux activity. The results of the analysis shown in Figure 38B indicate that membrane ABCA1 stability is positively associated with their hydrophobic moment.

[0126] [000172]Figures 39A to 39E show examples of the results of the tolerance test of representative peptides in mice. T-087, T-101, T-112, and T-114 were prepared in sterile H2O + 0.001% Tween-80 to a concentration of 520 μM and delivered intravitreally (IVT) to 12-week-old C57BL / 6J mice (n = 3 mice per group). Seven days after IVT injection, the eyes were harvested and prepared for hematoxylin and eosin staining. A veterinarian performed a histopathological analysis of the tissue slices in a blinded manner to determine whether the peptides induced inflammation or retinal destruction. Representative images of each peptide are shown, indicating that all peptides except T-112 were well tolerated.

[0127] [000173]Figures 40A and 40B show apoE- / - Results of an in vivo study measuring the effects of peptides T-087 and T-101 in reducing sub-RPE BODIPY+ lipid deposition in mice are shown. Figure 40A shows results of a study in which 3-month-old apoE - / - mice (B6.129P2-apoE tm1Unc / J) were fed a high-cholesterol diet (Research Diets D12079B) for 2 months. Two weeks prior to sacrifice, mice were randomized (n = 6 mice per group) and received an IVT injection of 520 μM of peptide T-087 or peptide T-101, or 1 μL of vehicle control. At sacrifice, eyes were removed, stained with BODIPY to visualize neutral lipids, and then imaged at 20x magnification. ImageJ was used to quantify the area of sub-RPE BODIPY+ staining, and then the area of interest (AOI) was divided by the length of the area to calculate the average width of lipid deposition. Nine images were averaged for each mouse. Data are shown as individual data points representing values for individual mice. Differences were tested by one-way ANOVA and Tukey's post hoc test. *, different from vehicle (P < 0.05). In a separate study summarizing the results in Figure 40B, 3-month-old apoE - / - mice (B6.129P2-apoE tm1Unc / J) were fed a high-cholesterol diet (Research Diets D12079B) for 2 months. Two weeks prior to sacrifice, mice were randomized (n = 4 mice per group) and received an IVT injection of 260 μM of peptide T-087 or peptide T-101, or 1 μL of vehicle control. At sacrifice, eyes were removed, stained with BODIPY to visualize neutral lipids, and then imaged and quantified as above. Twelve images were averaged for each mouse. Data are shown as individual data points representing values for individual mice. Differences were tested by one-way ANOVA and Tukey's post hoc test. *, different from vehicle (P < 0.05). In both Figure 40A and Figure 40B, treatment with test peptide T-087 or test peptide T-101 resulted in a statistically significant decrease in sub-RPE lipid deposits compared to vehicle-treated mice.

[0128] Examples of therapeutic peptides [000174]Therefore, the therapeutic peptides described herein can solubilize lipids, induce lipid efflux from cells, and receive and transport lipids released from within cells. These therapeutic peptides can be engineered mimetics of one or more apolipoproteins, such as apoA1, which have structural homology to endogenous apoA1 such that the structure and function of the therapeutic peptide is essentially amphiphilic and effectively enhances the activity of cholesterol efflux regulatory proteins or transporter proteins (e.g., ABCA1, ABCG1, SR-B1, or related proteins) involved in lipid efflux or lipid processing mechanisms. The therapeutic peptides described herein can interact with one or more molecules involved in lipid transport and processing pathways, such as lipoproteins, lipid efflux transporters, and lipoprotein particle modifiers. For example, if the therapeutic peptide provides mimetics that are structurally similar to apoA1, since apoA1 is associated with lipid processing mechanisms and cholesterol efflux pathways, the therapeutic peptide can associate with HDL and support HDL function. Many of the therapeutic peptides listed in FIGS. 1B-1I demonstrate these properties. Generally, the therapeutic peptides can be 80 amino acids or less (e.g., 75 or less amino acids, 70 or less amino acids, 65 or less amino acids, 60 or less amino acids, 55 or less amino acids, 50 or less amino acids, 49 or less amino acids, 48 or less amino acids, 47 or less amino acids, 46 or less amino acids, 45 or less amino acids, 44 or less amino acids, 43 or less amino acids, 42 or less amino acids, 41 or less amino acids, 40 or less amino acids, 35 or less amino acids, 30 or less amino acids, 25 or less amino acids, 22 or less amino acids, 18 or less amino acids, etc.).

