Cyclin-dependent kinase 5 (CDK5) inhibitory peptides

Abstract

To provide methods and compositions for promoting cognitive function and / or treating cognitive function disorders and impairments.SOLUTION: In particular the methods are accomplished by administering to a subject a specific CDK peptide inhibitor and a pharmaceutically acceptable carrier.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of U.S. Provisional Application No. 62 / 559,824, filed September 18, 2017, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Background of the Invention Brain atrophy occurs during normal aging and is a common feature of neurological diseases associated with impaired cognitive function and memory loss. Alzheimer's disease, Huntington's disease, frontotemporal dementia, and other related dementias cause significant loss of cognitive function, often weakening the afflicted person to a frail state. There is no known cure for Alzheimer's disease and related dementias, and the causes of these diseases are not fully understood. Furthermore, preclinical research has yet to explore strategies for restoring lost memory after substantial neuronal loss has occurred. Cyclin-dependent kinase 5 (CDK5), a member of the tau kinase and CDK family, plays multiple roles in brain development and has been implicated in several neurological disorders. Specifically, CDK5, a proline-specific serine / threonine kinase, is activated by the p25 protein. This occurs when intracellular levels of calcium are elevated, activating pain receptors, and then cleaves p35 to p25, which then binds to CDK5. Unlike p35, p25 is not immediately degraded, and its binding to CDKs consistently activates CDK5, leading to the hyperphosphorylation of tau. Deregulation of CDK5 activity has been shown to result in neuronal death, elevated amyloid-β (Aβ) accumulation, decreased synaptic plasticity, cytoskeletal disruption, morphological degeneration, apoptosis, and learning disabilities. Summary of the Invention

[0003] SUMMARY OF THE INVENTION In one aspect, the present invention relates to the discovery of methods and compositions for promoting cognitive function and thus treating memory loss and cognitive dysfunction / deficiency. Consequently, one aspect of the present invention relates to a method for treating cognitive dysfunction or deficiency in a subject in need thereof. The method comprises administering to the subject a cyclin-dependent kinase 5 inhibitor (CDK5i) peptide and a pharmaceutically acceptable carrier.

[0004] In one aspect of the invention, the pharmaceutical composition has the following structure: ARAFGX1PVRC X2S * (X1=I or V and X2=Y or F), and a pharmaceutically acceptable carrier. In some embodiments, the peptide comprises SEQ ID NO: 2. In one embodiment, the peptide comprises one or more conservative substitutions within the sequence of SEQ ID NO: 2.

[0005] In some embodiments, the pharmaceutically acceptable carrier comprises a polymer. In one embodiment, the polymer comprises a hydrophilic block and an endosomolytic block. In another embodiment, the hydrophilic block comprises polyethylene glycol methacrylate, and wherein the endosomolytic block comprises diethylaminoethyl methacrylate-butyl methacrylate copolymer. In some embodiments, the polymer is a stimuli-responsive polymer that responds to one or more stimuli selected from the group consisting of pH, temperature, UV-visible light, light irradiation, exposure to an electric field, ionic strength, and the concentration of a certain chemical by exhibiting a change in properties.

[0006] In some embodiments, the peptide has a deletion of 1 to 2 amino acids of SEQ ID NO: 1. In one embodiment, the peptide includes one or more additional amino acids at the N-terminus of SEQ ID NO: 1. In another embodiment, the peptide includes one or more additional amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the peptide is linked to a non-peptide molecule through a linker. In one embodiment, the non-peptide molecule is PEG or TEG. In some embodiments, the peptide has an enhanced or stabilized secondary structure.

[0007] In another aspect, the present invention provides a compound having the following structure: ARAFGX1PVRC X2S * A peptide consisting essentially of 5 to 25 amino acids in length having at least 50% amino acid sequence identity over its length to the amino acid sequence of SEQ ID NO: 1, where X1=I or V and X2=Y or F.

[0008] In some embodiments, the peptide contains one or more conservative substitutions. In one embodiment, the peptide has at least 60%, 70%, 80%, 90%, or 95% amino acid sequence identity over its length compared to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the peptide contains one or more conservative substitutions within the sequence of SEQ ID NO: 1. In some embodiments, the peptide has a deletion of one to two amino acids of SEQ ID NO: 1. In one embodiment, the peptide contains one or more additional amino acids at the N-terminus of SEQ ID NO: 1. In another embodiment, the peptide contains one or more additional amino acids at the C-terminus of SEQ ID NO: 1.

[0009] In some embodiments, the peptide is linked to a non-peptide molecule through a linker. In one embodiment, the non-peptide molecule is PEG or TEG. In another embodiment, the peptide has a reinforced or stabilized secondary structure. In another aspect, the present invention provides a peptide having a length of 12 to 50 amino acids and the following structure: ARAFGX1PVRC X2S * A peptide comprising the amino acid sequence of SEQ ID NO: 1, with (X1=I or V and X2=Y or F).

[0010] In another aspect, the present disclosure provides a recombinant nucleic acid encoding any one of the above-described peptides. Another aspect of the present disclosure includes a recombinant expression vector comprising the above-described nucleic acid operably linked to a promoter. A further aspect of the present disclosure provides a recombinant host cell comprising the above-described recombinant expression vector.

[0011] In another aspect, the present invention provides a method for treating a neurodegenerative condition in a subject, comprising administering to the subject a nucleic acid encoding a specific CDK5 peptide inhibitor or a peptide inhibitor that interferes with the CDK5-p25 / p35 interaction without interfering with the basal activity of CDK5 or interacting with CDK1 or CDK2.

[0012] In some embodiments, the subject also receives an additional therapy to treat the disorder. In another embodiment, the specific CDK5 peptide inhibitor is administered orally, transdermally, intravenously, cutaneously, subcutaneously, intranasally, intramuscularly, intraperitoneally, intracranially, or intracerebroventricularly.

[0013] In some embodiments, the neurodegenerative condition is Alzheimer's disease, Huntington's disease, frontotemporal dementia, seizure-induced memory loss, schizophrenia, Rubinstein-Taybi syndrome, Rett syndrome, fragile X, dementia with Lewy bodies, vascular dementia, ADHD, dyslexia, social, cognitive and learning disorders associated with bipolar disorder and autism, traumatic head injury, or attention deficit disorder. In other embodiments, the cognitive dysfunction / failure is associated with anxiety, conditioned fear response, panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, phobias, social anxiety disorder, or substance dependence recovery.

[0014] In some embodiments, the method further comprises exposing the subject to cognitive behavioral therapy (CBT), psychotherapy, behavioral exposure therapy, virtual reality exposure (VRE), or cognitive remediation therapy. In one embodiment, the CDK5 peptide inhibitor is administered once a day, every two days, every three days, every four days, every five days, every six days, or every seven days.

[0015] In some embodiments, the peptide has the following structure: ARAFGX1PVRC X2S * (X1=I or V and X2=Y or F) and has a length of 5 to 50 amino acids that has at least 50% amino acid sequence identity over its length to the amino acid sequence of SEQ ID NO: 1.

[0016] In some embodiments, the peptide contains one or more conservative substitutions. In other embodiments, the peptide has at least 60%, 70%, 80%, 90%, or 95% amino acid sequence identity over its length relative to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the peptide contains at least conservative substitutions within the sequence of SEQ ID NO: 1. In some embodiments, the peptide has a deletion of one to two amino acids of SEQ ID NO: 1. In another embodiment, the peptide contains one or more additional amino acids at the N-terminus of SEQ ID NO: 1. In other embodiments, the peptide contains one or more additional amino acids at the C-terminus of SEQ ID NO: 1.

[0017] In some embodiments, the peptide is linked to the non-peptide molecule via a linker. In one embodiment, the non-peptide molecule is PEG or TEG. In another embodiment, the peptide has an enhanced or stabilized secondary structure. In some embodiments, the peptide has a length of 8 to 25 amino acids, in other embodiments, the peptide has a length of 8 to 20 amino acids, and in other embodiments, the peptide has a length of 10 to 15 amino acids.

[0018] Each of the limitations of the present invention may encompass various aspects of the invention. Accordingly, it is expected that each of the limitations of the present invention involving any one element or combination of elements may be encompassed in each aspect of the present invention. The present invention is not limited in its application to the details of construction and to the arrangement of components set forth in the following description or illustrated in the drawings. The present invention is capable of other embodiments and of being practiced or carried out in various ways. Also, the phraseology and terminology used herein are for descriptive purposes and should not be considered limiting. The words "comprise," "include," "have," "contain," "involve," and variations thereof herein are meant to encompass the items listed thereafter and equivalents thereof, as well as additional items. [Brief explanation of the drawings]

[0019] Brief description of the diagram The figures are merely illustrative and are not required for practicing the invention disclosed herein. [Figure 1A-1B] Figures 1A-1B show details of the p25 / CDK5 complex and CDK5 inhibitory peptides. Figure 1A shows the structure of the p24 / CDK5 complex. Figure 1B shows the sequences of exemplary CDK inhibitory peptides. The sequences, from top to bottom, correspond to SEQ ID NOS: 5-13. The sequence of the CDK5 inhibitory peptide (boxed in the first row) is SEQ ID NOS: 2.

