Peptide drug conjugates for the treatment of neurodegenerative diseases

Abstract

The present invention relates to a conjugate molecule for use in the treatment and / or prevention of a neurodegenerative disease in a subject in need thereof, comprising a peptide and an N-methyl-D-aspartate receptor (NMDAR) antagonist, said peptide being a glucagon superfamily peptide, said peptide being linked to said NMDAR antagonist directly or via a chemical linker.

Description

[Technical field]

[0001] The present invention relates to the field of therapeutic compounds exhibiting agonistic activity on receptors for glucagon superfamily peptides and antagonistic activity on N-methyl-D-aspartate receptors for use in the treatment and / or prevention of neurodegenerative diseases in a subject. [Background technology]

[0002] Neurodegeneration, also known as neurodegenerative diseases, is characterized by complex and serious pathologies that primarily affect neurons in the human brain. Neurodegeneration occurs at various levels of the brain's neuronal circuits, from the molecular to the systemic level. Neurodegenerative diseases are considered incurable, as there is no known way to reverse the progressive degeneration of neurons. Neurodegenerative diseases, such as dementia, Alzheimer's disease, Parkinson's disease, and Huntington's disease, all occur as a result of the neurodegenerative process.

[0003] Alzheimer's disease is the most common neurodegenerative disorder, characterized by the loss of neurons and synapses in the cerebral cortex and certain subcortical regions. Most drug development programs for Alzheimer's disease have aimed to alleviate symptoms, with disappointing results of poor efficacy with little or no impact on disease reversal. Currently, two main drug classes are approved and used to treat Alzheimer's disease: acetylcholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists.

[0004] NMDA receptors (NMDARs) are a subclass of glutamate receptors whose activation requires both glutamate binding and postsynaptic depolarization, and upon activation, they induce Ca 2+The involvement of NMDARs in various neurological disorders has been of interest for many years, as NMDAR dysfunction, resulting for example from alterations in receptor channel activity, subunit expression, trafficking or localization, can contribute to a variety of neurological and psychiatric conditions.

[0005] For example, NMDAR antagonists such as memantine under the trade name Ebixa® are currently used to treat Alzheimer's disease, are available as generic drugs, and are used to treat millions of people worldwide. Memantine, a noncompetitive NMDAR antagonist, belongs to the group of open channel blockers of the NMDA receptor.

[0006] US2004102525A1 describes methods for the acute, chronic and prophylactic treatment of neurological and neurodegenerative diseases, including Alzheimer's disease, with memantine.

[0007] The glucagon superfamily consists of peptides such as glucagon, secretin, vasoactive inhibitory peptide (VIP), gastric inhibitory peptide (GIP), growth hormone releasing factor (GHRF), and glucagon-like peptide 1 (GLP-1) and GLP-2. These peptides exert diverse actions on nutrient intake, gastrointestinal motility, pancreatic islet hormone secretion, cell proliferation and apoptosis, nutrient absorption, and nutrient assimilation. Recent studies have further indicated that these peptides may also play a neuroprotective role in the brain, thus drawing increasing attention to their relevance in the treatment of neurodegenerative diseases.

[0008] GIP is derived from a 153 amino acid long proprotein encoded by the GIP gene and circulates as a biologically active 42 amino acid long peptide. The GIP receptor (GIPR) is expressed in various tissues and has been found in several regions of the brain with high expression levels in the olfactory bulb, hippocampus and large pyramidal neurons of the cerebral cortex. Activation of the GIPR leads to proliferation of neural progenitor cells and may therefore contribute to neurogenesis.

[0009] GLP-1 is a 30- or 31-amino acid-long peptide hormone derived from tissue-specific post-translational processing of the proglucagon peptide. GLP-1 receptor agonists are widely known for their antidiabetic and antiobesity effects. Recently, it has been demonstrated that long-acting GLP-1 receptor (GLP-1R) agonists have neuroprotective properties, and several companies are currently investigating the utility of GLP-1R agonists in the treatment of Alzheimer's disease in clinical Phase III trials.

[0010] WO2021 / 089678A1 describes the use of GLP-1R agonists, in particular semaglutide, for treating dementia or reducing the risk of developing dementia in subjects with metabolic syndrome.

[0011] WO2011143208A1 discloses a glucagon superfamily peptide agonist complexed with a G protein-coupled receptor (GPCR) ligand for use in the treatment of metabolic syndrome, diabetes, obesity, fatty liver and neurodegenerative diseases.The GPCR ligand may be the eicosanoid anandamide.Hampson AJ et al., Journal of neurochemistry, 70, 2: 671-676 (1998) describes the role of the eicosanoid anandamide in regulating NMDA receptor activity.