[0129] [000175]Therefore, the therapeutic peptides described herein can solubilize lipids and transport the captured lipids to cells. The therapeutic peptides can utilize GAG-dependent, GAG-independent, and / or lipid import receptors (e.g., LDLR, SR-B1, and related receptors) to facilitate lipid import into cells. The therapeutic peptides may have the property of binding to oxidized lipids, which may enable the clearance of pro-inflammatory and / or toxic oxidized lipid species. The therapeutic peptides may have the property of binding to LDLR in a lipid-dependent manner to ensure that the peptide does not bind to LDLR in the absence of lipids (binding of the peptide to LDLR in the absence of lipids can inhibit LDLR function and disrupt cholesterol homeostasis). Many of the peptides listed in FIGS. 1B-I and shown in the sequence listing as SEQ ID NOs: 5-30, 35-80, and 84-178 demonstrate these properties. Some of the therapeutic peptides include a terminal peptide region that is homologous (in some cases identical, and in some examples identical except for a few mismatches out of 11 amino acids) to the peptide sequence shown in SEQ ID NO: 8 (e.g., HLRKLRKRLLR). A second region can be linked to the terminal region. Alternatively, the D-conformation of SEQ ID NO: 8 can be positioned at the C-terminus of the peptide. The central variable region shown is similar to the region of SEQ ID NO: 16 (shown appended to the terminal region as peptide SEQ ID NO: 27). The therapeutic peptides can include this sequence and an extended peptide region of up to an additional 16 amino acids. The therapeutic peptides may have the property of binding and activating ABCA1 and maintaining the presence of ABCA1 on the cell membrane in a pro-inflammatory environment. Many of the peptides listed in FIGS. 1B-I and shown in the sequence listing as SEQ ID NOs: 5-30, 35-80, and 84-178 demonstrate these properties. Also, the therapeutic peptides may have N- and C-terminal modifications including acetylation and amidation.

[0130] [000176]In some examples, similar peptides may vary five or fewer of the 18 amino acids in the central variable region. For example, up to five of these amino acids may vary. Substitutions or deletions may be made. In some examples, these substitutions may be conservative substitutions. In some examples, the same terminal region is included coupled to a second central variable region. The central variable region may be similar to the sequence of SEQ ID NO: 20. The peptide comprising the terminal region and the central variable region is shown in SEQ ID NO: 29.

[0131] Compositions [000177]Any of the therapeutic peptides described herein may be used as part of a pharmaceutical composition. A pharmaceutical composition comprising a therapeutic peptide described herein may comprise one or more pharmaceutically acceptable carriers. The pharmaceutical composition may be suitable for any mode of administration, for example, a mode of administration by intravitreal administration.

[0132] [000178]In some examples, the composition comprises the polypeptides of SEQ ID NOs: 5-30, 35-80, and 84-178. For example, in some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 27. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 25. In some examples, the pharmaceutical composition comprises a peptide (or a modified form thereof) having the sequence of SEQ ID NO: 29. In some examples, the pharmaceutical composition comprises a peptide (or a modified form thereof) having the sequence of SEQ ID NO: 87. In some examples, the pharmaceutical composition comprises a peptide (or a modified form thereof) having the sequence of SEQ ID NO: 101. In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 8 at the N-terminus (or at the C-terminus in the D configuration) and having a total length of less than 80 amino acids.

[0133] [000179]In some examples, the composition comprises a polypeptide of SEQ ID NO: 35-80 or 84-120. For example, in some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 101 (or a modified form thereof). In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 87 (or a modified form thereof). In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 100 (or a modified form thereof). In some examples, the pharmaceutical composition comprises a peptide having the sequence of SEQ ID NO: 8 at the N-terminus (or at the C-terminus in the D configuration) and having a total length of less than 80 amino acids.