[0020] [Figure 2] FIG. 2 shows a computational model predicting the possible interactions of CDK5i peptides with CDK5 (left) and p25 (right). [Figure 3] FIG. 3 shows the reduction of CDK5 activity by CDK5i peptides. [Figure 4] FIG. 4 shows that the CDK5i peptide interacts with CDK5 and p25, but not with CDK1 or CDK2. [Figure 5A]Figures 5A-5B show some of the effects of the CDK5i peptide in Figure 5A, which shows that the CDK5i peptide does not affect basal CDK5 activity, while Figure 5B shows that the CDK5i peptide significantly reduces CDK5 activity in brains from P301S mice. [Figure 5B] Figures 5A-5B show some of the effects of the CDK5i peptide in Figure 5A, which shows that the CDK5i peptide does not affect basal CDK5 activity, while Figure 5B shows that the CDK5i peptide significantly reduces CDK5 activity in brains from P301S mice.

[0021] [Figures 6A-6B] Figures 6A-6B show neural progenitor cells (NPCs) from fAD iPSCs, demonstrating upregulation of HDAC2 and increased γH2AX signal, indicating DNA damage (Figure 6A) compared to cells from healthy neural iPSCs (Figure 6B). [Figure 7] Figure 7 shows neural progenitor cells (NPCs) treated with CDK5i peptide or its scrambled peptide. CDK5i significantly reduced the levels of HDAC2 and γH2AX signaling (indicative of DNA damage), while the scrambled protein had no effect.

[0022] [Figure 8] FIG. 8 presents a schematic of the fluorescence polarization high-throughput screen used to identify potential CDK5 inhibitory compounds using CDK5i peptides. [Figure 9A-9B] Figure 9A shows SEQ ID NO: 3 linked at the N-terminus to fluorescein isothiocyanate (FITC) with a linker (aminohexanoic acid; Ahx) for imaging studies. Figure 9B shows the results of human iPSC-derived neural progenitor cells treated with FITC-Ahx-Cdk5i-Tat (1 μM) (SEQ ID NO: 3) for 2 hours and imaged.

[0023] Detailed Description In one aspect, the present invention relates to the discovery of methods and compositions for treating neurodegenerative disorders. Illustratively, the compounds of the present invention are useful for promoting cognitive function and thus for treating memory loss and cognitive dysfunction / dysfunction. Consequently, one aspect of the present invention involves a method for treating cognitive dysfunction / dysfunction by administering an effective amount of a CDK5 peptide inhibitor to a subject in need thereof.

[0024] Surprisingly, according to the present invention, it has been discovered that CDK5 peptide inhibitors selectively block the interaction between CDK5 and p25 / p35 without interacting with basal CDK5 activity or other CDKs highly homologous to CDK5. This is in stark contrast to drugs that have been used to inhibit CDK5 hyperactivity because they exhibit poor specificity and affect the activity of other CDKs. Consequently, prior art CDK5 inhibitors inhibit basal CDK5 activity under normal conditions, potentially causing abnormal neuronal function and leading to serious side effects. The CDK5 peptide inhibitors of the present invention demonstrate high specificity and thus avoid the serious side effects associated with blocking basal CDK5 activity. In addition, it has been demonstrated that the peptides can cross the blood-brain barrier.

[0025] As shown in the examples below, peptides that disrupt the binding of CDK5 and p25 / p35 by interacting with one or more of these proteins in specific regions have significant therapeutic properties. Such disruption prevents complex formation, thereby avoiding the effects of CDK5 / p25 granules, such as neuronal cell death, increased amyloid-β accumulation, reduced synaptic plasticity, cytoskeletal disruption, morphological degeneration, apoptosis, and learning impairment. While disrupting specific complex formation, the peptide inhibitors described in this invention avoid the side effects associated with nonspecific binding to structurally similar molecules. Illustratively, in the examples provided herein, peptides can be designed to specifically inhibit the binding of CDK5 to p25 / p35. It has been determined that CDK5 peptide inhibitors, such as the CDK5 inhibitory (CDK5i) peptide of SEQ ID NO: 2, will interact with both CDK5 and p25, thus disrupting the formation of the p25 / CDK5 complex (CDK5i). In vitro studies have shown that CDK5 peptide inhibitors significantly reduce CDK5 kinase activity compared with scrambled peptide controls. Using a mouse model of neurodegenerative brain, it was found that CDK5 peptide inhibitors interacted with CDK5 and p35, while not binding to CDK1 or CDK2, which are highly homologous to CDK5. Furthermore, surprisingly, it was found that CDK5 peptide inhibitors did not affect basal CDK5 activity in control mice, but significantly reduced CDK5 kinase activity in brains of Alzheimer's disease (AD) patients and in brains of frontotemporal dementia (FTD) mouse models (P301S mice). These findings demonstrate that the peptide inhibitors of the present invention provide valuable therapeutic effects.

[0026] Furthermore, a human AD-related pathology model system using induced pluripotent stem cells (iPSCs) created from fibroblasts of patients with familial AD (fAD) has been employed. Neural progenitor cells (NPCs) derived from fAD iPSCs exhibited several pathological phenotypes, including upregulation of histone adenosine deacetylase 2 (HDAC2), which negatively regulates the transcription of genes associated with learning and memory; however, NPCs treated with a CDK5 peptide inhibitor exhibited significantly reduced levels of HDAC2 and γH2AX signaling (indicative of DNA damage) compared with scrambled peptides. Thus, some methods of the present invention include administering a CDK5 peptide inhibitor described herein to a subject in need thereof.

[0027] The compositions of the present invention include cyclin-dependent kinase 5 (CDK5) peptide inhibitors that may be used to treat neurodegenerative disorders.

[0028] Cyclin-dependent kinase 5 (CDK5) has been implicated in numerous pathological phenotypes in neurodegenerative disorders. Pharmacological inhibition or targeted knockdown of CDK5 has been shown to reverse neurotoxicity and tau pathology in selected neurological disorders, including Alzheimer's disease, Parkinson's disease, spinal muscular atrophy, and frontotemporal dementia (Qu et al., 2007; Piedrahita et al., 2010; Zhang et al., 2013; Miller et al., 2014). (Also see Amini et al., 2013; Rao et al., 2014; Seo et al., 2014). Abnormal CDK5 activity under pathological conditions is mediated by p25, a proteolytic fragment of p25, which has been shown to induce various pathological phenotypes in neurodegenerative brains, including neuronal and intracranial inflammation, tau hyperphosphorylation, and neuronal cell death. Inhibition of the p25 / CDK5 complex has been shown to be beneficial in certain disease states; for example, in 5xFAD mice (a mouse model of Alzheimer's disease), p25 blockade attenuates amyloid plaque and Aβ-induced synaptic depression, glial cell activation, neuronal and intracranial inflammation, and neuronal cell death (Amini et al., 2013; Rao et al., 2014; Seo et al., 2014). Blockade of p25 has also been shown to restore synaptic plasticity and cognitive performance in AD mice.

[0029] The CDK5 peptide inhibitors of the present invention are peptides that interact with the binding region of CDK5 and p25 and disrupt the formation of the CDK5 / p25 complex. The binding of CDK5 peptide inhibitors is specific in that it does not significantly affect basal CDK5 activity or effectors or pathways, such as CDK1 or CDK2, that the peptide is involved in. A peptide is considered to not significantly affect basal CDK5 activity if the basal CDK5 activity is substantially similar before and after exposure to the CDK5 peptide inhibitor. The level of basal CDK5 activity can be assessed by in vitro or in vivo assays. The level of basal CDK5 activity that remains substantially similar may be within 10%, 5%, 4%, 3%, 2%, or 1% of the level before exposure.

[0030] A peptide is considered not to significantly affect CDK1 or CDK2 binding if, after exposure to a CDK5 peptide inhibitor, no significant levels of the peptide bind to CDK1 and / or CDK2. The level of binding can be assessed by in vitro or in vivo assays known to those skilled in the art, including the assays presented in the Examples section below. A level of binding of a peptide inhibitor to CDK1 and / or CDK2 that is considered not significant may be less than or equal to 10%, 5%, 4%, 3%, 2%, or 1% of the bound complex within a mixture of the CDK5 peptide inhibitor and CDK1 and / or CDK2.

[0031] The length of the CDK5 peptide inhibitor may vary. For example, the CDK5 peptide inhibitor is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In select embodiments, the peptide inhibitor is 8-20, 10-20, 8-15, 10-15, 8-12, 10-12, or 12 amino acids in length. Peptides with such short peptide chains have high structural stability (eg, resistance to proteases) and excellent handling and storage properties.

