Peptides for suppressing inflammation

Abstract

Provided herein are peptides that exhibit ApoE biological activity, as well as compositions and pharmaceutical formulations comprising the peptides.The peptides, compositions, and methods disclosed herein have broad applications in that they can be used to treat a wide spectrum of injuries, diseases, disorders, and clinical indications.

Description

[0001] This application is a divisional application of U.S. Provisional Patent Application No. 61 / 454,342, filed on March 18, 2011, which is hereby incorporated by reference in its entirety. Technical Field

[0002] The present disclosure relates to peptides, methods, and compositions for reducing or inhibiting inflammation, reducing or inhibiting neuroinflammation, and treating neurological disorders.

[0003] Sequence Listing

[0004] A sequence listing is provided with this application (in electronic form only), which is incorporated herein by reference. The sequence listing file 9111-WO00_ASFILED_SequenceListing_Text.txt was created on March 15, 2012, and is 10,938 bytes in size. Background Art

[0005] Apolipoprotein E ("ApoE") is a 299 amino acid (34 kDa) glycoprotein produced primarily in the liver and brain that exhibits a variety of biological functions. First recognized for its role in cholesterol transport and metabolism, ApoE is present in a complex with very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL), and ApoE is able to bind to the low-density lipoprotein (LDL) receptor, LDL receptor-related protein (LRP), and the VLDL receptor. Weisgraber, (1994) Adv. Protein Chem. 45:249-302. ApoE is also known to have immunomodulatory properties, Laskowitz et al., (2001) Exp. Neurol. 167:74-85 and plays a role in neurological diseases and responses to brain injury, Laskowitz and Vitek, (2007) Pharmacogenomics 8:959-69.

[0006] The tertiary structure of ApoE consists of an amino-terminal region with four α-helical motifs, which includes the receptor binding domain; and a carboxyl-terminal region that is primarily responsible for lipid binding. The receptor binding region of ApoE has been mapped to the helical domain of residues 130-150 of the mature full-length protein, and this region of ApoE controls its ability to inhibit glial cell activation and CNS inflammation. Summary of the Invention

[0007] In one aspect, the present disclosure provides an isolated peptide of 5, 6, 7, 8 or 9 amino acid residues or a salt thereof comprising Formula I:

[0008] X1-X2-X3-X4-X5 (SEQ ID NO: 1)

[0009] wherein X1 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain; X2 is selected from an amino acid having a hydrophobic side chain, an amino acid having a positively charged side chain, or an amino acid having a polar uncharged side chain; X3 is selected from an amino acid having a positively charged side chain; X4 is selected from an amino acid having a positively charged side chain; and X5 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain. In some embodiments of this aspect, a peptide of Formula II is provided:

[0010] X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO:17);

[0011] wherein X1, X2, X3, X4 and X5 are as described above, and each of X6, X7, X8 and X9 is independently selected from any amino acid, and is optionally absent.

[0012] In one aspect, the present disclosure provides an isolated peptide of Formula I or a salt thereof:

[0013] X1-X2-X3-X4-X5 (SEQ ID NO: 1)

[0014] wherein X1 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain; X2 is selected from an amino acid having a hydrophobic side chain, an amino acid having a positively charged side chain, or an amino acid having a polar uncharged side chain; X3 is selected from an amino acid having a positively charged side chain; X4 is selected from an amino acid having a positively charged side chain; and X5 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain.

[0015] In embodiments of this aspect, the present disclosure provides a peptide or a salt thereof according to SEQ ID NO: 1, wherein X1 is V or R; X2 is S, A or H; X3 is K or R; X4 is K or R; and X5 is R, L or K. In some embodiments of this aspect, the present disclosure provides a peptide or a salt thereof comprising

[0016]

[0017] .

[0018] In one aspect, the present disclosure provides a composition comprising an isolated peptide of Formula I, and a carrier, diluent, vehicle, or adjuvant. Embodiments of this aspect provide a composition comprising a peptide according to SEQ ID NO: 1, or a salt thereof, wherein X1 is V or R; X2 is S, A, or H; X3 is K or R; X4 is K or R; and X5 is R, L, or K. In some embodiments of this aspect, the present disclosure provides a composition comprising

[0019]

[0020]

[0021] peptide or a salt thereof.

[0022] In one aspect, the present disclosure provides a method of reducing inflammation in a subject in need thereof, comprising administering to the subject an effective amount of a peptide of Formula I or a salt thereof:

[0023] X1-X2-X3-X4-X5 (SEQ ID NO: 1)

[0024] wherein X1 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain; X2 is selected from an amino acid having a hydrophobic side chain, an amino acid having a positively charged side chain, or an amino acid having a polar uncharged side chain; X3 is selected from an amino acid having a positively charged side chain; X4 is selected from an amino acid having a positively charged side chain; and X5 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain.

[0025] In embodiments of this aspect, the method comprises a peptide according to SEQ ID NO: 1, or a salt thereof, wherein X1 is V or R; X2 is S, A, or H; X3 is K or R; X4 is K or R; and X5 is R, L, or K. In some embodiments of this aspect, the method provides a peptide or a salt thereof comprising

[0026]

[0027]

[0028] .

[0029] In one aspect, the present disclosure provides a method of treating a neurological disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a peptide of Formula I or a salt thereof:

[0030] X1-X2-X3-X4-X5 (SEQ ID NO: 1)

[0031] wherein X1 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain; X2 is selected from an amino acid having a hydrophobic side chain, an amino acid having a positively charged side chain, or an amino acid having a polar uncharged side chain; X3 is selected from an amino acid having a positively charged side chain; X4 is selected from an amino acid having a positively charged side chain; and X5 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain.

[0032] In embodiments of this aspect, the method comprises a peptide according to SEQ ID NO: 1, or a salt thereof, wherein X1 is V or R; X2 is S, A, or H; X3 is K or R; X4 is K or R; and X5 is R, L, or K. In some embodiments of this aspect, the method provides a peptide or a salt thereof comprising

[0033]

[0034]

[0035] . In some embodiments, the method comprises treating a neurological disorder selected from at least one of traumatic CNS injury, subarachnoid hemorrhage, intracranial hemorrhage, stroke, experimental allergic encephalomyelitis, multiple sclerosis, neuroinflammation, chronic neurological disease, ALS, dementia, neuropathy, epilepsy, Parkinson's disease, and Alzheimer's disease.

[0036] In another aspect, the present disclosure provides medicaments comprising at least one peptide of Formula I, methods for preparing the medicaments, and methods comprising administering the medicaments as described herein.

[0037] In various embodiments of the aspects discussed above, the present disclosure relates to and provides peptides consisting essentially of, or consisting of, the recited sequences and structural formulas.

[0038] Other aspects and embodiments provided by the present disclosure will be apparent to those of ordinary skill in the art based on the following drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Peptide inhibition of microglial TNF-α secretion is described. Cultured BV-2 murine microglial cells were incubated with the indicated concentrations of a peptide (SEQ ID NO: 3) or a negative control peptide (SEQ ID NO: 13) and stimulated with lipopolysaccharide (LPS) (100 ng / mL) for 6 hours. After 6 hours, supernatants were collected and secreted TNF-α concentrations were measured by ELISA (** p < 0.01; ANOVA).

[0040] Figure 2Described is the improved vestibulomotor function of mice treated with peptides after traumatic brain injury. Equilibrium motor function is assessed based on rotorod latency. Baseline rotorod latency was assessed before (day 0) and every day after traumatic brain injury was induced by controlled pneumatic impact on the intact skull. At 2 hours and 6 hours after traumatic brain injury, mice received peptide (SEQ ID NO: 3; 0.05 mg / kg or 0.2 mg / kg) or saline vehicle by intravenous tail vein injection. As reflected by the increase in rotorod latency, animals treated with peptides showed improved equilibrium motor ability (* p < 0.05; ANOVA) relative to those treated with vehicle throughout the test period.

[0041] Figure 3 Described after traumatic brain injury, the balance motor function of mice treated with peptides is improved. Balance motor function is assessed according to the rotarod delay time. Baseline rotarod delay time is assessed before (day 0) and every day after the traumatic brain injury is induced by controlled pneumatic impact on the intact skull. At 2 hours and 6 hours after the traumatic brain injury, mice receive peptide (SEQ ID NO:4; 0.05 mg / kg) or saline vehicle by intravenous tail vein injection. As reflected by the increase in rotarod delay time, animals treated with peptides showed improved balance motor ability throughout the test period (* p < 0.05; repeated measures ANOVA).