[0134] [000180]In some examples, a pharmaceutical composition comprising a peptide described herein (including, but not limited to, peptides of SEQ ID NO: 5-30, 35-80, or 84-178) and a pharmaceutically acceptable carrier is suitable for administration to a human subject. Such carriers are well known in the art (see, e.g., Remington’s Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580). In some examples, the pharmaceutical composition is suitable for intravitreal injection. In some examples, the pharmaceutical composition is suitable for subretinal delivery. Such pharmaceutically acceptable carriers may be sterile liquids, such as water and oils, and the oils include oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, etc. Also, saline solutions and aqueous dextrose, polyethylene glycol (PEG), and glycerol solutions can be used as liquid carriers, especially for injectable solutions. The pharmaceutical composition may further comprise additional components, such as preservatives, buffers, isotonic agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity increasing agents, etc. The pharmaceutical compositions described herein may be packaged in single unit dosages or in multiple dosage forms. The composition is generally formulated as a sterile and substantially isotonic solution.

[0135] [000181]In one example, the therapeutic peptides described herein are formulated into a pharmaceutical composition intended for subretinal or intravitreal injection. Such formulations include pharmaceutically and / or physiologically acceptable media or carriers, particularly media or carriers suitable for administration to the eye, such as by subretinal injection, for example, buffered saline or other buffers, such as HEPES, to maintain the pH at an appropriate physiological level, and optionally, the use with other pharmaceutical agents, formulations, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier is typically liquid. Exemplary physiologically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free phosphate buffered saline. In one example, the carrier is an isotonic sodium chloride solution. In another example, the carrier is a balanced salt solution. In one example, the pharmaceutically acceptable carrier includes a surfactant, such as Tween-80, Tween-20, or perfluorooctane (Perfluoron solution). If the peptide solution is intended for long-term storage, it can be frozen in the presence of glycerol or Tween-20.

[0136] [000182]In certain examples of the methods described herein, the pharmaceutical composition described above is administered to a subject by subretinal injection. In other examples, the pharmaceutical composition is administered by intravitreal injection. Other forms of administration that may be useful in the methods described herein include direct delivery to the desired organ (e.g., the eye), oral, inhalation, intranasal, intratracheal, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration, but are not limited thereto. The routes of administration can be combined, if desired. In certain examples, the pharmaceutical composition of the present disclosure is administered after administration of an initial loading dose of a complement system protein.

[0137] [000183]In some examples, the route of administration is selected such that the route of administration reduces the risk of retinal detachment in the patient (e.g., intravitreal rather than subretinal). In some examples, when the vector / composition is intended to be administered to an elderly person (e.g., at least 60 years old), intravitreal administration is selected. In certain examples, any of the vectors / pharmaceutical compositions disclosed herein are administered intravitreally to a subject. The technique of intravitreal injection is known in the art (see, e.g., Peyman, G.A. et al. (2009) Retina 29(7):875-912; and Fagan, X.J. and Al-Qureshi, S. (2013) Clin. Experiment. Ophthalmol. 41(5):500-7). Briefly, for intravitreal injection, the subject can be prepared for the procedure by mydriasis, eye disinfection, and administration of an anesthetic. Any suitable mydriatic known in the art can be used for mydriasis. Adequate mydriasis can be confirmed prior to treatment. Disinfection can be achieved by applying an eye disinfection procedure, e.g., an iodide-containing solution such as povidone iodine (Betadine®). Also, a similar solution can be used to clean the eyelids, eyelashes, and any other adjacent tissues (e.g., skin). Any suitable anesthetic such as lidocaine or proparacaine can be used at any suitable concentration. The anesthetic can be administered by any method known in the art, including, but not limited to, topical eye drops, gels or jelly, and subconjunctival application of the anesthetic. Prior to injection, a sterilized eyelid speculum can be used to remove the eyelashes from the area. The injection site can be marked with a syringe. The injection site can be selected based on the patient's lens. For example, the injection site can be 3-3.5 mm from the limus in pseudophakic or aphakic patients and 3.5-4 mm from the limus in phakic patients. The patient may look in the direction opposite to the injection site. During injection, the needle can be inserted perpendicular to the sclera and directed towards the center of the eye. The needle can be inserted such that the tip stops in the vitreous body rather than in the subretinal space. Any suitable volume known in the art for injection can be used. After injection, the eye can be treated with a disinfectant such as an antibiotic.Also, the eye may be rinsed to remove excess disinfectant.