[0032] In some embodiments, the CDK5 peptide inhibitor has the following sequence: ARAFGX1PVRCX2S *(X1=I or V and X2=Y or F) (SEQ ID NO: 1). In some embodiments, the CDK5 peptide inhibitor has 50-100% (i.e., 50-60%, 50-70%, 50-80%, 50-90%, 60-70%, 60-80%) identity to the amino acid sequence set forth in SEQ ID NO: 1. %, 60-90%, 60-100%, 70-80%, 70-90%, 70-100%, 80-90%, 80-100%, or 90-100%) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. For example, a CDK5 peptide inhibitor may have greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0033] In one particular embodiment, the CDK5 peptide inhibitor is a peptide comprising an amino acid sequence having at least 50% amino acid sequence identity over its length to the amino acid sequence of ARAFGIPVRCYS (SEQ ID NO:2; FIG. 1B). In some embodiments, the CDK5 peptide inhibitor has 50-100% (i.e., 50-60%, 50-70%, 50-80%, 50-90%, 60-70%, 60-80%, 60-90%, 60-100%, 70-80%, 70-90%, 70-100%, 80-90%, 80-100%, or 90-100%) sequence identity to the amino acid sequence set forth in SEQ ID NO:2. For example, a CDK5 peptide inhibitor may have greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

[0034] The peptides disclosed herein may be modified through the addition of one or more components at either end of the peptide. Illustratively, peptides may be modified to add a label, such as FITC, at the C-terminus and / or N-terminus, with or without a linker, such as aminohexanoic acid (Ahx). Peptides may also include functional components at either or both ends of the peptide. Illustratively, functional components include a targeting peptide or domain, such as an antibody or antibody fragment, a translocation peptide or domain, such as a transactivator of transcription (TAT) peptide, or a stabilizing peptide or domain, with or without a linker. An exemplary modified peptide is ARAFGIPVRCYSYGRKKRRQRRR (SEQ ID NO:3), which comprises the Cdk5i peptide (ARAFGIPVRCYS) (SEQ ID NO:2) directly linked to the HIV TAT peptide (YGRKKRRQRRR) (SEQ ID NO:4).

[0035] In one particular embodiment, the CDK5 peptide inhibitor is a peptide comprising amino acids having at least 50% amino acid sequence identity over its length to the amino acid sequence of ARAFGIPVRCYSYGRKKRRQRRR (SEQ ID NO:3 - SEQ ID NO:2 + YGRKKRRQRRR (SEQ ID NO:4)). In some embodiments, the CDK5 peptide inhibitor has 50 to 100% (i.e., 50 to 60%, 50 to 70%, 50 to 80%, 50 to 90%, 60 to 70%, 60 to 80%, 60 to 90%, 60 to 100%, 70 to 80%, 70 to 90%, 70 to 100%, 80 to 90%, 80 to 100%, or 90 to 100%) sequence identity to the amino acid sequence set forth in SEQ ID NO:3. For example, a CDK5 peptide inhibitor may have greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3.

[0036] A "translocation peptide" or "translocation domain" refers to any sequence of amino acids that directs a peptide contained therein to a desired intracellular destination. For example, a translocation domain, such as a polyarginine sequence, can direct or facilitate the penetration of a peptide across a biological membrane, such as a phospholipid membrane, a mitochondrial membrane, or a nuclear membrane. For example, a translocation sequence directs a peptide from outside a cell through a cell membrane and into the cytoplasm or to a desired location within the cell, such as the nucleus, ribosomes, mitochondria, ER, lysosomes, or peroxisomes. Alternatively, or in addition, a translocation sequence can direct a peptide across physiological barriers, such as the blood-brain barrier, transmucosal barriers, or hematoencephalic, blood-retinal, gastrointestinal, and pulmonary barriers.

[0037] The CDK5 peptide inhibitors described herein are not naturally occurring peptides. Most peptides have non-naturally occurring sequences where one or more amino acids differ from the naturally occurring sequence. In some embodiments, the CDK5 peptide inhibitors include one or more amino acids not contained within the complete CDK5 polypeptide. In cases where a peptide has an amino acid sequence (SEQ ID NO: 2) that forms part of a naturally occurring polypeptide (complete CDK5), the peptide itself is not naturally occurring because it is less than the entire peptide. Not all complete CDK5 polypeptides are peptide inhibitors of the present invention because they do not specifically interfere with CDK5 binding to p25 / p35.

[0038] The peptide may be 8 amino acids long, or it may be longer. Illustratively, the peptide may have additional amino acids at the N- and / or C-terminus. At either end, the amino acids may be anywhere between 1 and 100 amino acids. In some embodiments, the peptide may range from 1 to 50, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or any integer in between. Peptides can be cyclic or acyclic. Cyclic peptides in some instances have improved stability properties. Those skilled in the art know how to produce cyclic peptides.

[0039] The peptides of the present invention may include conservative substitutions. As used herein, "conservative amino acid substitution" refers to an amino acid or nucleic acid that does not alter or substantially alter the function or other characteristics of the peptide or polynucleotide. A given amino acid can be replaced with a residue having similar physicochemical characteristics, such as replacing one fatty acid residue with another (such as Ile, Val, Leu, or Ala for each other) or one polar residue with another (such as Lys with Arg; Glu with Asp; or Gln with Asn). Other such conservative substitutions, such as replacing entire regions with similar hydrophobic characteristics, are well known. Peptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity is retained.

[0040] Amino acids can be grouped according to similarities in the properties of their side chains: (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser(s), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Instead, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions would involve exchanging a member of one of these classes for another. Specific conservative substitutions include, for example; Ala for Gly or Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly for Ala or Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Ile or Val; Lys for Arg, Gln, or Glu; Met for Leu, Tyr, or Ile; Phe for Met, Leu, or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp; and / or Phe for Val, Ile, or Leu, etc.

[0041] As used throughout this application, the term "peptide" is used in its broadest sense to refer to a sequence of subunit amino acids. The peptides of the present invention may contain L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids. The peptides described herein may be chemically synthesized or recombinantly expressed. The peptides may be linked to other compounds that promote increased in vivo half-life, such as by PEGylation, HESylation, PASylation, or glycosylation, or may be produced as Fc fusions or deimmunized variants. As will be appreciated by those skilled in the art, such linkages may be covalent or non-covalent.

[0042] Peptides may also be linked to other molecules: the peptide and molecule may be linked directly to each other (e.g., via a peptide bond), linked via a linker molecule, which may or may not be a peptide, or indirectly linked to each other, illustratively by attachment to a common carrier molecule.

[0043] Thus, a linker molecule ("linker") may optionally be used to link the peptide to another molecule. The linker may be a peptide consisting of one to multiple amino acids, or a non-peptide molecule. Examples of peptide linker molecules useful in the present invention include glycine-rich peptide linkers, in which the majority of amino acid residues are glycine. Preferably, such glycine-rich peptide linkers consist of about 20 or fewer amino acids.

[0044] In some embodiments, the linker consists of glycine and serine. Illustratively, the linker may be Gly-Ser or Gly-Gly-Ser. Alternatively, it may be any of these sequences with 1-4 Gly at one or both termini and / or 1-2 Ser at either termini. Sequences containing 2-10 or 2-5 consecutively linked copies of any one of these amino acids may be employed as peptide linkers.

[0045] In another aspect of the present invention, peptides are covalently linked to polyethylene glycol (PEG) molecules, notably 1500 or 4000 MW PEG, via their C-terminus or lysine residues to reduce urinary clearance and therapeutic dose and increase plasma half-life. However, in another embodiment, the half-life of the peptide is increased by incorporating the peptide into a biodegradable and biocompatible polymeric material for a drug delivery system that forms microspheres. Polymers and copolymers, illustratively poly(D,L-lactide-co-glycolide) (PLGA), are illustratively linked to PEG or TEG molecules. Such molecules are referred to as PEGylated or TEGylated peptides.

[0046] Peptides may be derived from SEQ ID NO: 1 or 2 by any chemical modification that improves resistance to proteolysis; and substantially homologous peptides may be derived from SEQ ID NO: 1 or 2 by substitution of one or more amino acids. By "substituted" or "modified," the present invention encompasses amino acids that are changed or modified from naturally occurring amino acids.

[0047] In particular, the N- and / or C-termini of the peptides described at the beginning of this article can be optionally protected against proteolysis. Illustratively, the N-terminus can be in the form of an acetyl group and / or the C-terminus can be in the form of an amino group. Internal modifications of peptides that are resistant to proteolysis are also envisioned. For example, in which at least the --CONH-- peptide bond is modified and replaced by a (CH2NH) reduced bond, a retro-inverso bond, a (CO--CH2 cetomethylene bond, a (CHOH--CH2) hydroxyethylene bond, an (N--N) bond, an E-alcene bond or a -CH=CH- bond.

[0048] By way of example, peptides may be modified by acetylation, acylation, amidation, cross-linking, cyclization, disulfide bond formation, covalent cross-link formation, cysteine ​​formation, pyroglutamic acid formation, formylation, gamma carboxylation, iodination, methylation, myristylation, oxidation, phosphorylation, etc.

[0049] The peptides of the present invention may consist of an amino acid(s) in the D configuration, which makes them resistant to proteolysis. They may also be stabilized, for example, by modifying at least two amino acid residues with an olefin side chain, preferably a C3-C8 alkenyl chain, preferably a penten-2-yl chain, followed by chemical cross-linking of the chain, according to the so-called "stapling" technique. For example, amino acids at positions i and i+4 to i+7 may be replaced with unnatural amino acids, which replace reactive olefin residues. All such proteolytically resistant, chemically modified peptides are encompassed by the present invention. Two amino acids are "homologous," "substantially homologous," or "substantially similar" if one or more amino acid residues are replaced by biologically similar residues, or if more than 80% of the amino acids are identical, or if more than about 90%, preferably more than about 95%, of the amino acids are similar (functionally identical).

[0050] Preferably, similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or any of the programs known in the art (BLAST, FASTA, etc.). The peptides described herein can be synthesized using standard synthetic methods known to those of skill in the art. In a preferred embodiment, the peptides are obtained by stepwise condensation of amino acid residues by condensation of a pre-formed fragment already containing the amino acids in the appropriate order, or by condensation of several previously prepared fragments, while protecting amino acid functional groups other than those involved in the peptide during condensation. In particular, the peptides can be synthesized according to the method originally described by Merrifield.