[0042] Figure 4 Described are the improved neurocognitive outcomes of mice treated with peptides after traumatic brain injury was induced by controlled pneumatic impacts to the skull. At 2 hours and 6 hours after the traumatic brain injury, mice received peptide (SEQ ID NO: 4; 0.05 mg / kg or 0.2 mg / kg) or saline vehicle by intravenous tail vein injection. Neurocognitive ability was assessed based on Morris water maze latency. Water maze latency was assessed by the submerged platform test starting 28 days after the traumatic brain injury, and ability was further assessed on consecutive days 29, 30, and 31 after the injury. As reflected by the shortening of the water maze latency, animals treated with peptides showed improved neurocognitive outcomes throughout the testing period (* p<0.05; ANOVA).

[0043] Figure 5Described after intracerebral hemorrhage, the balance motor function of mice treated with peptides is improved. Balance motor function is assessed according to the rotarod delay time. Baseline rotarod delay time is assessed before (day 0) and every day after the intracerebral hemorrhage is induced by stereotactic collagenase injection. At 2 hours and 6 hours after the collagenase injection, mice receive peptide (SEQ ID NO:4; 0.05 mg / kg) or saline vehicle by intravenous tail vein injection. As reflected by the increase in rotarod delay time, animals treated with peptides showed improved balance motor ability throughout the test period (* p < 0.05; repeated measures ANOVA).

[0044] Figure 6 Depicted are the percent inhibition of LDH release following NMDA exposure in cultures treated with an ApoE mimetic peptide (SEQ ID NO: 4).

[0045] Figure 7 A-7B depicts a cross section of a shock tube shock wave model apparatus.

[0046] Figure 8 Depicted is the effect of an ApoE mimetic peptide (SEQ ID NO: 4) on neurocognitive performance in mice with blast injury, as measured by Morris water maze latency (trend difference in learning, p = 0.7).

[0047] Figure 9 Depicted are the mean and individual amounts of VSRRR (SEQ ID NO: 4) peptide in mouse plasma over time following a single dose of 0.8 mg / kg.

[0048] Figure 10 Described are the temporal changes in CNS penetration of the VSRRR (SEQ ID NO: 4) peptide in brain tissue from mice.

[0049] Figure 11 A-11B describes the inhibition of TNF-α secretion by microglia by VR-55 (SEQ ID NO: 4), VL-5-1 (SEQ ID NO: 5), and VL-5-3 (SEQ ID NO: 10).

[0050] Figure 12 A-12B describes balance and motor function in mice treated with the peptide following modified middle cerebral artery occlusion. DETAILED DESCRIPTION

[0051] It will be understood that the various aspects and embodiments described herein are provided for illustration only and are not intended to limit the scope of the claims.

[0052] As used herein, the articles "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" refers to at least one element and may include more than one element. Unless otherwise specified, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0053] The present disclosure generally relates to peptides that can exert ApoE activity (also referred to herein as "ApoE mimetic activity"), including isolated and / or synthetic peptides. As used herein, ApoE refers to peptides that are derived from APOE Any of the various isoforms (e.g., alleles) of the genetically encoded human apolipoprotein-E protein. Non-limiting examples of ApoE include ApoE3 (SEQ ID NO: 14), ApoE2 (SEQ ID NO: 15), and ApoE4 (SEQ ID NO: 16). ApoE activity includes any functional biological activity, in vitro or in vivo, or a combination of biological activities, associated with the ApoE protein. ApoE activity may involve, for example, cholesterol metabolism, binding to physiological ApoE receptor proteins, neuroprotective activity, antioxidant activity, anti-excitotic activities, regulation of glial cell activity, inflammation, regulation of neuroinflammation, and the like. Recent studies have demonstrated that peptides with apoE mimetic activity can exert beneficial effects in a variety of animal models, including, for example, Alzheimer's disease (Laskowitz et al., (2010) J Neurotrauma 27:1983-1995), multiple sclerosis (Li et al., JPET, 2006), subarachnoid hemorrhage (Gao et al., 2006; Mesis et al., 2006), stroke (Tukhovskaya, J Neurosci Res, 2006), and neuropathy (Li et al., JPET, 2010). Therefore, the peptides, compositions, and methods disclosed herein have broad applications due to their potential for treating a range of ApoE-related diseases, disorders, and clinical indications.

[0054] ApoE is a known ligand for receptors including scavenger receptors such as the LDL receptor, VLDL receptor, LRP / α2M receptor, ER-2 receptor, LR8 receptor, ApoE receptor 2 (apoER2), and megalin / gp330 (collectively referred to as "ApoE receptors"). One region of ApoE known to be involved in receptor binding interactions is the α-helical domain between residues 130-150 of the native ApoE polypeptide (SEQ ID NO: 14). Active ApoE fragments comprising this helical domain have shown ApoE mimetic activity (see U.S. Patent Nos. 7,319,092 and 7,205,280, incorporated herein by reference in their entirety). These peptides retain the native ApoE primary amino acid sequence in the receptor binding helical domain and maintain the native α-helical secondary structure (Laskowitz et al., (2001)). Exp. Neurol. 167:74-85). The activity of these peptides has been shown to be dependent on the retention of the native α-helical secondary structure (Laskowitz et al. (2006) Acta Neurol. Scand. 114 (Supp. 185):15-20). As described in more detail below, it has been unexpectedly discovered that small peptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues in length) that have no primary sequence identity to ApoE effectively modulate, induce, and / or mimic ApoE biological activity and are useful in the treatment of various diseases, disorders, or conditions involving the biological function of ApoE.

[0055] As used herein, "peptide" refers to a compound comprising at least a single amino acid residue or a derivative thereof, or a compound comprising at least one amino acid mimetic. Amino acids are well known in the art and include, for example, isoleucine, leucine, alanine, asparagine, glutamine, lysine, aspartic acid, glutamic acid, methionine, cysteine, phenylalanine, threonine, tryptophan, glycine, valine, proline, serine, tyrosine, arginine, histidine, norleucine, ornithine, taurine, selenocysteine, selenomethionine, lanthionine, 2-aminoisobutyric acid, dehydroalanine, hypusine, citrulline, 3-aminopropionic acid, gamma-aminobutyric acid, nitroarginine, N-methylated leucine, homoarginine, dimethylarginine, acetyl lysine, azalysine, pyrrolysine, and the like. "Amino acid side chain" refers to the various organic substituent groups that distinguish one amino acid from another. Amino acids with hydrophobic side chains include the following non-limiting examples: alanine (A), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), and valine (V). Amino acid side chains with positively charged side chains under typical physiological conditions include the following non-limiting examples: arginine (R), histidine (H), and lysine (K). Amino acids with negatively charged side chains under typical physiological conditions include the following non-limiting examples: aspartic acid (D) and glutamic acid (E). Amino acids with polar uncharged side chains include the following non-limiting examples: serine (S), threonine (T), asparagine (N), and glutamine (Q). Considering these non-limiting examples, those skilled in the art will understand and be able to determine the characteristics of other amino acid side chains not explicitly exemplified above (e.g., hydrophobicity, positive / negative charge, and polar uncharged, etc.). A "derivative" of an amino acid side chain refers to an amino acid side chain that has been structurally modified (e.g., by chemical reaction to form a new species, covalently linked to another molecule, etc.). Some embodiments provide peptides comprising modifications, including but not limited to glycosylation, side chain oxidation, acetylation, amidation, or phosphorylation, as long as the modification does not destroy the biological activity of the peptide as described herein. For example, in some embodiments, the peptide can be modified by N-terminal acetylation and / or C-terminal amidation.

[0056] As used herein, "amino acid mimetics" are intended to include peptide mimetics, peptidomimetics (poly-N-substituted glycines), and β-peptides (i.e., peptides comprising one or more amino acid residues having an amino group attached to the β-carbon rather than the α-carbon). Suitably, the amino acid mimetics include altered chemical structures designed to favorably adjust molecular properties (e.g., stability, activity, reduced immunogenic response, solubility, etc.). Such altered chemical structures are generally not considered to occur in nature (e.g., incorporation of modified backbones, non-natural amino acids, etc.). Thus, non-limiting examples of amino acid mimetics include D-peptides, retro-peptides, retro-inverso peptides, β-peptides, peptidomimetics, and compounds comprising one or more D-amino acids, poly-N-substituted glycines, or β-amino acids, or any combination thereof.