[0138] [000184]Furthermore, in certain examples, it is desirable to perform non-invasive retinal imaging and functional studies to identify specific regions of eye cells to be targeted for treatment. In these examples, clinical diagnostic tests are used to determine the exact location for one or more subretinal injections. These tests may include fundus examination, RPE function, electroretinogram (ERG) (particularly, b-wave measurement, c-wave measurement), visual field measurement, confocal scanning laser ophthalmoscopy (cSLO), and optical coherence tomography (OCT) for mapping the tissue distribution of retinal layers and measuring the thickness of those layers, mapping the tissue distribution of cone density by adaptive optics (AO), functional eye examination, and the like. In some examples, one or more injections are performed in the same eye to target various regions of retained bipolar cells.

[0139] [000185]The composition can be delivered in a volume of from about 0.1 μL to about 1 mL, including all numbers within the range, depending on the size of the area to be treated, the route of administration, and the desired effect of the method. In one example, the volume is about 50 μL. In another example, the volume is about 70 μL. In one example, the volume is about 100 μL. In another example, the volume is about 125 μL. In another example, the volume is about 150 μL. In another example, the volume is about 175 μL. In yet another example, the volume is about 200 μL. In another example, the volume is about 250 μL. In another example, the volume is about 300 μL. In another example, the volume is about 450 μL. In another example, the volume is about 500 μL. In another example, the volume is about 600 μL. In another example, the volume is about 750 μL. In another example, the volume is about 850 μL. In another example, the volume is about 1000 μL.

[0140] [000186]For example, the dosage can be from about 100 ng / eye to about 10 mg / eye (e.g., about 100 ng / eye, about 150 ng / eye, about 200 ng / eye, about 250 ng / eye, about 300 ng / eye, about 400 ng / eye, about 500 ng / eye, about 600 ng / eye, about 700 ng / eye, about 800 ng / eye, about 900 ng / eye, about 1 μg / eye, about 2 μg / eye, about 3 μg / eye, about 5 μg / eye, about 10 μg / eye, about 15 μg / eye, about 20 μg / eye, about 25 μg / eye, about 30 μg / eye, about 35 μg / eye, about 40 μg / eye, about 50 μg / eye, about 60 μg / eye, about 70 μg / eye, about 80 μg / eye, about 90 μg / eye, about 100 μg / eye, about 120 μg / eye, about 150 μg / eye, about 175 μg / eye, about 200 μg / eye, about 250 μg / eye, about 300 μg / eye, about 350 μg / eye, about 400 μg / eye, about 500 μg / eye, about 750 μg / eye, about 1 mg / eye, about 1.5 mg / eye, about 2 mg / eye, about 2.5 mg / eye, about 3 mg / eye, about 3.5 mg / eye, about 4 mg / eye, about 4.5 mg / eye, about 5 mg / eye, or any range among these).

[0141] [000187]Still other dosages and volumes within these ranges can be selected by the attending physician taking into account the subject to be treated, preferably the general condition of a human, the age of the subject, the particular eye disorder, and the extent to which the disorder has progressed if it is progressive. For extraocular delivery, e.g., oral delivery and / or intravitreal delivery, the dosage can be increased according to a scale-up from the retina.

[0142] Methods of treatment / prevention [000188]Various methods for preventing, treating, arresting, or improving eye disorders and associated retinal changes are described herein. Any of these methods can include identifying patients who may benefit from one or more of these treatments and / or identifying which one or more of the treatments described herein may be most beneficial for a particular patient. Assays described herein that can generate a patient score or characterization based on one or more biomarkers described herein can be used to identify patients who may benefit from one or more of these treatments and / or which treatment should be applied. Any of these methods can include determining the dosage to be delivered, the route of delivery, and / or the schedule for delivering one or more doses. Assays described herein that score and / or characterize based on one or more biomarkers can be used to determine the dosage to be delivered, the route of delivery, and / or the schedule for delivering one or more doses.