[0051] In some instances, peptides may include only natural amino acids; however, as is known in the art, unnatural amino acids (i.e., compounds that do not occur in nature but can be inserted into a peptide chain) and / or amino acid analogs may alternatively be employed.

[0052] There are many known unnatural amino acids, any of which may be incorporated into the peptides of the invention. Some examples of unnatural amino acids include 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta-cyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine, 1-amino-cyclopropanecarboxylic acid, 1-amino-2-phenyl-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-1-methylpyrrole-2-carboxylic acid, Examples of amino acids include 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta-, and / or para-substituted phenylalanines (e.g., substituted with --C(=O)C6H5; --CF3; --CN; -halo; --NO2; CH3), disubstituted phenylalanines, substituted tyrosines (e.g., further substituted with --Q=O)C6H5; --CF3; --CN; -halo; --NO2; CH3), and statines. Derivatization can be performed to include hydroxylated, phosphorylated, sulfonated, acylated, and glycosylated amino acid residues.

[0053] Additionally, one or more of the amino acids in a peptide or peptide may be modified by the addition of a chemical entity, such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a covalent linker, functionalization, or other modification. A peptide or peptide may also be a single molecule or a multi-molecular complex, such as a protein. A peptide or peptide may be only a fragment of a naturally occurring protein or peptide. A peptide or peptide may be naturally occurring, recombinant, synthetic, or any combination thereof.

[0054] In some examples, the peptide can include (e.g., comprise, consist essentially of, or consist of) at least 7 (e.g., 7, 8, 9, 10, 11, 12) consecutive amino acids of the sequence of SEQ ID NO: 1 or 2.

[0055] Examples of chemical synthesis techniques are solid-phase synthesis and liquid-phase synthesis. In solid-phase synthesis, for example, the amino acid corresponding to the C-terminus of the peptide to be synthesized is bound to a support soluble in an organic solvent, and one amino acid having an amino group and a side chain functional group protected by an appropriate protecting group is condensed one by one from the C-terminus to the N-terminus, and the amino acid bound to the resin or the protecting group of the amino group of the peptide is released by repeated alternating reactions, thereby extending the peptide chain in this manner. Solid-phase synthesis methods are roughly classified into the tBoc method and the Fmoc method depending on the type of protecting group used. Typically used protecting groups include tBoc (t-butoxycarbonyl), Cl-Z (2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl (benzyl), Fmoc (9-fluorenyloxycarbonyl), Mbh (4,4'-dimethoxydibenzhydryl), Mtr (4-methoxy-2,3,6-trimethylbenzenesulfonyl), Trt (trityl), Tos (tosyl), Z (benzyloxycarbonyl), and Clz-Bzl (2,6-dichlorobenzyl) for amino groups; NO2 (nitro) and Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) for guanidino groups; and tBu (t-butyl) for hydroxyl groups. After synthesis of the desired peptide, it is deprotected and cleaved from the solid support. Such peptide cleavage reactions may be catalyzed by hydrogen fluoride or trifluoromethanesulfonic acid for the Boc method, and by TFA for the Fmoc method.

[0056] Alternatively, peptides may be synthesized using recombinant technology. CDK5 peptide inhibitors may be expressed as isolated nucleic acids encoding the peptides. Isolated nucleic acid sequences include RNA or DNA. As used herein, "isolated nucleic acids" are those that are removed from their normal surrounding nucleic acid sequences in a genome or DNA sequence. Such isolated nucleic acid sequences may contain additional sequences useful for facilitating expression and / or purification of the encoded peptides, including, but not limited to, polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export and secretion signals, nuclear localization signals, and cell membrane localization signals.

[0057] Thus, in another aspect, the present invention provides a recombinant expression vector comprising an isolated nucleic acid of any aspect of the present invention operably linked to a suitable control sequence. A "recombinant expression vector" includes a vector in which a nucleic acid coding region or gene is operably linked to any control sequence capable of affecting expression of the gene product. A "control sequence" operably linked to a nucleic acid sequence of the present invention is capable of effecting expression of the nucleic acid molecule. Control sequences need not be contiguous with the nucleic acid sequence, so long as they function to direct its expression. Thus, for example, intervening untranslated but transcribed sequences can be present between a promoter sequence and a nucleic acid sequence and the promoter sequence can still be considered "operably linked" to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including, but not limited to, plasmids and virus-derived expression vectors. The control sequences used to drive expression of the disclosed nucleic acid sequences in mammalian systems are either basal (driven by any of a variety of promoters, including, but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of an inducible promoter, including, but not limited to, tetracycline, steroid-responsive). The construction of expression vectors for use in transfecting host cells is well known in the art and can thus be accomplished by standard techniques.

[0058] The method of producing a peptide may optionally include the step of purifying the peptide and / or chemically modifying the peptide.

[0059] The methods of the present invention may be used to promote cognitive function in normal subjects or to treat subjects with cognitive impairment. As used herein, a normal subject is a subject who has not been diagnosed with a disorder associated with impaired cognitive function.

[0060] "Cognitive function" refers to the mental processes of an animal or human subject related to gathering and / or processing information; understanding, reasoning, and / or applying information and / or ideas; summarizing or describing ideas and / or information; creative, problem-solving, and possibly insightful acts; and learning, perceiving, and / or being aware of ideas and / or information. Mental processes are distinct from their beliefs, desires, and the like. In some embodiments, cognitive function may be assessed, and optionally defined, by one or more tests or assays for cognitive function. Non-limiting examples of tests or assays for cognitive function include the CANTAB. (e.g., Fray et al. "CANTAB battery: proposed utility in neurotoxicology."Neurotoxicol Teratol. 1996;18(4):499-504), Stroop Test, Trail Making, Wechsler Digit Span, or the CogState computerized cognitive test (see also Dehaene et al. "Reward-dependent learning in neural networks for planning and decision making. Brain Res. 2000;126:21729; see Iverson et al. "Interpreting change on the WAIS-III / WMS-III in clinical samples." Arch Clin Neuropsychol. 2001;16(2):183-91; and Weaver et al. "Mild memory impairment in healthy older adults is distinct from normal aging." Cogn. 2006;60(2):146-55).

[0061] Impaired cognitive function refers to cognitive function that is not as robust as that observed in age-matched normal subjects, and includes a state in which cognitive function is reduced.In some cases, cognitive function is reduced by about 5%, about 10%, about 30% or more compared to the cognitive function measured in age-matched normal subjects.Cognitive function may be promoted to any detectable degree, but in humans, it is preferably promoted sufficiently to allow the impaired subject to perform the daily activities of normal life.

[0062] In some embodiments, a method for treating cognitive impairment or dysfunction is provided. The method comprises administering an effective amount of a CDK5i peptide to a subject in need thereof. The CDK5 peptide inhibitor may be administered at an effectively low dosage to maintain an effective cumulative CDK5 peptide inhibitor serum concentration in the subject. The CDK5 peptide inhibitor may be administered once every other day. The CDK5 peptide inhibitor may be administered once, twice, three times, four times, or five times a day, and / or every other day, every third day, every fourth day, every fifth day, every sixth day, every seventh day, etc. The CDK5 peptide inhibitor may also be administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0063] In some embodiments, the cognitive dysfunction or dysfunction is associated with, but is not limited to, Alzheimer's disease, Huntington's disease, frontotemporal dementia, seizure-induced memory loss, schizophrenia, Rubinstein-Taybi syndrome, Rett syndrome, fragile X, dementia with Lewy bodies, vascular dementia, ADHD, dyslexia, social, cognitive, and learning disorders associated with bipolar disorder and autism, traumatic head injury, stroke-induced cognitive and motor deficits, traumatic brain injury, cognitive dysfunction-mediated neuronal degeneration and neurodegenerative loss, or attention deficit disorder. In some embodiments, the cognitive dysfunction or dysfunction is associated with, but is not limited to, anxiety, conditioned fear response, panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, phobias, social anxiety disorder, substance dependence recovery, or Age Associated Memory Impairment (AAMI), and Age Related Cognitive Decline (ARCD). One skilled in the art will understand that the methods of the present invention may be used to treat any condition associated with cognitive dysfunction or dysfunction.

[0064] Alzheimer's disease (AD) is a degenerative brain disorder characterized by cognitive and non-cognitive neuropsychiatric symptoms, accounting for approximately 60% of all cases in patients over the age of 65. In AD, the cognitive system controlling memory is damaged. Long-term memory is often preserved, while short-term memory is lost; conversely, memory may become disorganized, resulting in errors in recognizing familiar people and places. Psychiatric symptoms are common in AD, and psychotic disturbances (auditory hallucinations and delusions) are present in many patients. The psychiatric symptoms of AD may involve shifts in dopamine or acetylcholine concentrations, which may increase the dopamine / cholinergic balance. For example, it has been proposed that increased dopamine release may be responsible for the positive symptoms of schizophrenia. This may result in dopaminergic / cholinergic disruption. In AD, a reduction in cholinergic neurons effectively reduces acetylcholine release, resulting in a negative disruption of the dopaminergic / cholinergic balance. Indeed, antipsychotic agents used to treat the psychotic disorders of schizophrenia are also useful in reducing the psychotic disorders in Alzheimer's disease patients and may be combined with the compositions described herein for use in the methods of the present invention.