[0057] Typically, the peptide comprises a sequence of at least 3 amino acids (amino acid residues) or amino acid mimetics. Embodiments of the present disclosure relate to small peptides of at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid residues, mimetics or combinations thereof. Some embodiments described herein provide peptides of less than 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues and / or mimetics. Some embodiments relate to peptides of 5 amino acids in length. The peptides described herein can be provided in a charged form, typically with a net positive charge, and can be generated and used as salts (e.g., alkali metal salts, basic or acidic addition salts). The selection and formation of such salts are within the capabilities of those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy , 21st edition, Lippincott Williams & Wilkins, A Wolters Kluwer Company, Philadelphia, Pa (2005).

[0058] Embodiments of the present disclosure provide synthetic peptides with ApoE mimetic activity. Although they can exhibit ApoE mimetic activity, the disclosed peptides do not share primary protein sequence identity with the ApoE polypeptide (SEQ ID NO: 13). In other words, the disclosed peptide sequences do not appear in the primary amino acid sequence of the ApoE polypeptide, nor do they exhibit an α-helical secondary structure similar to the native ApoE receptor binding domain. In one embodiment, the synthetic peptides are optionally isolated and / or purified to a single active species.

[0059] In one aspect of the present disclosure, the peptide comprises Formula I or a salt thereof:

[0060] X1-X2-X3-X4-X5 (SEQ ID NO: 1)

[0061] wherein X1 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain; X2 is selected from an amino acid having a hydrophobic side chain, an amino acid having a positively charged side chain, or an amino acid having a polar uncharged side chain; X3 is selected from an amino acid having a positively charged side chain; X4 is selected from an amino acid having a positively charged side chain; and X5 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain. Some embodiments provide peptides wherein X1 is V or R; X2 is S, A, or H; X3 is K or R; X4 is K or R; and X5 is R, L, or K.

[0062] Some embodiments of this aspect provide a peptide of Formula II:

[0063] X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO:17);

[0064] Wherein X1, X2, X3, X4 and X5 are as described above, and each of X6, X7, X8 and X9 is independently selected from any amino acid, and is optionally absent. In such embodiments, the peptide of formula II may comprise 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues or 9 amino acid residues.

[0065] In another aspect, the present disclosure provides a peptide of Formula I or a salt thereof:

[0066] X1-X2-X3-X4-X5 (SEQ ID NO: 1)

[0067] wherein X1 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain; X2 is selected from an amino acid having a hydrophobic side chain, an amino acid having a positively charged side chain, or an amino acid having a polar uncharged side chain; X3 is selected from an amino acid having a positively charged side chain; X4 is selected from an amino acid having a positively charged side chain; and X5 is selected from an amino acid having a hydrophobic side chain or an amino acid having a positively charged side chain. Some embodiments provide peptides wherein X1 is V or R; X2 is S, A, or H; X3 is K or R; X4 is K or R; and X5 is R, L, or K.

[0068] Several non-limiting embodiments of peptides according to Formula I are disclosed in Table 1. In some embodiments, the peptide may comprise

[0069]

[0070] In some embodiments, the peptide according to Formula I is

[0071]

[0072] .

[0073] In some embodiments of all aspects described herein, the peptide consists essentially of the amino acid sequence and structural formula disclosed herein. In some embodiments of all aspects described herein, the peptide consists essentially of the amino acid sequence and structural formula disclosed herein.

[0074] Table 1

[0075]

[0076] In some embodiments, the peptide can show at least one ApoE mimetic activity. In some embodiments, for example, the disclosed peptide can bind to one or more physiological ApoE receptors, for example, cell surface receptors expressed by glial cells, and receptors that function as follows: inhibiting neuronal cell death and calcium influx (excitotoxicity) associated with N-methyl-D-aspartate (NMDA) exposure; protecting against LPS-induced TNF-α and IL-6 production (for example, in an in vivo sepsis model); preventing, treating or delaying inflammatory conditions, such as atherosclerosis, arthritis or inflammatory bowel disease; inhibiting glial or microglial activation; inhibiting macrophage activation; inhibiting lymphocyte activation; inhibiting inflammation; inhibiting CNS inflammation; treating neuropathy; and / or improving neurodegenerative diseases (for example, mild cognitive impairment, dementia, Parkinson's disease or Alzheimer's disease) and / or neural damage in acute CNS trauma (for example, traumatic brain injury).

[0077] In some embodiments, the peptide binds to a specific receptor with an affinity similar to that of ApoE. In some embodiments, the peptide binds to a specific receptor with an affinity similar to that of a previously disclosed longer 20 amino acid ApoE mimetic peptide that binds to macrophages with a dissociation constant (K d ) is approximately 50 nM (Misra et al., (2001) J. Leukocyte Biol. 70:677-683). For example, the peptide binds to the receptor's K dThe peptide may have a K of about 100 μM, about 90 μM, about 80 μM, about 70 μM, about 60 μM, about 50 μM, about 40 μM, about 30 μM, about 20 μM, about 10 μM, about 5 μM, about 1 μM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, about 5 nM, about 1 nM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM, about 5 pM, or about 1 pM. The peptide may have a K of about 100 μM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM, about 5 pM, or about 1 pM. d The peptide may have a K of greater than or equal to about 1 pM, about 5 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 1 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 1 μM, about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, or about 100 μM. dThe concentration of the active ingredient may be about 1 pM to about 10 pM, about 5 pM to about 15 pM, about 10 pM to about 20 pM, about 20 pM to about 30 pM, about 30 pM to about 40 pM, about 40 pM to about 50 pM, about 50 pM to about 60 pM, about 60 pM to about 70 pM, about 70 pM to about 80 pM, about 80 pM to about 90 pM, about 90 pM to about 100 pM, about 100 pM to about 1 nM, about 1 nM to about 10 nM, about 5 nM to about 15 nM, about 10 nM to about 20 nM, about 20 nM to about 30 nM, about 30 nM to about 40 nM, about 40 nM to about 50 nM, about 50 nM to about 60 nM, about 60 nM to about 70 nM, about 70 nM to about 80 nM, about 80 nM to about 90 nM, about 90 nM to about 100 nM, about 100 nM to about 1 μM, about 1 μM to about 10 μM, about 5 μM to about 15 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 40 μM, about 40 μM to about 50 μM, about 50 μM to about 60 μM, about 60 μM to about 70 μM, about 70 μM to about 80 μM, about 80 μM to about 90 μM, about 90 μM to about 100 μM, about 100 μM to about 1 μM. For example, the peptide binds to macrophages with a K d It can be less than or equal to about 100 μM, about 90 μM, about 80 μM, about 70 μM, about 60 μM, about 50 μM, about 40 μM, about 30 μM, about 20 μM, about 10 μM, about 5 μM, about 1 μM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, about 5 nM, about 1 nM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM, about 5 pM, or about 1 pM.

[0078] The extent of binding to the ApoE receptor can be assessed using any technique known in the art, such as, for example, typical binding assays (e.g., competitive binding assays), ELISA, functional assays (e.g., as described in the Examples), etc. In some embodiments, the size of the peptide can confer improved pharmacokinetics, facilitate crossing the blood-brain barrier, allow intranasal administration, reduce production costs, increase efficacy (e.g., on a per gram basis), and / or reduce the immunogenicity of the peptide.

[0079] The peptides can be produced using any method known in the art for preparing polypeptides, including, for example, synthetic and recombinant methods. For example, in some embodiments, the peptides can be synthesized using synthetic chemistry techniques, such as solid phase synthesis, Merrifield-type solid phase synthesis, t-Boc solid phase synthesis, Fmoc solid phase synthesis, BOP solid phase synthesis, and solution phase synthesis. See, for example, Stewart and Young, Solid Phase Peptide Synthesis , 2nd ed., (1984) PierceChem. Co., Rockford Ill.; The Peptides: Analysis, Synthesis, Biology, Grossand Meienhofer, Eds., vols. 1-2 (1980) Academic Press, New York; Bodansky, Principles of Peptide Synthesis , (1984) Springer-Verlag, Berlin. In other embodiments, the peptides can be produced by expressing the peptides from nucleic acids encoding the peptides in cells or cell-free systems, for example, according to recombinant techniques familiar to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual , (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology , (2002) John Wiley & Sons, Somerset, NJ; each of which is incorporated herein by reference in its entirety. The peptides may incorporate any of the various modifications and protecting groups described herein or known to those skilled in the art, for example, as described in McOmie, Protective Groups in Organic Chemistry , (1973) Plenum Press, New York.