[0012] WO2011094337A1 discloses a complex of a peptide combination including a GIP agonist peptide and a glucagon antagonist peptide for treating metabolic disorders such as diabetes and obesity. The peptides described in WO2011094337A1 can also be administered in combination with a therapeutic agent for neurodegenerative diseases.

[0013] US2012329707A1 discloses glucagon / GLP-1 receptor co-agonist complexes and their use in the treatment of metabolic syndrome, diabetes, obesity, hepatic steatosis and neurodegenerative diseases.

[0014] Zheng Liu et al., Med. Chem. Commun. 8, 1:135-147 (2016) discloses the biological evaluation of newly synthesized memantine nitrate as a potential therapeutic agent for neurodegenerative diseases, including Alzheimer's disease.

[0015] WO2021245199A1 discloses complexes of GLP1 receptor agonists and NMDA receptor antagonists, and the use of these complexes in the treatment of obesity and obesity-related diseases.

[0016] As the population ages, the incidence of neurodegeneration is increasing dramatically without effective therapeutic interventions or a clear understanding of the pathophysiology of distinct neurodegenerative disease states.

[0017] Thus, there remains an unmet need for improved pharmacological treatments for neurodegenerative diseases that are more effective and safer (with less toxic effects). Summary of the Invention

[0018] Against this background, it is an object of the present invention to provide improved therapies for use in the treatment and / or prevention of neurodegenerative diseases, including administering therapeutic agents that have fewer side effects and are better tolerated by the subject.

[0019] Thus, a first aspect of the present invention relates to a combination molecule for use in the treatment and / or prevention of a neurodegenerative disease in a subject in need thereof, comprising a peptide and an N-methyl-D-aspartate receptor (NMDAR) antagonist, said peptide being a glucagon superfamily peptide, said peptide being linked directly or via a chemical linker to said NMDAR antagonist, said NMDAR antagonist being selected from the group of open channel blockers of the NMDA receptor.

[0020] The inventors have surprisingly found that the complex of the present invention is effective in treating or preventing neurodegenerative diseases in subjects, particularly in treating and preventing Alzheimer's disease. It is hypothesized that this novel medical strategy utilizes the advantages of both molecular entities, glucagon superfamily peptide receptor agonism and NMDA receptor antagonism, in a single molecule. The inventors have demonstrated that the combination of the two neuroprotective modalities in the complex molecule results in improved neuroprotective effects in in vitro and in vivo models of NMDA-induced cytotoxicity, and in the most commonly used transgenic AD mouse model (5×FAD), respectively. To the best of the inventors' knowledge, no existing therapeutic strategy for neurodegenerative diseases, either preclinical or clinical, combines NMDA receptor antagonism with glucagon superfamily peptide receptor agonism.

[0021] Without being bound to any particular theory, the inventors speculate that the neuroprotective effect of the conjugate molecule of the present invention is achieved by accumulation of the NMDAR antagonist at and / or near glucagon superfamily peptide receptor sites in the body due to the affinity of the peptide for the glucagon superfamily peptide receptor.

[0022] Peptides have an amino terminus and a carboxyl terminus. In the context of the present invention, the amino terminus and the carboxyl terminus of a peptide are also referred to as the N-terminus and C-terminus, respectively, and the corresponding derived forms.

[0023] Peptides of the invention may be composed of amino acids encoded by the genetic code, or may include amino acids encoded by the genetic code and naturally occurring amino acids not encoded by the genetic code, such as hydroxyproline, gamma-carboxyglutamic acid, ornithine, phosphoserine, D-alanine (dAla) and D-glutamine. Additionally, peptides of the invention may incorporate synthetic amino acids such as D-alanine and D-leucine, or alpha-aminoisobutyric acid (Aib), d-serine (dSer), N-methylserine, etc.

[0024] In one embodiment, the amino acid at position 2 (counting from the N-terminus) of the peptide is D-alanine, D-serine, α-aminoisobutyric acid, N-methyl-serine, glycine, or valine.

[0025] The peptides of the present invention may have one or more modifications to stabilize secondary structure, such as a cyclization between glutamic acid at amino acid position 15 and lysine at amino acid position 20 counting from the N-terminus of the peptide.

[0026] The peptides can be obtained from any source and, if desired, produced, for example, they can be isolated from tissues or they can be recombinantly produced or synthesized by methods well known to those of skill in the art.