[0143] [000189]Generally, the methods include administering to a mammalian subject in need thereof an effective amount of any of the compositions described herein. For example, treatment of age-related macular degeneration can include localized delivery of a therapeutic composition described herein to the retina of a patient. The cells that are the therapeutic targets in these diseases can include photoreceptor cells in the retina, or cells of the RPE underlying the neurosensory retina, or cells of the choroidal capillaries or Bruch's membrane. In certain embodiments, the disclosure provides a method of treating a subject having age-related macular degeneration (AMD), the method comprising administering to the subject any of the compositions described herein.

[0144] [000190]In certain examples, methods are provided for preventing, arresting, or improving vision loss associated with an eye disorder in a subject. Vision loss associated with an eye disorder refers to any decrease in peripheral vision, central (reading) vision, night vision, daytime vision; loss of color vision; loss of contrast sensitivity; or reduced visual acuity. The methods and compositions described herein may be directed to increasing photoreceptor function. As used herein, "increasing photoreceptor function" means improving the function of photoreceptors, or increasing the number or proportion of functional photoreceptors, compared to an affected eye (having the same eye disorder), the same eye at an earlier time point, an untreated portion of the same eye, or the contralateral eye of the same patient. Photoreceptor function may be evaluated using functional studies conventional in the art, such as ERG or visual field measurements.

[0145] [000191]For each of the methods described, the treatment can be used to prevent the occurrence of retinal damage or to rescue an eye with a mild or advanced disease. As used herein, the term "rescue" means preventing the progression of the disease to complete blindness, preventing the spread of damage to the cells of an intact eye, improving the damage in the damaged cells of the eye, or providing an improvement in vision. In one example, the composition is administered before the disease becomes symptomatic or before photoreceptor loss. "Symptomatic" means any of the various retinal changes described above or the onset of vision loss. In another example, the composition is administered after the disease has become symptomatic. In yet another example, the composition is administered after the onset of photoreceptor loss. In yet another example, the composition is administered after the onset of outer nuclear layer (ONL) degeneration. In some examples, it is desirable for the composition to be administered while the bipolar cells that connect the ganglion cells and the optic nerve remain intact. In another example, the composition is administered after the onset of photoreceptor loss. In yet another example, the composition is administered when less than 90% of the photoreceptors are functional or remaining compared to an unaffected eye. In another example, the composition is administered when less than 80% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 70% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 60% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 50% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 40% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 30% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 20% of the photoreceptors are functional or remaining. In another example, the composition is administered when less than 10% of the photoreceptors are functional or remaining. In one example, the composition is administered only to one or more regions of the eye. In another example, the composition is administered to the entire eye. In yet another example, the method includes performing functional and imaging studies to determine the effectiveness of the treatment.These studies include ERG and in vivo retinal imaging, as described in the examples below. In addition, visual field studies, perimetry and microperimetry, pupillometry, mobility tests, visual acuity, contrast sensitivity, and color vision tests may be performed.

[0146] [000192]In another example, any of the methods described herein may be performed in combination with another or secondary treatment. The treatment may be any currently known or yet-to-be-discovered treatment that helps prevent, arrest, or improve any of the described retinal changes and / or vision loss.

[0147] [000193]In some examples, the methods of treatment described herein may incorporate the identification of a patient based on one or more diagnostic techniques. In particular, the identification of one or more genetic abnormalities associated with dysfunction of one or more components of the lipid transport and processing machinery may support the selection of one or more of the therapeutic peptides described herein. In certain examples, an abnormality in the genotype of one or more components of the lipid processing machinery may result in the observed dysregulation of lipid processing associated with the etiology of AMD. Based on the identified abnormality, one or more of the therapeutic peptides may be administered to support the lipid processing machinery or cholesterol homeostasis.

[0148] [000194]Any of the methods described herein (including the user interface) may be implemented as software, hardware, or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions executable by a processor (e.g., a computer, tablet, smartphone, etc.), the instructions causing the processor to perform any of the steps including, but not limited to, displaying, communicating with a user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, warning, etc.