[0065] Also provided according to the present invention is a method for recapturing memory in subjects with Alzheimer's disease by administering CDK5 peptide inhibitor or other compounds of the present invention.This method can optionally involve administering inhibitor and monitoring the subject to identify the recapturing of previously lost memory.Subjects can be monitored by routine tests known in the art.For example, some are described in books such as DSM mentioned above or in medical science literature.

[0066] The present invention also provides a method for treating Huntington's disease by administering an effective amount of a CDK5i peptide. Huntington's disease is a neurological disease that results in cognitive decline associated with a relentless progression to death. Null cognitive symptoms associated with Huntington's disease include loss of mental speed, attention, and short-term memory, and / or behavioral symptoms. In some embodiments, the method of treatment is not selected based on the expression level of a Huntington's disease biomarker gene selected from the group consisting of ANXA1, AXOT, CAPZA1, HIF1A, JJAZ1, P2Y5, PCNP, ROCK (p160ROCK), SF3B1, SP3, TAF7, and YIPPEE. In some embodiments, the method of diagnosis and treatment is not selected based on the expression level of a Huntington's disease biomarker gene disclosed in U.S. Patent Application No. US2007 / 0015183. In some embodiments, the method of diagnosis and treatment is selected based on medical history, family history, or imaging studies.

[0067] The present invention further provides a method for treating frontotemporal dementia (FTD) by administering an effective amount of CDK5i peptide.FTD is a neurological disease that typically results in cognitive decline associated with progressive neuronal loss in the frontal and temporal lobes of the brain.The cognitive symptoms associated with FTD are deficits in social and personal behavior, emotional blunting, and expressive and receptive language.Diagnostic and treatment methods may be selected based on medical history, family history, neuropsychological testing, or brain imaging testing.

[0068] As used herein, treating a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical trials. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms associated with a disorder involving cognitive impairment, reduction in the extent of the disease, delay or slowing of disease progression, recovery, alleviation or stabilization of the condition, and other beneficial results such as improvement of cognitive function or reduction in the rate of cognitive decline.

[0069] In some embodiments, the subject may receive additional treatment for the disorder in addition to the CDK5i peptide. The combined treatment may be any type of treatment suitable for treating a particular disease. For example, the combined treatment may be behavioral therapy or medication. Behavioral therapy includes, but is not limited to, electroconvulsive seizure therapy, exercise, group therapy, talk therapy, or conditioning. In another embodiment, the behavioral therapy is cognitive behavioral therapy. Examples of behavioral therapy that may be used in the ongoing method are described below. For example, Cognetive-Behavioral Therapies by K. Dobson, ed., Guilford Publications, Inc., 2002; The New Handbook of Cognetive Therapy Techniques by Rian E. McMullin; Norton, WW & Company, Inc., 2000; and Cognetive Therapy: Basics and Beyond by Judith SS Beck, Guilford Publications, Inc., 1995, all of which are incorporated herein by reference in their entirety.

[0070] The CDK5i peptide inhibitor may be administered repeatedly, such as daily, weekly, or monthly, in one or more doses. It may also be administered prophylactically; that is, before symptoms appear, on a regular basis (daily, weekly, monthly, etc.), and in some cases, irregularly, for example, whenever symptoms begin.

[0071] Specific CDK5 peptide inhibitors may be administered orally, transdermally, intravenously, dermally, subcutaneously, intranasally, intramuscularly, intraperitoneally, intracranially, or intracerebroventricularly.

[0072] The present invention also relates to improving cognitive function in normal subjects by administering an effective amount of a CDK5 peptide inhibitor. Improving cognitive function includes promoting cognitive function in a subject so that it more closely resembles or exceeds the function of an age-appropriate, normal, non-impaired subject. A normal subject is one who has not been diagnosed with any disorder or condition associated with impaired cognitive function. A subject's cognitive performance is affected by various factors, and the method of the present invention can be performed to counteract any factor, such as sleep deprivation, mental fatigue, physical fatigue, or over-fatigue.

[0073] A subject is intended to mean a human or vertebrate, or mammal, including, but not limited to, dogs, cats, horses, cows, pigs, sheep, goats, turkeys, chickens, and primates, such as monkeys. In some embodiments, the subject is one that is otherwise not in need of a CDK5 and / or p25 inhibitor. Human subjects are preferred.

[0074] The term "effectiveness" of a compound of the present invention refers to an amount necessary or sufficient to realize a desired biological effect. For example, a therapeutically effective amount of a compound of the present invention is an amount sufficient to restore memory access. In combination with the teachings provided herein, by choosing between various active compounds and weighing factors such as efficacy, comparative bioavailability, patient weight, severity of side effects, and preferred mode of administration, a preventive or therapeutic treatment regimen that does not cause substantial toxicity and is overall effective in treating a particular subject can be designed. The effective amount for any particular application may vary depending on such factors as the disease or condition being treated, the specific therapeutic compound being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can empirically determine the effective amount of a particular therapeutic compound of the present invention without undue experimentation.

[0075] In one example, an effective amount of a CDK5 peptide inhibitor of the present disclosure can range from about 100 μg to 100 mg per kg body weight when administered intravenously. In some embodiments, a therapeutically effective amount is 10 to 100 mg / kg body weight administered intraperitoneally, including 20 to 80 mg / kg body weight, 30 to 70 mg / kg body weight, or 40 to 60 mg / kg, such as about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, 70 mg / kg, about 80 mg / kg, or about 90 mg / kg. Doses may be given in single or divided doses. The amount of CDK5 peptide inhibitor to be administered will be determined in light of various relevant factors, such as the condition to be treated, the selected route of administration, the age, sex, and weight of the subject, and the severity of the subject's symptoms, among other factors, all of which are known to those of skill in the art. The exemplified doses should not limit the scope of the invention in any way.

[0076] The target dose for delivery of the compounds described herein may typically be administered once daily for a series of consecutive days, and depending on the application, may be administered daily, weekly, or monthly, and any amount of time in between. In one embodiment, the composition is administered once daily for at least two consecutive days. In another embodiment, the therapeutic compound (CDK5 peptide inhibitor and a pharmaceutically acceptable carrier) is administered once every other day, or once daily with at least two days between administrations. Because there is some human variation in a given dose, the dosage may be customized in some cases. Such manipulation is within the skill of one of ordinary skill in the art in light of the teachings found herein.

[0077] The desired biological effect may be a reduction or inhibition of one or more symptoms associated with a neurodegenerative disease. One or more symptoms do not have to be completely (i.e., 100%) eliminated to achieve the desired biological effect. Compared to symptoms without treatment, administration of a CDK5 peptide inhibitor may reduce the symptom(s) by a desired degree, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% (i.e., completely). Alternatively, the desired biological effect may be a reduction or inhibition of CDK5 kinase in the brain.

[0078] The formulations of the present invention are administered in pharmaceutically acceptable solutions which may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, and any other therapeutic ingredients.

[0079] For use in therapy, an effective amount of the therapeutic compound of the present invention can be administered to a subject by any method that delivers the therapeutic agent or compound to the desired surface, e.g., mucosal, systemic, etc. Administering the pharmaceutical composition of the present invention can be accomplished by any method known to those skilled in the art. The present invention includes any embodiment or combination of peptides of the present invention and a pharmaceutically acceptable carrier. In addition to the peptide of the present invention, the pharmaceutical composition may contain (a) a lyoprotectant, b) a surfactant; (c) a bulking agent; (d) an osmolality adjusting agent; (e) a stabilizer; (f) a preservative, and / or (g) a buffer. Illustratively, the lyoprotectant may include, for example, sucrose, sorbitol, or trehalose. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule that, when combined with the peptide, substantially prevents or reduces the chemical and physical instability of the peptide in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0080] For oral administration, the therapeutic compounds of the present invention can be easily formulated by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers allow the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the subject to be treated. If the pharmaceutical preparation for oral use can be obtained as a solid excipient, and if it is desired to obtain a tablet or dragee core, the resulting mixture can be optionally milled and the granular mixture can be processed after adding suitable excipients. Suitable excipients are, inter alia, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; for example, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof. Optionally, the oral preparations may also be formulated in saline or buffer, i.e., administered without EDTA or any carrier to neutralize internal acidic conditions.

[0081] Also specifically contemplated are oral dosage forms of the above component(s) or components. The component(s) may be chemically modified to enable effective oral delivery of the derivative. Generally, contemplated chemical modifications include attachment of one or more moieties to the component molecule itself, such that the moieties (a) inhibit proteolysis; and (b) allow uptake into the bloodstream from the stomach or intestine. Also desired is increased overall stability and circulation time of the component(s) in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and polyproline. Other polymers that may be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane, and polyethylene glycol moieties.