[0027] The peptides of the complexes of the invention belong to the glucagon superfamily of peptides, which is a group of peptides that are related in structure in their N- and C-terminal regions (see, e.g., Sherwood et al., Endocrine Reviews 21: 619-670 (2000), which is incorporated herein by reference). Members of this group include all glucagon-related peptides, as well as growth hormone releasing hormone, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide 27, secretin, modified GLP-1 (SEQ ID NO: 1), unmodified GLP-1 (SEQ ID NO: 2), exendin-4 (SEQ ID NO: 3), gastric inhibitory peptide (GIP) (SEQ ID NO: 4), GLP-1 / GIP coagonist (SEQ ID NO: 5 and SEQ ID NO: 6), GLP-1 / GIP / glucagon (Gcg) triagonist (SEQ ID NO: 7), and analogs, derivatives or conjugates in which up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids have been modified relative to the native peptide.

[0028] In one embodiment, the peptide of the present invention is an incretin hormone. The incretin family of peptide hormones includes, but is not limited to, GLP-1, GIP, combinations and / or analogs thereof.

[0029] The peptides of the present invention retain the ability to interact as agonists with receptors of the glucagon receptor superfamily. As used herein, an "agonist" is a molecule, such as a hormone or drug, that attaches or binds to a cellular receptor and exerts an effect on the cell. When an agonist binds to a particular receptor, a biological response is elicited.

[0030] In one embodiment, the peptide is a GLP-1 peptide, a GIP peptide, a combination thereof, and / or any derivative or analogue thereof that interacts with the GLP-1 and / or GIP receptor as an agonist.

[0031] The peptides of the present invention may have at least 70% amino acid sequence identity with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. Also, the peptides of the composite molecules may have at least 70% amino acid sequence identity with SEQ ID NO:6 or SEQ ID NO:7. In certain embodiments, the peptides of the composite molecules have the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In other specific embodiments, the peptides of the composite molecules have the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.

[0032] Peptides with co-agonist and triagonist activity are also contemplated. Peptides with co-agonist or triagonist activity exhibit the ability to bind to two or more different receptors of the glucagon receptor superfamily. In one embodiment, the co-agonist is a GLP-1 / GIP receptor co-agonist. In one embodiment, the GLP-1 / GIP receptor co-agonist is as set forth in SEQ ID NO:5. In one embodiment, the GLP-1 / GIP receptor co-agonist is as set forth in SEQ ID NO:6. Peptides with co-agonist activity for the GLP-1 / Gcg receptor or the GIP / Gcg receptor are also contemplated. In another embodiment, the triagonist is a GLP-1 / GIP / Gcg receptor triagonist. In one embodiment, the triagonist is as set forth in SEQ ID NO:7.

[0033] In one embodiment, the peptide of the conjugate molecule of the invention has at least 70% amino acid sequence identity with SEQ ID NO: 1. For example, the peptide may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, or more than about 90% identity with SEQ ID NO: 1. In one embodiment, the peptide has at least 80% amino acid sequence identity with SEQ ID NO: 1. In a further embodiment, the peptide has at least 95% amino acid sequence identity with SEQ ID NO: 1.

[0034] In one embodiment, the peptide is as set forth in the conservative amino acid sequence of SEQ ID NO: 8. In the context of the present invention, the peptide of SEQ ID NO: 8 comprises, in addition to the conservative amino acids, natural or synthetic amino acids at amino acid positions 1, 10, 12, 15, 16, 23, 24, 27, 28, 30, 31, counting from the N-terminus of the peptide of SEQ ID NO: 8. Such peptides are exemplified by the GLP-1 / GIP co-agonist peptide of the amino acid sequence set forth in SEQ ID NO: 5, see FIG. 6. In a further embodiment, the peptide is as set forth in SEQ ID NO: 8 and has at least 70% amino acid sequence identity with SEQ ID NO: 1.

[0035] In one embodiment, the peptide of the present invention is gastric inhibitory peptide (GIP) as set forth in SEQ ID NO: 4, which has GIP activity at the GIP receptor.

[0036] In one embodiment, the peptide of the invention has the amino acid sequence shown in SEQ ID NO: 1. Such peptides may have significantly increased GLP-1 activity at the GLP-1 receptor as compared to native GLP-1 (unmodified GLP-1, SEQ ID NO: 2) at the GLP-1 receptor. Also contemplated are GLP-1 receptor peptides having at least 70% amino acid sequence identity with SEQ ID NO: 1, but which do not have an exendin-4 extension at the C-terminus of the peptide. In the context of the present invention, the exendin-4 extension has the amino acid sequence GPSSGAPPPS (SEQ ID NO: 9).