[0149] [000195]When a feature or element is referred to in this specification as being "above" another feature or element, it can be directly on the other feature or element, or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly above" another feature or element, no intervening features or elements are present. Also, when a feature or element is referred to as being "connected to", "coupled to", or "linked to" another feature or element, it is understood that it may be directly connected, coupled, or linked to the other feature or element, or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected to", "directly coupled to", or "directly linked to" another feature or element, no intervening features or elements are present. Although one example is described or shown, the features and elements so described or shown may apply to other examples. Also, it is understood by those skilled in the art that a reference to a structure or feature "adjacent to" another feature may have portions that overlap with or are beneath the adjacent feature.

[0150] [000196]The terms used in this specification are for the purpose of describing particular examples only and are not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprising" and / or "comprises" as used herein, when used, specify the presence of the stated feature, step, operation, element, and / or component, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0151] [000197]Spatial relative terms, such as "under", "below", "lower", "over", "upper", etc., may be used herein for ease of explanation to describe the relationship of one element or feature to another element or feature as illustrated in the drawings. It is understood that spatial relative terms are intended to encompass various orientations of the device in use or operation in addition to the orientation depicted in the drawings. For example, if the device in the drawings is turned over, an element described as "under" or "beneath" another element or feature can be oriented "over" the other element or feature. Thus, the exemplary term "under" can encompass both upward and downward orientations. The device may be oriented in another way (rotated 90 degrees or in other orientations), and the spatial relative expressions used herein are to be interpreted accordingly. Similarly, terms such as "upwardly", "downwardly", "vertical", "horizontal", etc. are used herein only for purposes of explanation unless specifically indicated otherwise.

[0152] [000198]The terms "first" and "second" may be used herein to describe various features / elements (including steps), but these features / elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another. Thus, without departing from the teachings of the present invention, a first feature / element described below may be referred to as a second feature / element, and similarly, a second feature / element described below may be referred to as a first feature / element.

[0153] [000199]Throughout this specification and the following claims, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising", are to be interpreted as indicating that the various components can be used together in a method and an article (e.g., a composition, an apparatus including a device, and a method). For example, the term "comprising" is understood to imply the inclusion of any indicated element or step, but not the exclusion of any other element or step.

[0154] [000200]Generally, any of the apparatus and methods described herein should be understood to be inclusive, but all or subsets of components and / or steps may alternatively be exclusive and may be expressed as "consisting of" or alternatively "consisting essentially of" various components, steps, sub-components, or sub-steps.

[0155] [000201]In this specification and the claims, as used herein, including when used in examples and unless otherwise expressly specified, all numbers can be interpreted as if the term "about" or "approximately" were prefixed thereto even if the term is not expressly shown. The phrase "about" or "approximately" can be used to describe a magnitude and / or a position to explain that the value and / or position being described is within a reasonable range predicted for the value and / or position. For example, a numerical value can have a value that is ±0.1% of the value shown (or range of values), ±1% of the value shown (or range of values), ±2% of the value shown (or range of values), ±5% of the value shown (or range of values), ±10% of the value shown (or range of values), etc. Also, any numerical value given herein should be understood to include about or approximately that value unless the context indicates otherwise. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. Also, as would be appropriately understood by one of ordinary skill in the art, when a value is disclosed, it is understood that "less than that value", "greater than that value", and the possible ranges between values are also disclosed. For example, if the value "X" is disclosed, "less than X" and "greater than X" (here, for example, X is a numerical value) are also disclosed. Also, throughout this application, it is understood that the data is provided in several different formats and that this data represents ranges of endpoints and starting points, as well as any combination of data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that points greater than 10 and 15, greater than or equal to 10 and 15, less than 10 and 15, less than or equal to 10 and 15, and equal to the points between 10 and 15, as well as points between 10 and 15 are disclosed. Also, each unit between two particular units is also understood to be disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0156] [000202]Although various exemplary examples are described above, any of several variations can be made to the various examples without departing from the scope of the invention as described by the claims. For example, the order in which the various described method steps are performed may often vary in alternative examples, and in other alternative examples, one or more method steps may be omitted entirely. Optional features of the various device and system examples may be included in some examples but not in others. Therefore, the above description is provided primarily for illustrative purposes, and since the scope of the invention is set forth in the claims, the above description should not be construed as limiting the scope of the invention.