[0080] In some embodiments, the peptides can be designed to mimic the physical, chemical, and / or structural features of the alpha-helical receptor binding domain located between residues 130-150 of the native ApoE polypeptide (SEQ ID NO: 14). For example, in some embodiments, the peptides can be designed using a "linear walk" peptide design method to mimic the physical, chemical, and / or structural features of the polar face extending along the outer surface of the three-dimensional structure of the native ApoE receptor-binding helix, wherein the amino acids at each consecutive position in the peptide are selected by attempting to emulate one or more properties (e.g., relative size / steric hindrance, polarity, non-polarity, charge, uncharge, hydropathic index (e.g., hydrophobicity, hydrophilicity), acidity, basicity, ability to form bonds (e.g., covalent bonds, hydrogen bonds, van der Waals interactions), etc.) of each consecutive residue exposed on the polar face of the native ApoE receptor binding helix at each descending periodic turn of the helix. In some embodiments, the peptides can be designed based on the physical, chemical, and / or structural features of the ApoE binding domain of one or more ApoE receptors. For example, for an ApoE receptor having a binding pocket or surface that interacts with ApoE in an intermolecular receptor-ligand binding interaction, the peptide can be designed by selecting peptide amino acid residues expected to exhibit binding interactions along the ApoE binding pocket / surface of the receptor to maximize the expected binding affinity between the peptide and the ApoE binding pocket / surface of the receptor.

[0081] In one aspect, the present disclosure provides a method of treating a neurological disorder in a subject in need thereof, comprising administering to the subject an effective amount of a peptide of Formula I or Formula II, or a composition or formulation comprising an effective amount of a peptide of Formula I or Formula II, or a combination thereof.

[0082] In another aspect, the present disclosure provides a method of reducing inflammation in a subject in need thereof, comprising administering to the subject an effective amount of a peptide of Formula I or Formula II, or a composition or formulation comprising an effective amount of a peptide of Formula I or Formula II, or a combination thereof.

[0083] In embodiments relating to the above aspects, the peptide and / or composition can be used to treat, improve or prevent certain signs, symptoms and / or harmful neurological effects of acute and / or chronic CNS damage. Acute CNS damage used herein includes but is not limited to apoplexy (caused by thrombosis, embolism or vasoconstriction), closed craniocerebral injury, traumatic brain injury, global cerebral ischemia (e.g., ischemic stroke, global cerebral hypoxia, focal cerebral ischemia, and coronary artery bypass grafting), ischemic stroke, global cerebral hypoxia (global anoxia), focal cerebral ischemia (focal ischemia), subarachnoid hemorrhage and intracranial hemorrhage. Ischemic damage to the central nervous system can be caused by whole brain or focal ischemic conditions. Global cerebral ischemia occurs when the blood flow to the entire brain stops for a period of time, such as during cardiac arrest. Focal ischemia occurs when part of the brain lacks normal blood flow, such as during thrombotic occlusion of cerebral blood vessels, traumatic brain injury, edema and brain tumors. Most CNS damage caused by cerebral ischemia occurs within a few hours or even days after the ischemic condition and is secondary to the release of cytotoxic products from the injured tissue. Chronic CNS damage includes, but is not limited to, Alzheimer's disease (AD), Parkinson's disease, epilepsy and HIV-related encephalopathy. The discovery that ApoE peptides can inhibit glial cell activation provides the disclosed methods, peptides and compositions with a role in treating any neurological disease involving microglial activation. For example, microglia express activation markers in AD, indicating that key inflammatory events in AD involve microglia. These activated microglia cluster near amyloid plaques. In epilepsy, microglia are also activated.

[0084] In some embodiments, the peptides and / or compositions can be used to prevent, treat or improve clinical neurological signs and symptoms associated with inflammatory conditions affecting the nervous system (e.g., CNS). Non-limiting examples include multiple sclerosis, vasculitis, acute disseminated encephalomyelitis, and Guillain-Barre syndrome. In this regard, the disclosed ApoE mimetic peptides can be used alone or in combination with other known anti-inflammatory drugs or cytokines to formulate pharmaceutical compositions for the treatment of CNS inflammatory conditions.

[0085] In some embodiments, the peptide and / or composition can be used for preventing, treating or improving the patient's condition related to NMDA excitotoxicity. NMDA excitotoxicity has been associated with neurotic Lathyrus chinensis poisoning (neurolathyrism), amyotrophic lateral sclerosis (ALS), schizophrenia, HIV dementia and encephalopathy (encephalopy), Huntington's chorea, Parkinson's disease, bipolar disorder, multiple sclerosis in humans and experimental allergic encephalomyelitis (EAE) in animals, pain, depression, stroke, epilepsy, hereditary d-2-hydroxyglutaric aciduria, AD and traumatic brain injury. In some embodiments, the peptide and / or composition can block NMDA receptor-mediated excitotoxicity and provide neuroprotection. NMDA antagonists are also used for clinical anesthesia, and have been shown to suppress chronic pain, drug tolerance and alcohol dependence. Therefore, in some embodiments, disclosed method, peptide and composition can be used as an anesthetic preparation and the composition of the combined therapeutic composition comprising other known compounds for treating the patient's condition.

[0086] In some embodiments, the peptides and / or compositions can be used to protect against LPS-induced cytokine production in sepsis. Intact ApoE has been shown to protect mice from bacterial LPS-induced death. Other possible combination therapies for sepsis include the administration of anti-inflammatory cytokines, including IL-10, transforming growth factor-β, granulocyte colony-stimulating factor, interferon-φ, macrophage migration inhibitory factor, high mobility group box 1 protein, and monoclonal antibodies, including anti-endotoxin antibodies, anti-tumor necrosis factor antibodies, and anti-CD14 antibodies. Thus, embodiments provide for the use of peptides, alone or in combination with other known anti-inflammatory cytokines and antibodies, in compositions and methods for treating sepsis.

[0087] The effects of the disclosed methods, peptides, and compositions can be assessed at the cellular or tissue level (e.g., histologically or morphometrically), or by assessing the neurological state of the subject. The inhibition or reduction of glial activation can be assessed by various methods readily apparent to those skilled in the art; one such method is to measure the production or presence of a compound known to be produced by activated glial cells and compare this measurement to the level of the same compound in a control. Alternatively, the effects of the methods and compounds of the present invention in inhibiting, reducing, or preventing microglial activation can be assessed by comparing the signs and / or symptoms of a CNS disease in treated subjects to those in control subjects, wherein such signs and / or symptoms are associated with or secondary to the activation of microglial cells.

[0088] The terms "treating" and "treatment" when used in reference to a disease or a subject in need of treatment generally include, but are not limited to, interrupting or slowing the progression of a disease, alleviating the disease, preventing or alleviating symptoms and / or clinical signs, reducing the severity of the disease and / or symptoms, or shortening the duration of the disease, as compared to an untreated subject and / or in the absence of treatment. In some embodiments, the treatment method can alleviate or improve one or more clinical signs of the specific disease being treated. Certain embodiments of the method for treating a disease or condition associated with ApoE activity comprise administering a therapeutically effective amount of a peptide of Formula I, or a peptide of Formula II, or one or more peptides selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and pharmaceutical compositions thereof. In embodiments, the methods of treatment may involve any method that prevents further progression of, slows or reduces further progression of, or reverses diseases and / or clinical symptoms associated with ApoE activity.

[0089] Subjects to be treated by the methods described herein include mammalian subjects, including human subjects and non-human (animal) subjects, such as dogs, cats, rabbits, goats, horses, pigs, cattle, etc. (including male and female subjects, and subjects of all ages, including infants, teenagers, adolescents and adult subjects). Treatment of the subject may be for any purpose, such as reducing inflammation, inhibiting microglial activation, improving chronic diseases, etc. The term "simultaneous administration" as used herein means that the administration of two compounds is close enough in time to achieve the immunological effect of the combination. Therefore, simultaneous administration can be performed by sequential administration or simultaneous administration (e.g., simultaneous administration in a common or identical carrier).

[0090] In some embodiments, the disclosed peptides and compositions can be administered by any suitable route of administration, including but not limited to: injection (subcutaneous, intraperitoneal, intravenous, intrathecal, intramuscular, intracerebroventricular and spinal injection), intranasal, oral, transdermal, parenteral, inhalation, nasopharyngeal or transmucosal absorption. Administration includes providing at least one peptide as described herein (e.g., Formula I, Formula II and / or SEQ ID NOs: 1-12) formulated into a pharmaceutical composition. Direct administration of active agents (e.g., compounds, peptides, etc.) to the brain is known in the art. Intrathecal injection delivers the agent directly to the ventricles and spinal fluid. Surgically implantable infusion pumps are available to provide sustained-release administration of the agent directly into the spinal fluid. Spinal injection includes lumbar puncture and injection of the drug compound into the cerebrospinal fluid. Administration also includes targeted delivery (wherein the peptides of the present disclosure are active only in a targeted area of ​​the body (e.g., in brain tissue)), and sustained-release formulations (wherein the peptide is released in a controlled manner over a period of time). The method for sustained-release formulation and targeted delivery is known in the art, and includes, for example, using liposomes, biodegradable microspheres of loaded drugs, drug-polymer conjugates, drug-specific binding agent conjugates etc. Pharmaceutically acceptable carriers are well known to those skilled in the art, and include chitosan nanoparticles or other relevant enteric polymer preparations. Taking into account, for example, the stage and severity of the degree of treatment, disease and related symptoms of the patient's age, body weight, sex, race, organ (for example, liver and kidney) function, expectation, and the tolerance of the patient to the treatment, it is within the ability of those skilled in the art to determine the specific pharmaceutical preparation and therapeutically effective amount and dosage regimen for given treatment.