[0082] In some embodiments, the polymer is a block polymer and comprises a hydrophilic block and an endosomolytic block. Any suitable hydrophilic block and endosomolytic block may be used. In one embodiment, the hydrophilic block comprises polyethylene glycol methacrylate. In another embodiment, the endosomolytic block comprises diethylaminoethyl methacrylate butyl methacrylate copolymer. In further embodiments, the polymer is a stimuli-responsive polymer that responds to one or more stimuli selected from the group consisting of pH, temperature, UV-visible light, light irradiation, exposure to an electric field, ionic strength, and the concentration of a certain chemical by exhibiting a change in properties. As used herein, a "stimuli-responsive polymer" is a polymer that changes its coupling properties in response to a stimulus. A stimuli-responsive polymer responds to changes in external stimuli, such as pH, temperature, UV-visible light, light irradiation, exposure to an electric field, ionic strength, and the concentration of a certain chemical by exhibiting a change in properties. The chemical could be a multivalent ion such as calcium ion, a polyion of any charge, or an enzyme substrate such as glucose. For example, a temperature-responsive polymer may be responsive to changes in temperature by exhibiting a lower critical solution temperature in aqueous solution. A stimuli-responsive polymer may be a multi-responsive polymer that exhibits changes in properties in response to combined, simultaneous, or sequential changes in two or more external stimuli. A stimuli-responsive polymer may be a synthetic or natural polymer that exhibits reversible physical or physicochemical changes, such as folding / unfolding transitions, reversible precipitation behavior, or other physical changes in response to stimuli such as temperature, light, pH, ions, or pressure. Exemplary stimuli-responsive polymers are temperature-sensitive polymers, pH-sensitive polymers, and photosensitive polymers.

[0083] The site of release may be the stomach, the small intestine (the duodenum, jejunum, or ileum), or the large intestine. Those skilled in the art have formulations available that will not dissolve in the stomach, but will release the material in the duodenum or elsewhere in the small intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the therapeutic agent or by release of the biologically active material beyond the stomach environment, such as in the intestine.

[0084] To ensure complete gastric resistance, a coating that is impermeable to at least pH 5.0 is important. Examples of more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may also be used as mixed films.

[0085] For tablets not intended for gastric protection, a coating or mixture of coatings may also be used. This may include sugar coatings or coatings that make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutics or powders; for liquid forms, a soft gelatin shell may be used. The shell material for cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets, or powder tablets, moist mashing techniques may be used.

[0086] The therapeutic agent may be included in the formulation in the form of fine, multiparticulate granules or pellets with a particle size of about 1 mm. The formulation of the material for capsule administration may also be as a powder, lightly compressed plugs, or even tablets. The therapeutic agent may be prepared by compression.

[0087] All colors and flavors may be included. For example, a therapeutic agent may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage, containing the colors and flavors.

[0088] The volume of the therapeutic agent may be diluted or increased with an inert material. These diluents may include carbohydrates such as mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, and starch, among others. Certain inorganic salts may also be used as lubricants, including calcium triphosphate, magnesium carbonate, and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

[0089] Disintegrants may be included in formulating therapeutic agents into solid dosage forms. Materials used as disintegrants include, but are not limited to, starch, including the starch-based commercial disintegrant Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge, and bentonite may all be used. Another form of disintegrant is a soluble cation exchange resin. Powdered gums may be used as disintegrants and binders, and can include powdered gums such as agar, karaya, or sugar currant. Alginic acid and its sodium salt are also useful as disintegrants. Binders that may be used to hold the therapeutic agent together and form a hard tablet include materials from natural products such as acacia, hot currant, starch, and gelatin. Others include methylcellulose (MC), ethylcellulose (EC), and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) could both be used in granulating the therapeutic agent in an alcoholic solution.

[0090] Antifriction agents may be included in the formulation of the therapeutic agent to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic agent and the die wall, and may include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oil, and stearic acid wax. Soluble lubricants may also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000, etc.

[0091] Lubricants may be added which may improve the flow characteristics of the drug during the formulation and aid in rearrangement during compression. Lubricants may include starch, talc, pyrogenic silica, and silicoaluminate.

[0092] To aid in dissolving the therapeutic agent in an aqueous environment, a surfactant may be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents may also be used and may include benzalkonium chloride or benzathonium chloride. A list of possible nonionic detergents that may be included in the formulation as surfactants includes lauromacrogol 400, 40 polyoxyl stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. These surfactants may be present in the therapeutic agent formulation either alone or as a mixture in different ratios.

[0093] Orally usable pharmaceuticals include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Push-fit capsules may contain fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres are well defined in the art. All formulations for oral administration should be in a dosage appropriate for such administration.

[0094] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use herein may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, such as, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve which delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0095] Contemplated for use in the practice of the present invention are a wide variety of mechanical devices designed for pulmonary delivery of therapeutic products, including nebulizers, metered dose inhalers, and dry powder inhalers, all of which are familiar to those skilled in the art.

[0096] Some specific examples of commercially available devices for the practice of the present invention are the Ultravent Inhaler manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II Inhaler manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin Metered Dose Inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhalater Powder inhaler manufactured by Fisons Corp., Bedford, Massachusetts.

[0097] All such devices require the use of a suitable formulation for the delivery of the therapeutic agent. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material in addition to conventional diluents and / or carriers useful in therapy. The use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is also contemplated. Chemically modified therapeutic agents may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.

[0098] Formulations suitable for use in nebulizers, either jet or ultrasonic, will typically contain a therapeutic agent dissolved in water. The formulation may also include a buffer and a simple sugar (e.g., for stabilization and regulation of osmotic pressure). Nebulizer formulations also contain a surfactant to reduce or prevent surface-induced aggregation of the compound caused by atomization of the solution in aerosol formation. Formulations for use with metered-dose inhalers will generally contain a finely divided powder containing the therapeutic agent suspended in a propellant with a surfactant adjuvant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon, or hydrocarbon, or combinations thereof, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane. Suitable surfactants include sorbitan trioleate and soybean lecithin. Oleic acid may also be useful as a surfactant.

[0099] Formulations for dispensing from powder inhalation devices will comprise a finely divided powder containing the therapeutic agent, and may also include a bulking agent such as lactose, sorbitol, sucrose, or mannitol in an amount to facilitate dispensing of the powder from the device, e.g., 50 to 90% of the formulation. The therapeutic agent will most advantageously be formulated in the form of particles having an average particle size of 10 mm (or microns), and most preferably 0.5 to 5 mm for most effective delivery to the distal lung.

[0100] Nasal delivery of the pharmaceutical compositions of the present invention is also contemplated. Nasal delivery allows for direct passage of the therapeutic product of the pharmaceutical compositions of the present invention into the bloodstream after administration to the nose, without the need for product accumulation in the lungs. Formulations for nasal delivery include those with dextran or cyclodextran.

[0101] For nasal administration, an effective device is a small, rigid bottle fitted with a metered-dose sprayer. In one embodiment, the metered dose is delivered by drawing the solution of the present invention into a chamber of defined volume with an opening dimensioned to aerosolize the aerosol formulation by forming a spray when the liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present invention. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

[0102] Instead, a plastic squeeze bottle is used that has an opening or aperture that is sized to aerosolize the aerosol formulation by forming a spray when squeezed. The aperture is usually found at the top of the bottle, and the top is generally tapered to fit partially in the nasal passage for effective administration of the aerosol. Preferably, the nasal inhaler will provide a metered dose of the aerosol formulation for administration of a metered dose of the drug.

[0103] The compounds may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion, when it is desired to deliver them systemically. Formulations for injection may be provided in unit dose form, for example, in ampoules or multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0104] Pharmaceutical preparations for parenteral administration include aqueous solutions of active compounds in water-soluble form.In addition, suspensions of active compounds can be prepared as suitable oily injection suspensions.Suitable lipophilic solvents or vehicles include sesame oil, or ethyl oleate or triglycerides, or synthetic fatty acid esters such as liposomes.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound, allowing the preparation of highly concentrated solutions.

[0105] Alternatively, the active compound may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.In addition to the formulations described above, the compound may also be formulated as a depot preparation.Such long-acting preparations may be formulated with suitable polymers or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0106] The pharmaceutical compositions may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0107] Suitable liquid or solid pharmaceutical forms include, for example, aqueous or saline solutions for inhalation, microencapsulated, cochleated, coated with microscopic gold particles, contained in liposomes, sprayed, aerosolized, skin-implanted pellets, or dried on a sharp object for scratching into the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or preparations with delayed release of active compounds, in which excipients and additives and / or adjuvants, disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweeteners, and dissolving agents are commonly used as described above. Pharmaceutical compositions are suitable for use in various drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.

[0108] The therapeutic compounds of the present invention, and optionally other therapeutic agents, may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, although non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, malic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts may also be prepared as alkaline earth salts, such as sodium, potassium, or calcium salts of the alkali metal or carboxylic acid groups thereof.

[0109] Suitable buffering agents include acetic acid and salts (e.g., 1-2% w / v), citric acid and salts (e.g., 1-3% w / v), and phosphoric acid and salts (e.g., 0.8-2% w / v). Suitable preservatives include benzalkonium chloride (e.g., 0.003-0.03% w / v), chlorobutanol (e.g., 0.3-0.9% w / v), parabens (e.g., 0.01-0.25% w / v), and thimerosal (e.g., 0.004-0.02% w / v).

[0110] The pharmaceutical compositions of the present invention contain an effective amount of a therapeutic compound of the present invention, optionally contained in a pharmaceutically acceptable carrier. The term pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans or other vertebrates. The term carrier refers to a natural or synthetic organic or inorganic component with which the active ingredient is combined to facilitate application. The components of the pharmaceutical compositions are also capable of being mixed with the compounds of the present invention and with each other in a manner such that there is substantially no interaction that would impair the desired pharmaceutical efficacy.