[0157] [000203]The examples and illustrations included herein are for illustration, not limitation, and show specific examples in which the subject matter may be practiced. As noted, other examples may be utilized and derived from them, provided that structural and logical substitutions and changes are made without departing from the scope of this disclosure. Such examples of the subject matter of the invention may be referred to herein individually or collectively by the term "invention," which is for convenience only and is not intended to spontaneously limit the scope of this application to any single invention or inventive concept. Thus, although specific examples are illustrated and described herein, any arrangement calculated to achieve the same purpose can replace the specific examples shown. This disclosure is intended to embrace any and all adaptations or variations of the various examples. Combinations of the above examples and other examples not specifically described herein will be apparent to those skilled in the art upon reading the above description.

Claims

1. A polypeptide comprising an amino acid sequence that is at least 80% or at least 90% homologous to the sequence of Sequence ID No.

87.

2. The polypeptide according to claim 1, wherein the polypeptide has ATP-binding cassette transporter stabilizing activity.

3. The polypeptide according to claim 1 or 2, wherein any peptide residue at positions 2-7, 9, and 10-17 that is different from the amino acid sequence of SEQ ID NO: 87 is a conservative substitution or conservative hydrophobic substitution having a hydrophobic value within 0.25 of the hydrophobic value of the peptide residue at the position corresponding to the sequence of SEQ ID NO: 87, calculated using the Fauchere and Pliska method, and any different peptide residue at positions 1, 8, 10, and 18 is any amino acid.

4. The polypeptide according to claim 1 or 2, wherein the polypeptide comprises a helix coil having a hydrophobic moment of 0.65 μH or more.

5. The polypeptide according to claim 1 or 2, wherein the amino acid sequence is the sequence of SEQ ID NOs: 87, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, or 177.

6. The polypeptide according to claim 1, wherein the amino acid sequence is the sequence of SEQ ID NOs: 87, 152, 160, 161, 163, or 172.

7. The polypeptide according to claim 1, wherein the amino acid sequence is the sequence of SEQ ID NO:

87.

8. The polypeptide according to claim 1, 2, 6, or 7, wherein the polypeptide has ATP-binding cassette transporter membrane stabilization and agonist activity, and transporter protein binding activity.

9. A pharmaceutical composition for the treatment of age-related macular degeneration (AMD) in a patient, comprising a polypeptide having an amino acid sequence that is at least 80% homologous to the sequence of Sequence ID No.

87.

10. The pharmaceutical composition according to claim 9, wherein the polypeptide has ATP-binding cassette transporter stabilizing activity.

11. The pharmaceutical composition according to claim 9 or 10, wherein the polypeptide comprises a helix coil having a hydrophobic moment of 0.65 μH or more.

12. The pharmaceutical composition according to claim 9 or 10, wherein any peptide residue at positions 2-7, 9, and 10-17 that is different from the amino acid sequence of SEQ ID NO: 87 is a conservative substitution or conservative hydrophobic substitution having a hydrophobic value within 0.25 of the hydrophobic value of the peptide residue at the position corresponding to the sequence of SEQ ID NO: 87, calculated using the Fauchere and Pliska method, and any different peptide residue at positions 1, 8, 10, and 18 is any amino acid.

13. The pharmaceutical composition according to claim 9 or 10, wherein the amino acid sequence is the sequence of SEQ ID NOs: 87, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, or 177.

14. The pharmaceutical composition according to claim 9, wherein the amino acid sequence is the sequence of SEQ ID NOs: 87, 152, 160, 161, 163, or 172.

15. The pharmaceutical composition according to claim 9, wherein the amino acid sequence is the sequence of Sequence ID No.

87.

16. A pharmaceutical composition for the treatment of age-related macular degeneration (AMD) in patients, comprising a polypeptide containing the amino acid sequence of Sequence ID No.

87.

17. A pharmaceutical composition according to claim 9, 10, 14, 15, or 16, for administration by intraocular injection.

18. The pharmaceutical composition according to claim 9, 10, 14, 15, or 16, for administration by intravenous (IV) injection.

19. A pharmaceutical composition according to claim 9, 10, 14, 15, or 16, for administration by subcutaneous (SC) injection.

20. The pharmaceutical composition according to claim 9, 10, 14, 15, or 16, wherein the patient is 40 years of age or older.

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