[0091] Some embodiments of the methods described herein provide for intranasal delivery of one or more peptides described herein or compositions comprising peptides for use in patients with chronic diseases, such as multiple sclerosis, Alzheimer's disease, epilepsy, Parkinson's disease, arthritis, inflammatory bowel disease, leukemia, or atherosclerosis. Formulations and methods suitable for intranasal and inhalation administration are known in the art.

[0092] In embodiments involving therapeutic applications, administration can be performed on subjects already suffering from the target condition. Suitably, subjects in the latent or acute stages of the disease can be treated by the methods described herein, alone or in combination with other treatments, depending on the specific disease / condition, patient, and combination. One skilled in the art will be able to determine whether a combination therapy is appropriate.

[0093] In therapeutic methods and applications, the peptides and compositions described herein can be administered to a subject in an amount sufficient to treat or at least partially inhibit symptoms and / or complications. An amount sufficient to achieve this is generally referred to as a "therapeutically effective dose." An amount effective for this use may depend, in part, on the peptide, composition, mode of administration, stage and severity of the condition being treated, age, weight, and general health of the patient, and the judgment of the prescribing physician.

[0094] In embodiments, an effective amount of the compositions and peptides disclosed herein may comprise less than about 100 mg / kg, less than about 50 mg / kg, less than about 25 mg / kg, less than about 10 mg / kg, less than about 1 mg / kg, less than about 0.1 mg / kg, less than about 0.05 mg / kg, less than about 0.01 mg / kg, less than about 0.005 mg / kg, and less than about 0.001 mg / kg of the peptide. In some embodiments, an effective amount of the compositions and peptides disclosed herein may comprise at least about 0.0001 mg / kg, at least about 0.001 mg / kg, at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 5 mg / kg, and at least about 10 mg / kg of the peptide. This includes, for example, peptides in amounts ranging from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 0.5 mg / kg, and from about 0.01 mg / kg to about 0.05 mg / kg. In some embodiments, the methods, peptides, and compositions described herein can be used in serious disease states, i.e., states that are life-threatening or potentially life-threatening. In such cases, it is possible and acceptable for the treating physician to deem it necessary to administer a large excess of these compositions. Furthermore, one of ordinary skill in the art will also know how to appropriately adjust or modify variables such as dosage, dosage schedule, and route of administration for a given subject.

[0095] The peptides and compositions of the present disclosure can be administered acutely (i.e., during or shortly after the onset of an event leading to a condition requiring treatment), prophylactically (e.g., prior to elective surgery, or before the onset of neurological signs or symptoms), or during the course of a degenerative disease to reduce or ameliorate the progression of symptoms that may occur. The timing and interval of administration vary depending on the subject's symptoms and can be administered over a period of hours, days, weeks, or longer, at intervals spanning minutes, hours, or days, as can be determined by one skilled in the art.

[0096] Some embodiments relating to pharmaceutical compositions for therapeutic or prophylactic treatment provide formulations specific for any of mucosal (oral, nasal, inhalation, rectal, vaginal, tracheal, etc.), parenteral, topical, or local administration. For the purposes herein, since mucosal administration refers to applying the vaccine to a mucosal surface, such as the surface of the respiratory tract, gastrointestinal tract, reproductive tract, etc., mucosal administration is different from topical administration. In some embodiments, the pharmaceutical composition is suitable for parenteral administration, such as intravenous, subcutaneous, intradermal, or intramuscular administration. Topical administration (i.e., non-mucosal) can be in any suitable form, such as aqueous or non-aqueous liquids (e.g., droplets), emulsions, pastes, ointments, creams, etc., for non-mucosal surfaces of a subject, such as eyes, ears, nails, hair, or skin. Therefore, the present disclosure provides a composition for topical (mucosal or non-mucosal) or parenteral administration, comprising one or more small ApoE mimetic peptides dissolved or suspended in a suitable carrier (e.g., an aqueous carrier). In an embodiment, the pharmaceutical composition is administered nasally. Any various aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized by conventional known sterilization techniques, or can be aseptically filtered. The resulting solution can be packaged for use as is or lyophilized, and the lyophilized preparation is combined with a sterile solution before administration. The composition can include pharmaceutically acceptable auxiliary substances required for close physiological conditions, such as buffers, osmotic regulators, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. Alternatively, the pharmaceutical composition as described herein can also be in a dry powder formulation. In embodiments relating to dry powder vaccine formulations, typically in the presence of at least one filler (e.g., trehalose or other sugars), the liquid vaccine is rapidly frozen in a vacuum and dried (e.g., lyophilized) to provide a vaccine formulation with excellent temperature stability. Such dry powder vaccine formulations can be administered to the host as a dry powder, thereby eliminating the need for liquid recovery.

[0097] In aspects of the compositions described herein (including pharmaceutical compositions and formulations), some embodiments provide compositions comprising at least one peptide according to SEQ ID NO: 1 (e.g., SEQ ID NOs: 1-12) in combination with an acceptable carrier, vehicle, diluent, or adjuvant. In further embodiments, the composition comprises a peptide selected from any one of SEQ ID NOs: 2-12, or any combination of two or more thereof, in combination with a carrier, vehicle, diluent, or adjuvant.

[0098] In some embodiments, the present disclosure provides a composition consisting essentially of a peptide of SEQ ID NO: 1 or SEQ ID NO: 17 and a carrier, vehicle, diluent, or adjuvant. In further embodiments, the composition consists essentially of a peptide selected from any one of SEQ ID NOs: 2-12, or any combination of two or more thereof, and a carrier, vehicle, diluent, or adjuvant.

[0099] In one aspect, the present disclosure provides a medicament for treating a neurological condition in a subject in need thereof, wherein the medicament comprises an effective amount of a peptide of Formula I and / or Formula II.

[0100] In one aspect, the present disclosure provides a medicament for treating inflammation in a subject in need thereof, wherein the medicament comprises an effective amount of a peptide of Formula I and / or Formula II.

[0101] While the following examples provide further detailed descriptions of certain aspects and embodiments of the present disclosure, these examples should be considered illustrative only and not limiting the scope of the claims in any way. Example

[0102] Example 1: Materials and methods for evaluating glial cell activation based on cell culture

[0103] In vitro methods have been developed to assess the ability of compounds (e.g., peptides) to inhibit glial cell activation using cultured cells. Laskowitz et al., (2001) Exp. Neurol., 167:74-85. Glial cell cultures are grown and maintained under standard conditions. Cultured glial cells can include primary mouse mixed glial cell cultures, mouse BV-2 microglia, and human C3 microglia. Adherent glial cells are washed with OptiMEM® medium (available from Invitrogen Corp.) to remove serum and overlay with fresh OptiMEM® medium containing the peptide.

[0104] The peptide of 5 amino acids in length is applied to one or more cell samples in parallel with a concentration range of about 0 to about 50 μM, for example 0.3, 3 and 30 μM. Using longer peptides (each 12 amino acids), peptide AL-10-1 (SEQ ID NO:18) and VL-17-9 (SEQ ID NO:19) are used as negative and positive controls respectively. Using Aono et al., Neuroscience (2003) 116:437 and Aono et al., Neurobiology of Disease (2002) 11:214 (both are incorporated by reference in their entirety), a series of peptides (see Table 2) are screened in the primary rat neurocortical cultures exposed to NMDA. The neuroprotective effect of resisting the cell death mediated by NMDA in primary neural cultures is expressed as, compared with the culture treated with vehicle, 24 hours after being exposed to NMDA, the reduction percentage of LDH (table 2). LDH release shows that the neuronal cell death after NMDA exposure. Peptide VR-55 (SEQ ID NO:4) reduced NMDA-mediated excitotoxic cell death by approximately 19% at a concentration of 1 μM. The effect was specific, and not all peptides exhibited neuroprotective effects. The positive control peptide VL-17-9 reduced LDH release by 31%. In contrast, the shorter VL-5-2 reduced LDH release by 31%, indicating that the shorter peptide also reduced NMDA-mediated excitotoxic cell death.