[0037] The peptide of the present invention, when complexed with NMDAR antagonist, can accumulate at a high rate at glucagon superfamily peptide receptor site, resulting in improved efficacy of NMDAR antagonist.The peptide of the present invention has at least 80% amino acid sequence identity with SEQ ID NO:1 as an example shown in FIG.6.The peptide of the complex has at least 70% amino acid sequence identity with SEQ ID NO:1 as an example shown in FIG.7.The alignment of GLP-1 / GIP (peptide of SEQ ID NO:5) and GLP-1 (peptide of SEQ ID NO:1) shown in FIG.6 is further shown below. [ka]

[0038] The peptides of the composite molecule are of sufficient length that the peptide (in free form) exhibits at least 0.1% of the activity of the native glucagon superfamily peptide at the glucagon superfamily peptide receptor. Generally, this is observed for peptides containing at least 10 amino acids, and activity may not be exhibited if the peptide contains more than 60 amino acids. Thus, in one embodiment, the peptide has a length ranging from 10 to 60 amino acids, for example 20 to 50 amino acids. The amino acid sequence of the present invention should be a certain percentage identical to other peptide sequences and sufficiently contain the amino acid sequence of the peptide, for example at least 10 amino acids, to allow putative identification of the peptide by manual evaluation of the sequence by a person skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool) (for reviews, see Altschul, et al., Meth Enzymol. 266: 460,1996; and Altschul, et al., Nature Genet. 6: 119, 1994).

[0039] In the context of the present invention, peptides may differ in % identity by having substitutions, insertions of natural or synthetic amino acids, and / or deletions of amino acids.

[0040] In one embodiment, the peptide is modified by acetylation, fatty acid conjugation, diacid conjugation, albumin conjugation, small molecule albumin conjugating agents and / or PEG conjugation. Also contemplated are peptides modified by conjugation to carrier proteins such as antibodies. The modifications are preferably made at positions 16, 17, 20, 21, 24, 29, 40 (counting from the N-terminus), in the C-terminal region or at the C-terminal amino acid of the peptide. The conjugation can be made by any suitable linker, such as disulfide, maleimide, alpha ketone or click chemistry based conjugation. The skilled artisan knows how to prepare such conjugates. Preferably, the PEG molecule may be larger than 1 kDa and the fatty acid and diacid may contain more than 12 carbon atoms. In general, it is preferred to add a spacer between the modification (PEG / fatty acid / diacid) and the peptide, the linker being a gamma-Glu linker, preferably a short PEG chain.

[0041] An NMDA receptor antagonist binds to an NMDA receptor, and an NMDAR antagonist, for example, in its free form, has a dissociation constant K d NMDAR antagonists generally have dissociation constants in the nanomolar range; for example, the dissociation constant of MK801 (dizocilpine) with NMDA receptors of different species is K d = 6.3 nM, and K in mouse brain homogenate d = 10 nM, K in pig brain d The dissociation constant is 1.3 nM. The determination of the dissociation constant is well known to those skilled in the art. In one embodiment, the NMDAR antagonist in free form has a dissociation constant K d The K of the NMDA receptor is in the range of 0.5 to 1000 nM, for example, in the range of 0.5 to 100 nM. The NMDA receptor is, for example, a human NMDA receptor, and the NMDAR antagonist, for example, has a K dIn the context of the present invention, an NMDA receptor antagonist in free form refers to an antagonist that is not bound to any chemical group, in particular is not chemically linked, and is therefore in its natural, unmodified form. A person skilled in the art will appreciate that inter-species differences between NMDA receptors are expected to be small. Thus, the K measured in rodents, such as mice and rats, or in higher mammals, such as pigs, is d Values ​​are the K values ​​measured at human NDMA receptors or at NMDA receptors in other relevant animals and mammals. d is expected to be similar to the value.

[0042] Any NMDAR antagonist can be used. As used herein, an "antagonist" is a type of receptor ligand or drug that binds to and blocks the receptor, thereby blocking or attenuating a biological response, rather than activating the receptor as an agonist would. Channel blockers are antagonists of the respective ion channels. The NMDAR antagonists in the present invention are selected from the group of open channel blockers of the NMDA receptor. Open channel blockers of the NMDA receptor can function by non-competitively binding to a site in the channel pore near the extracellular entrance. Their binding requires the channel to be open, and therefore their presence physically blocks the flow of ions through the channel.