[0111] Therapeutic agents may be delivered to the brain using formulations capable of delivering therapeutic agents across the blood-brain barrier. One obstacle to delivering therapeutic agents to the brain is the physiology and structure of the brain. The blood-brain barrier consists of specialized capillaries lined by a single layer of endothelial cells. The intercellular spaces are sealed by tight junctions, so the only access from the blood to the brain is through the endothelial cells. The barrier allows only certain substances, such as lipophilic molecules, to pass through, while other harmful compounds and pathogens remain. Thus, lipophilic carriers are useful for delivering non-lipophilic compounds to the brain. For example, DHA, a fatty acid naturally occurring in the human brain, has been found to be useful for delivering covalently attached drugs to the brain (U.S. Patent No. 6,407,137). U.S. Patent No. 5,525,727 describes a redox system of a dihydropyridine pyridinium salt carrier for specific and sustained delivery of drug species to the brain. U.S. Patent No. 5,618,803 describes enhanced drug delivery via phosphonate derivatives. U.S. Patent No. 7,119,074 describes amphiphilic prodrugs of therapeutic compounds conjugated to PEG-oligomers / polymers for delivering the compounds across the blood-brain barrier. The compounds described herein may be modified by covalent attachment to or formulated with lipophilic carriers. Others will be known to those skilled in the art.

[0112] The therapeutic agents of the present invention may be delivered with other therapeutic agents to enhance memory recovery or treat other symptoms or causes of disorders associated with memory loss. Illustratively, environmental enrichment (EE) has been used to enhance memory. EE involves creating a stimulating environment around a subject. Other therapeutic agents may also be combined to treat the underlying disorder or enhance memory recall.

[0113] Examples of combinations of the compounds of the present invention with other drugs in unit dose or kit form include the following: anti-Alzheimer's drugs, beta-secretase inhibitors, gamma-secretase inhibitors, HMG-CoA reductase inhibitors, NSAIDs including ibroprofen, N-methyl-D-aspartate (NMDA) receptor antagonists such as memantine, cholinesterase inhibitors such as galantamine, rivastigmine, donepezil, and tacrine, vitamin E, CB-1 receptor antagonists or inverse agonists, antibiotics such as doxycycline and rifampin, anti-amyloid antibodies, or drugs that affect receptors or enzymes to either increase the efficacy, safety, or convenience of the compounds of the present invention, or to reduce unwanted side effects or toxicity. The above list of combinations is illustrative only and is not intended to be limiting in any way.

[0114] The present invention encompasses articles of manufacture that refer to any one or collection of components. In some embodiments, the articles are kits. The articles include pharmaceutical or diagnostic grade compounds of the present invention in one or more containers. The articles may include instructions or labels that promote or describe the use of the compounds of the present invention.

[0115] As used herein, "promoted" includes all methods of doing business, including methods of education, hospital and other clinical instruction, pharmaceutical industry activities, including the sale of pharmaceutical products, and pharmaceutical industry activities, including any advertising or other promotional activities, including written, oral, and electronic communications, related to the compositions of the present invention associated with the treatment of cognitive disorders such as Alzheimer's disease.

[0116] "Instructions" may define a promotional component and typically involve written instructions associated with the compositions of the invention or packaging of the compositions of the invention. Instructions may also encompass any oral or electronic instructions provided in any manner.

[0117] Thus, in some embodiments, the agents described herein may be assembled into pharmaceutical, diagnostic, or research kits that facilitate their use in therapeutic, diagnostic, or research applications. The kits may include one or more containers housing the components of the invention and instructions for use. Specifically, such kits may include one or more agents described herein along with instructions describing the intended therapeutic application and appropriate administration of the agents. In some embodiments, the agents in the kits may be in pharmaceutical formulations and in pharmaceutical formulations and dosages suitable for the particular application and method of administration of the agents.

[0118] The kits may be designed to facilitate the use of the methods described herein by a physician and may take many forms. Each of the components of the kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), as applicable. In some cases, some of the compositions may be configurable or otherwise processable (e.g., into an active form), for example, by the addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. As used herein, "instructions" may define instructional and / or promotional components and typically involve written instructions or associated packaging of the present invention. Instructions may also include any oral or electronic instructions provided in any manner, for example, audiovisually (e.g., videotape, DVD, etc.), so that the user will clearly recognize that the instructions are associated with the kit. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, and the instructions may also reflect approval by the agency of the manufacture, use, or sale for human administration.

[0119] The kit may contain any one or more of the components described herein in one or more containers. For example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or for separating and mixing a sample and applying to a subject. The kit may include a container containing an agent described herein. The agent may be sterilely prepared, packaged in a syringe, and shipped refrigerated. Alternatively, it may be stored in a vial or other container for storage. A second container may contain another agent that is sterilely prepared. Alternatively, the kit may include a premixed active agent and be shipped in a syringe, vial, tube, or other container.

[0120] The kit may take various forms, such as a blister pouch, shrink-wrap pouch, vacuum-sealable pouch, sealable thermoformed tray or similar pouch, or a tray format in which the accessories are loosely packaged within one or more tubes, containers, boxes, or bags. The kit may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to remain unpackaged. The kit may be sterilized using any suitable sterilization technique known in the art, such as radiation sterilization, heat sterilization, or other sterilization methods. The kit may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, cloths such as gauze for applying or removing disinfectants, disposable gloves, aids for administering the agent before administration, etc., depending on the specific application.

[0121] The compositions of the kit may be provided in any suitable form, for example, as a liquid solution or as a dry powder. If the composition provided is a dry powder, the powder may be reconstituted by adding a suitable solvent, and may also be provided. If a liquid form of the composition is used, the liquid form may be concentrated or ready-to-use. The solvent will depend on the compound and the mode of use or administration. Suitable solvents for drug compositions are well known and available in the literature. The solvent will depend on the compound and the mode of use or administration.

[0122] In one series of embodiments, the kit comprises one or more container means, such as carrier means, vials, tubes, etc., compartmentalized to receive in adjacent confinement, each of the container means containing one of the separate elements to be used in the method. For example, one of the containers may contain a positive control for the assay. In addition, the kit may include containers for other components, such as buffers useful in the assay.

[0123] The present invention also encompasses completed, packaged, and labeled pharmaceutical products. This article of manufacture includes a suitable unit dose form in a suitable vessel or container, such as a hermetically sealed glass vial or other container. In the case of dosage forms suitable for parenteral administration, the active ingredient is suitable for administration as a sterile and particle-free solution. In other words, the present invention encompasses both parenteral solutions and lyophilized powders, each of which is sterile, and the latter is suitable for reconstitution prior to injection. Alternatively, the unit dose form may be a solid suitable for oral, transdermal, topical, or mucosal delivery.

[0124] In another embodiment, the compositions of the present invention are stored in a container with biocompatible detergents, including but not limited to lecithin, taurocholate, and cholesterol; or with other proteins, including but not limited to gamma globulin and serum albumin. More preferably, the compositions of the present invention are stored with human serum albumin for human use and with bovine serum albumin for veterinary use.

[0125] As with any pharmaceutical product, the packaging materials and containers are designed to protect the safety of the product during storage and shipping. Additionally, the products of the present invention include instructions for use or other informational material that advises a physician, technician, or patient on how to properly prevent or treat the disease or disorder in question. In other words, the article of manufacture includes instruction means that suggest or suggest a dosing regimen, including, but not limited to, actual doses, monitoring procedures, and other monitoring information.

[0126] More specifically, the present invention provides articles comprising packaging material such as a box, bottle, tube, vial, container, spray, syringe, intravenous (iv) bag, envelope, etc.; and one or more unit dosage forms of a pharmaceutical agent contained within the packaging material. The present invention also provides articles of manufacture comprising packaging material such as a box, bottle, tube, vial, container, spray, syringe, intravenous (iv) bag, envelope, etc.; and one or more unit dosage forms of a pharmaceutical agent contained within the packaging material. The present invention further provides articles of manufacture comprising packaging material such as a box, bottle, tube, vial, container, spray, syringe, intravenous (iv) bag, envelope, etc.; and one or more unit dosage forms of a pharmaceutical agent contained within the packaging material. The present invention further provides articles of manufacture comprising a needle or syringe for injection of a pharmaceutical agent, preferably packaged in sterile form, and / or a packaged alcohol pad.

[0127] In a specific embodiment, the article of manufacture includes packaging material, and a pharmaceutical product and instructions contained within the packaging material, wherein the pharmaceutical product is a CDK5 peptide inhibitor and a pharmaceutically acceptable carrier, and the instructions indicate a dosing regimen for preventing, treating, or managing a subject with a cognitive disorder, such as Alzheimer's disease.

[0128] In some embodiments, the subject is therapeutically monitored. For example, the appropriateness of treatment parameters such as dosage, schedule, etc. can be determined by conventional methods for memory monitoring. In addition, the clinical condition of the patient can be monitored for desired effects, for example, an increase in cognitive function. If an insufficient effect is achieved, the patient can then be boosted by further treatment, and treatment and treatment parameters can be modified, such as by increasing the amount of the composition of the present invention and / or other active agents, or by changing the route of administration.