[0105] Table 2

[0106]

[0107] The dose response of the ApoE mimetic peptide VR-55 (SEQ ID NO: 4) in protecting against NMDA excitotoxicity in primary neural cell cultures was determined. Figure 6 The percentage inhibition of LDH release as a function of VR-55 (SEQ ID NO: 4) exposure was shown to be dose-dependent. Table 3 shows the cumulative data for all peptides in the related family (all at 1 μM concentration) screened for their inhibitory ability in the same bioassay. "0" indicates less than 5% inhibition; "+" indicates 5-10% inhibition; "++" indicates 11-20% inhibition; and "+++" indicates greater than 20% inhibition. In addition to VL-17-9, there are a number of other candidates with greater than 20% inhibition, including VR-55, VL-5-1, VL-5-2, and VR-52.

[0108] Table 3: List of ApoE mimetic peptides

[0109]

[0110] Larger peptides or proteins may need to be tested at higher concentrations to observe measurable inhibition of glial activation. If using primary mouse cells and BV-2 cells, stimulate the cells with 100 ng / ml E. coli LPS (available from Sigma-Aldrich Co.), collect the supernatant 6 hours after LPS stimulation, and analyze nitrite (using the colorimetric Greiss reagent system, available from Promega Corp.) and / or TNF-α (using a solid phase ELISA kit, available from Invitrogen Corp.). If using human C3 cells, stimulate the cells with 200 μg / ml polyinosinic acid 5' (available from Sigma-Aldrich Co.), collect the supernatant 5 days after stimulation, and analyze TNF-α using a solid phase ELISA kit (available from Invitrogen Corp.).

[0111] Example 2: Inhibition of microglial activation by ApoE mimetic peptides

[0112] Murine BV-2 cultures were prepared and used to assess inhibition of microglial activation as described in Example 1. Replicate samples of BV-2 cells were incubated without peptide, with 0.3 μM, 3 μM, or 30 μM of an ApoE mimetic peptide (VSKRR; SEQ ID NO: 3), or with 0.3 μM, 3 μM, or 30 μM of a negative control peptide (VSKKR; SEQ ID NO: 13). Each sample was stimulated with LPS, and TNF-α production was assessed as described in Example 1. Figure 1 As shown in , treatment with each dose of the ApoE mimetic peptide (SEQ ID NO: 3) tested resulted in a decrease in TNF-α production relative to untreated cells or cells treated with a negative control peptide (SEQ ID NO: 13). This data suggests that the peptides disclosed herein can be used to reduce the release of proinflammatory mediators.

[0113] ApoE mimetic peptides VR-55 (SEQ ID NO: 4), VL-5-1 (SEQ ID NO: 5), and VL-5-3 (SEQ ID NO: 10) were tested for their protective activity against brain injury by downregulating CNS inflammatory responses. Figure 11 A and 11B show that VR-55, VL-5-1, and VL-5-3 inhibited the release of the inflammatory cytokine TNF-α in mixed primary glial cell cultures after exposure to LPS.

[0114] Example 3: Materials and methods for in vivo testing of neurological deficits

[0115] Developed a mouse model of traumatic brain injury. Laskowitz et al. (2010) J. Neurotrauma , 27:1983-95. Male mice (12-14 weeks old) were anesthetized for 90 seconds using 4.3% isoflurane in oxygen in an anesthesia induction chamber. The trachea was intubated and the lungs were mechanically ventilated using 1.4% isoflurane in a 50 / 50 mixture of oxygen and nitrogen. Body temperature was maintained at 37°C using surface heating / cooling. The top of the skull was exposed through a midline incision to determine anatomical coordinates, and a 3-mm concave metal disk was fixed to the skull surface using adhesive in the midline and just caudal to the bregma. The disk diffuses impact energy and reduces the incidence of depressed skull fractures to less than 10%. After general anesthesia, the mouse was positioned in a stereotactic apparatus and the skull was exposed. A pneumatic impactor (2.0 mm diameter; available from Air-Power, Inc.) with a firing rate of 6.8 ± 0.2 m / s and a head displacement of 3 mm was used to deliver a single midline impact to the disk surface.

[0116] Also provided is an experimental mouse model for intracerebral hemorrhage. James et al. (2009) Stroke , 40:632-39. Male mice (16-20 weeks old) were anesthetized for 90 seconds using 4.6% isoflurane in oxygen in an anesthesia induction chamber. The trachea was intubated and the lungs were mechanically ventilated using 1.6% isoflurane in a 70 / 30 mixture of nitrogen / oxygen. Body temperature was maintained at 37°C using an underbody warming system. The animal's head was fixed in a stereotactic frame, local anesthetic was injected, and the scalp was incised. After exposing the skull, a burr hole was created 2 mm to the left of the bregma, and a 0.5 µL syringe needle (available from Hamilton Co.) was advanced to a depth of 3 mm from the cortex. Clostridial collagenase type IV-S (available from Sigma-Aldrich Co.) (0.1 U in 0.4 μL normal saline) was injected over a 5-minute period. Subsequently, the incision was closed, and the animal was allowed to resume spontaneous breathing and then extubated.

[0117] Developed an experimental method for measuring neurological impairment. Balance motor function was assessed using an automatic rotating rod (available from Ugo Basile North America, Inc.). One day before the experimental induction of neurological conditions or injury (for example, traumatic brain injury or intracerebral hemorrhage as described above), mice were subjected to 2 continuous training trials for 60 seconds at a set rotation speed (16 revolutions per minute), followed by 3 additional trials at an accelerated rotation speed. The average time from the rotation column falling in the latter 3 experiments was recorded as baseline delay time. After injury, mice were subjected to daily continuous trials with 3 accelerated rotation speed tests (inter-test intervals of 15 minutes). Records were taken of the average delay time from the rod, and the mice that could not catch the rod were counted as a delay time of 0 seconds.

[0118] Another experimental method for measuring neurological deficits was developed using the Morris water maze. This method uses a black aluminum pool containing a moving platform (7.5 cm in diameter) and filled with 25-27°C water emulsified with milk powder. Each training or testing session consists of 4 trials per day, with an interval of 20-30 minutes between trials. The day before the test, mice were trained using a visible platform (in each trial, the platform was marked and positioned in a different quadrant to minimize quadrant habituation, without additional maze visual cues) to accustom the mice to handling and swimming, and to make them aware of the goal of the test, which was to escape the water by climbing onto the platform. After the training day, mice were tested using a hidden platform submerged 1 cm below the water surface (for 4 consecutive days, for all trials, the platform was submerged in the western quadrant and with multiple additional maze visual cues). For each test, the mouse was placed in the pool, facing the periphery, and allowed to find the platform for a maximum of 90 seconds. For each trial, the mouse started in one of the 4 different quadrants, and the order of the starting quadrants was randomly defined each day. Latency to find the platform and swimming speed were recorded using a computerized video tracking system (Ethovision 2.2.14; available from Noldus Information Technology, Leesburg VA).

[0119] Example 4: Effects of ApoE mimetic peptides on neurological consequences after traumatic brain injury

[0120] Groups of mice received ApoE mimetic peptide (SEQ ID NO: 3; 0.05 mg / kg or 0.2 mg / kg) or vehicle (saline) by intravenous tail vein injection 2 hours and 6 hours (again) after induction of traumatic brain injury as described in Example 3. The balance motor function of each mouse was tested by the rotarod latency time measured before and after the injury as described in Example 3. As reflected by the increase in rotarod latency time, animals treated with 0.05 mg / kg or 0.2 mg / kg ApoE mimetic peptide (SEQ ID NO: 3) showed significantly improved balance motor performance compared to vehicle treatment. See Figure 2 This effect was persistent over the 5-day testing period. This data suggests that the peptides disclosed herein can be used to treat traumatic brain injury.

[0121] Example 5: Effects of ApoE mimetic peptides on neurological consequences after traumatic brain injury

[0122] Groups of mice received either ApoE mimetic peptide (SEQ ID NO: 4; 0.05 mg / kg) or vehicle (saline) by intravenous tail vein injection 2 hours and 6 hours (again) after induction of traumatic brain injury as described in Example 3. Each mouse was tested for balance motor function by measuring the rotarod latency time before and after injury as described in Example 3. Animals treated with 0.05 mg / kg ApoE mimetic peptide (SEQ ID NO: 4) had improved balance motor performance as reflected by an increase in the rotarod latency time. See Figure 3 . This effect was persistent over the 5-day testing period. Additional groups of mice received ApoE mimetic peptide (SEQ ID NO: 4; 0.05 mg / kg or 0.2 mg / kg) or vehicle (saline) by intravenous tail vein injection 2 hours and 6 hours (again) after induction of traumatic brain injury as described in Example 3. Neurocognitive performance of each mouse was tested using the Morris water maze on days 28, 29, 30, and 31 after injury as described in Example 3. Animals treated with 0.05 mg / kg or 0.2 mg / kg of ApoE mimetic peptide (SEQ ID NO: 4) showed improved neurocognitive outcomes as reflected by water maze latency times. See Figure 4 This data suggests that the peptides disclosed herein may be useful in treating traumatic brain injury.

[0123] Example 6: Effects of ApoE mimetic peptides on neurological consequences after intracerebral hemorrhage

[0124] Groups of mice received either ApoE mimetic peptide (SEQ ID NO: 4; 0.05 mg / kg) or vehicle (saline) by intravenous tail vein injection 2 hours and 6 hours (again) after induction of intracerebral hemorrhage as described in Example 3. Balance motor function of each mouse was tested by rotarod latency measured before and after induction of intracerebral hemorrhage as described in Example 3. Animals treated with 0.05 mg / kg ApoE mimetic peptide (SEQ ID NO: 4) had improved balance motor performance as reflected by increased rotarod latency. See Figure 5 This effect was persistent over the 5-day test period. This data suggests that the peptides disclosed herein can be used to treat intracerebral hemorrhage.

[0125] Example 7: Data on the Effects of ApoE Mimetic Peptides on Neurological Consequences Following Shock Wave Injury

[0126] The shock tube shock wave model was used to study shock wave injury in mice. A set of 3 shock tubes ( Figure 7 A) to provide a range of shock wave conditions with realistic peak overpressures, proportional durations and impulses. For the peptide tests, a shock tube 1240 mm long and 78 mm in diameter was used. The driving section was constant for all tests and consisted of a 25 mm thick intermediate flange bolted together with a corresponding blind flange and a sliding flange connected to the driven tube. The shape of the driving section can be changed to change the overpressure characteristics of the tube. A full-face gasket (Graphite / Buna-N material) was installed between each flange to prevent leakage. The diaphragm consisted of multiple pieces of polyethylene terephthalate (PET) film installed between the driving intermediate flange and the flange connected to the driven section. The shock wave was sent down to the driven tube section by filling the driving section with high-pressure helium through a device behind the blind flange until the diaphragm ruptured.

[0127] The shock tube was mounted vertically on an extruded aluminum frame using three vibration-damping U-bolts. Three flush-mounted piezoresistive pressure transducers (PTs) (Endevco 8530B; Endevco Corp., San Juan Capistrano, CA) were spaced 120° around the diameter and offset 6 mm from the open end of the shock tube. Because the tube wall thickness was less than the length of the PTs, a 6 mm thick collar (19 mm long) was placed over the end of the tube and welded in situ to provide additional mounting support for the PTs. An additional PT was mounted on the driver section to measure the burst pressure when the diaphragm ruptured. Chest protection for the mouse was provided using an aluminum fixture ( Figure 7B). In previous tests, the peak overpressure and impulse were reduced by more than a factor of 10. For each test, the peak incident overpressure, positive phase duration, and peak incident impulse in the three-terminal tube PT were recorded (data not shown). A range of diaphragm thicknesses (0.58 to 0.84 mm) was used to control the level of actuating burst pressure, and the actuating gas tank pressure was adjusted to 7.0 MPa. Atmospheric conditions (temperature, pressure, humidity) were recorded before each test. All sensors were sampled at 1 MHz using a 500 kHz anti-aliasing filter. Data were post-processed using an 8th-order low-pass Butterworth filter with a cutoff frequency of 40 kHz.

[0128] Two groups of 15 wild-type mice (Jackson Labs) were exposed to shock waves, one of which received a peptide injection (SEQ ID NO: 4) and the other received a vehicle-only control. Neurological deficits in injured mice were measured using the Morris water maze as described above. The efficacy of the ApoE mimetic peptide on neurocognitive abilities was examined in mice with shock wave injury using the Morris water maze assay ( Figure 8 ). Figure 8 ApoE mimetic peptides were shown to reduce neurocognitive deficits following blast wave injury, as shown by prolonged time in the quadrant with the previously learned hidden platform (i.e., as in Laskowitz et al., J. Neurotrauma, 24:1093-1107 (2007) for assessing retention ability), administration of the peptide produced a trend toward enhanced cognitive performance (data not shown). Animals treated with the vehicle spent 17.8 ± 1.9 seconds in the correct quadrant, compared to 21.8 ± 2.7 seconds in the correct quadrant for animals treated with the ApoE mimetic peptide (SEQ ID NO: 4), p = 0.24. This trend was toward improved learning in the Morris water maze (p = 0.07). This trend was toward improved performance in the probe test (p = 0.25).

[0129] The mouse apnea data is extendible to other species and can be a good model system. The blast wave injury model is unique compared to the blunt trauma model in that there is recovery of motor function and persistent and early cognitive deficits. Based on the rotarod results, the mice that received the blast wave appeared to recover motor function quickly. After the blast wave, there were no significant deficits between the sham and injury conditions. Based on the Morris water maze results, the mice that received the blast wave showed significant cognitive deficits throughout the water maze test.

[0130] Example 8: Pharmacokinetics of Intravenous Delivery in Blood and CNS

[0131] The amount of ApoE mimetic peptide VSRRR (SEQ ID NO: 4) in plasma and CNS was determined as follows:

[0132] Quantification of VSRRR in Mouse Plasma Using LC / Selected Ion Monitoring (SIM) / MS

[0133] 48 μL aliquots of mouse plasma were measured into wells of a 2 mL 96-well plate. 6 μL of the stable isotope labeled (SIL) form of the VSRRR peptide ("VSRRR

[10] "; SEQ ID NO:4) (5 pmoles / μL in 50 mM ammonium bicarbonate (pH 8) buffer) was added. For standards and quality controls (QCs), 6 μL of the synthetic form of the VSRRR peptide ("VSRRR"; SEQ ID NO:4) in 50 mM ammonium bicarbonate was added, and an equal volume of 50 mM ammonium bicarbonate was added to all wells containing mouse PK samples. 1140 μL of 50 mM ammonium formate (pH 10) was added to a final volume of 1200 μL. Salts and proteins were removed using the OASIS® HLB solid phase extraction (SPE) protocol as described below:

[0134] 1. 500 μL methanol (MeOH) passed through each well x 1

[0135] 2. 500 μL 25% acetonitrile (ACN) / 1% trifluoroacetic acid (TFA) x 1 (as pre-elution)

[0136] 3. 500 μL MeOH x 1

[0137] 4. 500 μL 50 mM ammonium formate x 2

[0138] 5. Pipette 1 mL of each sample mixture directly into the corresponding wells on an OASIS® HLB plate (hydrophilic-lipophilic balanced reversed-phase sorbent; Waters Corp.) and slowly vacuum through the wells.

[0139] 6. 500 μL 50 mM ammonium formate x 1

[0140] 7. 500 μL 10% ACN / 50 mM ammonium formate x 2

[0141] 8. 500 μL 25% ACN / 50 mM ammonium formate x 1

[0142] 9. Remove the plate to collect the wash / flow-through and place it in the collection plate

[0143] 10. Elute with 100 μL of 25% ACN / 1% TFA x 3, using slow vacuum elution. The final eluent should be approximately 300 μL.

[0144] The SPE eluate was dried using a vacuum centrifuge. The sample was reconstituted in 50 μL of buffer containing 1% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA), and 0.02% heptafluorobutyric acid (HFBA). Two microliters of the reconstituted sample were analyzed by nanocapillary LC coupled to a high-resolution, accurate mass tandem mass spectrometer. Specifically, an electrospray ionization source with NanoLockSpray™ (WatersCorp.) and a nanoAcquity UPLC® system (Waters Corp.) were used, along with a nanoLC column (1.7 μm BEH130 C18 150 μm ID x 100 mm length; Waters Corp.), a 10-minute gradient from 3% to 19% ACN and 0.1% formic acid (mobile phase A = 0.1% formic acid / 0.001% HFBA), and a total LC run time of 16.5 minutes, a flow rate of 1.8 μL / min, and a column temperature of 35°C. A SYNAPT™ G1 HDMS™ high-resolution mass spectrometer (Waters Corp.) was used to acquire full-scan MS data in the mass range of 50–4000 Da using an enhanced duty cycle scan of 360 Da.