[0129] The present invention is further illustrated by the following examples, which should not be construed as limiting in any way. The entire contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference. [Example]

[0130] example Example 1: Design of CDK5 inhibitory peptides The region in CDK5 that is essential for its p25 binding was determined. The amino acid sequence in this specific region is unique compared to other CDKs and is conserved across many species, including humans. Therefore, a 12-amino acid-long peptide (CDK5i, SEQ ID NO: 2) was designed from this region as a potential p25 / CDK5 inhibitor (Figures 1A-1B). Computational modeling predicted the expected strong interaction of this CDK5 inhibitory peptide (CDK5i) with both CDK5 and p25. This peptide also demonstrated that it could inhibit the formation of the p25 / CDK5 complex (Figure 2).

[0131] Knowledge of the binding site and activity of SEQ ID NO: 2 has guided the design of a set of peptides useful in accordance with the present invention. These CDK5 peptide inhibitors can also be used to identify other CDK5 inhibitors (fluorescence polarization high-throughput screens) using fluorescently labeled CDK5i, as shown in Figure 8.

[0132] Example 2. In Vitro Screening of CKD5i Peptides The CDK5 activity and the effect of the peptide (SEQ ID NO: 2) were examined. Purified recombinant p25 / CDK5 complexes were incubated with the CDK5i peptide, and then CDK5 kinase activity was measured using radiolabeled ATP and its substrate, HI. The results showed that the CDK5i peptide significantly reduced CDK5 kinase activity compared with that of the scrambled peptide-treated group (Figure 3).

[0133] To determine the effects of CDKi peptides in neurodegenerative brains, brain tissue samples from P301S and control mice were used. P301S mice were used as models of Alzheimer's disease (AD) and full-insular dementia (FTD). As shown in Figure 4, we found that CDK5i peptide physically interacted with CDK5 and p35, the precursor of p25, in the brain. However, it did not bind to CDK1 or CDK2, family members highly homologous to CDK5.

[0134] Basal CDK5 activity is essential during neurodevelopment. It is also required for various neuronal functions. Unlike pathological conditions, CDK5 activity under physiological conditions is largely mediated by p24, not p25. To test the effect of CDKi peptides on basal CDK5 activity, we incubated brain tissue from control mice with CDK5i and found no change in basal CDK5 activity (Figure 5A). However, we did show that CDK5i peptides significantly reduced CDK5 kinase activity in the brains of P301S mice, which typically reflects overactivation of the kinase by upregulated p25 (Figure 5B).

[0135] To validate the effects of CDK5i peptides on AD-related pathologies in a human model system, we used induced pluripotent stem cells (iPSCs) created from fibroblasts of patients with familial AD (fAD). Neural progenitor cells (NPCs) derived from fAD iPSCs exhibit multiple pathological phenotypes, including upregulation of histone deacetylase 2 (HDAC2), which negatively regulates the transcription of genes related to learning and memory (Figure 6A). Lines carrying the PSEN1 M146I mutation exhibited the strongest phenotype, and further characterization revealed increased DNA damage in these cells compared to NPCs derived from healthy iPSCs (Figure 6B).

[0136] We then treated NPCs with either the CDK5i peptide or a scrambled version of the peptide and examined the resulting pathological phenotype. We found that the CDK5i peptide significantly reduced HDAC2 and γH2AX (indicative of DNA damage) levels in PSEN 1 M146I NPCs, whereas the scrambled version had no effect (Figure 7).

[0137] Example 3: CDK5i peptide inhibitors with functional domains An exemplary peptide was designed (FIG. 9A) containing a CDK5 inhibitory domain and a functional domain (SEQ ID NO: 3). The functional domain enhances the delivery of the Cdk5i peptide into cells. In this example, it is a transactivator of transcription (TAT) peptide, a fragment of HIV conjugated at the C-terminus to enable the Cdk5i peptide to translocate across the cell membrane. To visualize the distribution of the peptide, fluorescein isothiocyanate (FITC) was conjugated at the N-terminus via a linker (aminohexanoic acid; Ahx).

[0138] Human iPSC-derived neural progenitor cells were treated with FITC-Ahx-Cdk5i-Tat (1 μM) (SEQ ID NO: 3) for 2 hours and then imaged.The results are shown in Figure 9B.The results show that FITC signal is located in the cell, demonstrating that the peptide can pass through the cell membrane.

[0139] Example 4. Cdk5i crosses the blood-brain barrier in mice Wild-type mice were intraperitoneally injected with a single dose of Cdk5i (40 mg / kg) and sacrificed 24 hours after injection. Before extracting the brain, the mice were transcardially perfused with 40 mL of cold phosphate-buffered saline (PBS) to ensure the elimination of any Cdk5i circulating in the blood that may not have crossed the blood-brain barrier. After perfusion, the brains were dissected into two hemispheres, and one hemisphere was lysed with cold lysis buffer (RIPA) to extract total protein. The total protein was then briefly run on a 12% acrylamide gel to separate large proteins, then digested with trypsin enzyme, and subjected to targeted mass spectrometry using the Cdk5i peptide sequence of SEQ ID NO: 3 linked to a GGG spacer / linker and FITC (Cdk5i sequence: FITC-GGG-SEQ ID NO: 3).

[0140] A substantial portion of the Cdk5i sequence (11 of 12 amino acids), including the spliced ​​tat protein sequence, was detected using mass spectrometry based on spectral number and peak area. The data are shown in Table 1. Using targeted mass spectrometry, brain lysates from mice injected with Cdk5i showed enrichment for Cdk5i based on spectral number and peak area (second row of data in Table 1). [Table 1]

[0141] The above written specification is believed to be sufficient to enable one skilled in the art to practice the invention. The present invention is not limited in scope by the examples provided, since the examples are intended as single illustrations of one aspect of the present invention, and of other functionally equivalent embodiments within the scope of the present invention. Various modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the above description and will fall within the scope of the appended claims. The advantages of the present invention are not necessarily exhaustive by each aspect of the present invention.

Claims

1. The following structure: ARAFGX １ PVRC X ２ S ＊ (X １ = I or V and X ２ 1. A pharmaceutical composition comprising a peptide of 12 to 50 amino acids in length comprising an amino acid sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 having the amino acid sequence (SEQ ID NO: 1) of SEQ ID NO: 1, wherein SEQ ID NO: 1 is a nucleotide sequence of SEQ ID NO: 1, and SEQ ID NO: 2 ... wherein the peptide disrupts the CDK5-p25 / p35 interaction, Optionally, wherein the peptide comprises at least one conservative substitution within the sequence of SEQ ID NO:

1. Optionally, wherein the peptide has an enhanced or stabilized secondary structure, and Optionally, wherein the peptide is linked to the non-peptide molecule via a linker. The pharmaceutical composition.

2. 2. The pharmaceutical composition of claim 1, wherein the peptide comprises the amino acid sequence of SEQ ID NO:

2.

3. the pharmaceutically acceptable carrier comprises a polymer; Optionally, wherein the polymer is (a) comprising a hydrophilic block and an endosomolytic block, wherein the hydrophilic block optionally comprises polyethylene glycol methacrylate, and wherein the endosomolytic block optionally comprises diethylaminoethyl methacrylate-butyl methacrylate copolymer; or (b) a stimuli-responsive polymer that responds to one or more stimuli selected from the group consisting of pH, temperature, UV-visible light, light irradiation, exposure to an electric field, ionic strength, and the concentration of a chemical by exhibiting a change in properties; The pharmaceutical composition of claim 1.

4. The peptide comprises at least: (a) a deletion of 1 to 2 amino acids of SEQ ID NO: 1; (b) one additional amino acid at the N-terminus of SEQ ID NO: 1; and / or (c) one additional amino acid at the C-terminus of SEQ ID NO: 1 2. The pharmaceutical composition of claim 1, comprising:

5. The peptide is linked to a functional domain, Optionally, wherein the functional domain is a transactivation domain, a targeting domain, or a stabilization domain. The pharmaceutical composition according to any one of claims 1 to 4.

6. 6. The pharmaceutical composition of claim 5, wherein the transactivation domain is a peptide of the transactivator of transcription (TAT) or the peptide sequence YGRKKRRQRRR (SEQ ID NO: 4).

7. The following structure: ARAFGX １ PVRC X ２ S ＊ (X １ = I or V and X ２ = Y or F), wherein the peptide is 11 to 25 amino acids in length and comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the peptide disrupts the CDK5-p25 / p35 interaction, Optionally, wherein the peptide has an enhanced or stabilized secondary structure, and Optionally, wherein the peptide is linked to the non-peptide molecule via a linker. The peptide.

8. Contains at least one conservative substitution, Optionally, wherein at least one conservative substitution is within the sequence of SEQ ID NO:

1. The peptide of claim 7.

9. below: (a) a deletion of 1 to 2 amino acids of SEQ ID NO: 1; (b) at least one additional amino acid at the N-terminus of SEQ ID NO: 1; and / or (c) at least one additional amino acid at the C-terminus of SEQ ID NO: 1 8. The peptide of claim 7, having the formula:

10. Encoding the peptide of the pharmaceutical composition according to any one of claims 1 to 6 or the peptide according to any one of claims 7 to 9, Optionally, wherein the promoter is operably linked to Recombinant nucleic acids.

11. The composition of claim 1 for use in a method for treating a neurodegenerative disease.

12. The peptide of claim 7, comprising at least one amino acid analogue.

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