[0145] The amount of VSRRR and VSRRR

[10] was quantified by measuring the area under the curve (AUC) of the selective ion chromatogram of doubly charged ions (m / z 357.7 and 362.7) at high resolution. The final amount of VSRRR quantification was determined using the ratio of AUC (VSRRR / VSRRR

[10] ). The ratios from 5 animals were averaged for each time point, and a standard curve was generated using calibration standards. Duplicate aliquots of the QC samples were analyzed in triplicate by LC / MS to determine the reproducibility of the analysis.

[0146] Figure 9 The amount of peptide in plasma samples is shown over time following a single dose of 0.8 mg / kg of peptide. The lower limit of quantitation (LLOQ) is indicated. Table 4 shows the results for intravenous (IV) administration.

[0147] Table 4

[0148]

[0149] Mouse Brain PK of the Therapeutic Peptide VSRRR – Sample Preparation Methods

[0150] The mouse brain was weighed in a 1.5 mL Eppendorf tube. The entire brain was transferred to a 14 mL culture tube. 1 mL of 8 M urea in 50 mM ammonium formate (pH 10) and 2.5 pmol / mL SIL peptide were added to every 100 mg of wet tissue weight. For standard controls and QC, 10 μL of peptide standard was added to every 100 mg of brain tissue (1%), and 990 μL of the above buffer was used to reach a total volume of 1 mL for every 100 mg of tissue. Each sample was processed using a tissue tearer for approximately 20 seconds. 1.5 mL of the sample was transferred to a 2-mL Eppendorf tube. Each sample was subjected to three impact probe sonications, each impact for 5 seconds. Subsequently, the sample was heated at 37°C for 30 minutes. The sample was centrifuged at 15,000 rpm for 30 minutes. A very small amount of precipitate was visible at the bottom of each tube, and the precipitate was avoided when aspirating 1 mL of sample (out of a total volume of 1.5 mL) and placing it directly onto the OASIS® plate. For plasma samples, salts and proteins were removed using the OASIS® HLB solid phase extraction (SPE) protocol described above. After extraction, the samples were completely dried in a Speed ​​Vac and reconstituted in 25 μL of 1% ACN / 0.1% TFA / 0.02% HFBA. Using a full scan MS method, 3 μL of sample was injected into a SYNAPT™ G2 HDMS™ (Waters Corp.) with a total run time of 16.5 minutes. The target peptides were detected between 3 and 8 minutes.

[0151] Table 5 and Figure 10 Shown are the mean peptide amounts over time in CNS samples from five animals following a single dose of 0.8 mg / kg peptide. Figure 10 The lowest level of quantification was shown to be 1.4 pg / mg.

[0152] Table 5

[0153]

[0154] Table 6 shows the analyte concentrations in two 3-minute brains and two 10-minute brains. The analyte concentrations were calculated from single-point internal standard quantification. Preliminary data suggest CNS penetration. Analyte concentrations were calculated from a 7-point standard curve calibration curve formula generated using samples between 1.4 pg analyte / mg tissue and 89.2 pg / mg (2x serial dilutions). Inter-animal reproducibility for brain analysis was high (greater than 21% CV). The C of drug molecule in brain after a single dose was 0.04. max It is about 9 pg analyte / mg tissue.

[0155] Table 6

[0156]

[0157] Example 9: Effects of ApoE Mimetic Peptides in Mouse Stroke Studies

[0158] Focal ischemia-reperfusion model

[0159] A modified middle cerebral artery occlusion (MCAO) model (Huang et al., (1994) Science, 265:1883-1885; Laskowitz et al., (1997) J. Cereb. Blood Flow Metab., 17:753-758) was used to determine the effects of mimetic peptides on post-stroke neurological function and to evaluate the efficacy of the peptides as therapeutic agents for stroke. After induction of anesthesia with 4.6% isoflurane, mice were intubated endotracheally and the lungs were mechanically ventilated with 1.6% isoflurane in 30% O2 / 70% N2. The right common carotid artery was identified through a midline neck skin incision. The external carotid artery was ligated and transected. The internal carotid artery was dissected distally until the origin of the pterygopalatine artery was visible. A 6-0 nylon monofilament with a lightly silicone-coated blunt tip was inserted into the stump of the proximal external carotid artery and advanced 11 mm into the internal carotid artery to occlude the middle cerebral artery. After 90 minutes, the filament was removed to restore blood perfusion and the skin incision was closed with sutures. Isoflurane was stopped and the mouse was intubated after spontaneous breathing was restored. The injured mouse was placed in an oxygen-enriched environment (FIO2 = 50%) for 1 hour and then returned to its cage. Throughout the process, rectal temperature was continuously monitored and servo-regulated using 37°C surface heating / cooling.

[0160] Testing for motor deficits

[0161] Two groups of 10-12 week old male C57Bl / 6J mice: a control group (n=12) and a VR-55-treated group (n=9) were subjected to MCAO. Control mice received 100 μL of sterile saline vehicle via intravenous tail vein injection 30 minutes and 6 hours after reperfusion injury, while VR-55-treated mice received 100 μL of sterile saline vehicle and VR-55 (SEQ ID NO: 4; 0.05 mg / kg) via intravenous tail vein injection 30 minutes and 6 hours after reperfusion injury.

[0162] As described above, balance motor function was assessed using an automatic rotating rod (Ugo Basile, Comerio, Italy). One day before MCAO, mice were subjected to 2 continuous training trials for 60 seconds at a set rotation speed (16 revolutions per minute), followed by 3 additional trials at an accelerated rotation speed. The average time from the rotation column falling in the latter 3 experiments was recorded as baseline functional rotating rod delay time. From the 1st day after MCAO, mice were subjected to daily continuous trials with 3 accelerated rotation speed tests (inter-test intervals of 15 minutes) over a 3-day period. The average delay time from the rod was recorded. Mice that could not catch the rotating rod were set to a delay time of 0 seconds.

[0163] like Figure 12 As shown in Figures 12A and 12B, control and VR-55-treated mice had similar baseline functional rotarod latency times. Control mice had a baseline functional rotarod latency time of 217 + / - 20 seconds ("Saline"), while VR-55-treated mice had a baseline functional rotarod latency time of 214 + / - 22 seconds ("VR-55"). On day 1 after injury, control and VR-55-treated mice also had similar functional rotarod latency times. However, on day 3 after injury, VR-55-treated mice showed an improvement in functional rotarod latency time compared to control mice. VR-55-treated mice had a functional rotarod latency time of 216 + / - 26 seconds, while control mice had a functional rotarod latency time of 161 + / - 32 seconds. These results are consistent with a reduction in delayed neurological damage secondary to the inflammatory response.

[0164] Treated mice are expected to show improved histological endpoints.Other mimetic peptides described herein can be administered at varying doses and are also expected to show improved functional and histological endpoints in the MCAO model of stroke in mice.

Claims

An isolated peptide or a salt thereof, wherein the peptide is VARKL (SEQ ID NO: 5) or VARRL (SEQ ID NO: 10).

2. The peptide according to claim 1, which is modified by N-terminal acetylation and / or C-terminal amidation.

3. The peptide of claim 1, wherein the peptide has no primary polypeptide sequence identity with any region of 5 consecutive amino acids of the human ApoE protein as shown in SEQ ID NO:

14. 4 . The peptide of claim 3 , wherein the peptide has no primary polypeptide sequence identity with any five consecutive amino acids from residue 130 to residue 150 of the human ApoE protein as shown in SEQ ID NO:

14. The peptide of claim 1 , wherein the peptide inhibits the activation of microglia. The peptide of claim 5 , wherein the peptide inhibits TNF-α secretion from cultured microglial cells exposed to lipopolysaccharide.

7. The peptide of claim 1, wherein the peptide binds to an ApoE receptor on the surface of a cell.

8. The peptide of claim 1, wherein the peptide blocks NMDA receptor-mediated excitotoxicity.

9. Use of a peptide according to claim 1 or claim 2 or a salt thereof in the preparation of a medicament for reducing inflammation in a subject in need thereof.

10. The use according to claim 9, wherein the drug is administered by injection, inhalation, transdermal, intravenous, intranasal, intracranial and / or intrathecal routes.

11. Use of a peptide according to claim 1 or claim 2 or a salt thereof in the preparation of a medicament for treating CNS inflammation in a subject in need thereof.

12. A composition comprising the peptide according to claim 1 and a pharmaceutically acceptable carrier or adjuvant.

13. A composition comprising the peptide of claim 1 and a pharmaceutically acceptable vehicle.

14. A composition comprising the peptide of claim 1 and a pharmaceutically acceptable diluent.

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