[0043] Examples of open channel blockers of the NMDA receptor are disclosed, for example, in Traynelis et al., Pharmacological reviews, 62, 3: 405-496 (2010), and Ogden and Traynelis, Trends in pharmacological sciences, 32, 12: 726-733 (2011), and include, but are not limited to, dizocilpine, memantine, ketamine, norketamine, hydroxynorketamine, amantadine, dextromethorphan, levomethorphan, dextrorphan, levorphanol, phencyclidine (PCP), 1-phenylcyclohexylamine (PCA), CNS-1102 (aptiganel), remacimide, pentamidine, 9-aminoacridine, 1,2,3,4-tetrahydro-9-aminoacridine (tacrine), 7-methoxytacrine, lanicemine, and the like.

[0044] In the context of the present invention, NMDAR antagonist is a small molecule, for example up to 900 kDa.In one embodiment, NMDAR antagonist is selected from dizocilpine, also known as MK801, memantine, ketamine, phencyclidine, argionotoxin, such as argionotoxin-636, dextrorphan, aptiganel, alkaline, polyamine, such as diethylenetriamine and putrescine, neramexane and amantadine.In one embodiment, NMDAR antagonist is selected from memantine, neramexane and dizocilpine.Neramexane is a non-limiting example of a compound related to memantine.

[0045] The peptide and the NMDAR antagonist in the present invention are linked. In the context of the present invention, the conjugated molecule is also called peptide-drug conjugate (PDC). The peptide and the NMDAR antagonist can be linked or directly linked to each other. For example, the NMDAR antagonist can be linked via an amide bond, which is from the amino group of the NMDAR antagonist to the carboxylic acid group of the peptide. Such an amide bond can be formed at any residue on the peptide that has a carboxylic acid group, such as a glutamic acid residue, an aspartic acid residue, a synthetic residue with a carboxylic acid group, or a carboxylic acid at the C-terminus. In one embodiment, the NMDAR is memantine. For example, when the NMDAR antagonist is memantine, the amine of memantine can be linked to the carboxylic acid of the amino acid residue of the peptide.

[0046] In one embodiment, the conjugate molecule is for use in treating and / or preventing a neurodegenerative disease in a subject in need thereof, the conjugate molecule comprises a peptide having the amino acid sequence shown in SEQ ID NO: 1, the amino acid at amino acid position 2 (counting from the N-terminus) of the peptide is α-aminoisobutyric acid, the NMDAR antagonist is memantine, and the peptide is linked to the NMDAR antagonist directly or via a chemical linker. In another embodiment, the peptide is shown in SEQ ID NO: 1, the NMDAR is memantine, and the chemical linker is selected from linkers containing disulfide groups.

[0047] In the context of the present invention, directly linked means that the peptide is covalently bonded to the NMDAR antagonist, and there is no additional chemical group, such as a linker group, between the two molecules. The peptide and the NMDAR antagonist may be linked via a chemical linker. Any chemical linker can be used. However, the length of the chemical linker is usually preferably up to 30 atoms. A long chain has the advantage of keeping the NMDAR antagonist away from the peptide, so that the NMDAR antagonist exerts no or little steric hindrance on the peptide when the peptide and the glucagon superfamily peptide receptor interact. No or low steric hindrance of the peptide leads to a high affinity for the glucagon superfamily peptide receptor. A complex with high affinity for the glucagon superfamily peptide receptor may accumulate more at the site of the glucagon superfamily peptide receptor. The chemical linker is preferably a cleavable linker, such as an acid cleavable linker, an enzyme cleavable linker, a peptide cleavable linker, or a disulfide linker, which are generally well known in the art for use in peptide-drug conjugates.Examples of such cleavable linkers include compounds that contain glucuronides, β-galactosides, disulfides, hydrazones, and / or compounds that can be cleaved by galactosidases, glucuronidases, pyrophosphatases, phosphatases, arylsulfatases, proteases, or esterases.For example, the linker can include peptides that can be cleaved by cathepsins, such as GFLG.The linker can further include 4-aminobenzoic acid (PAB), which can be linked to the amino group of NMDAR antagonists via amide or carbamate bonds. The linker preferably releases the NMDAR antagonist in free form (i.e., in its native form), which can be achieved by many different linker chemistries, such as the disulfide linkers disclosed herein. These and additional linker chemistries are well known to those skilled in the art.

[0048] The NMDAR antagonist may be bound to the C-terminal region of the peptide. In the context of the present invention, the C-terminal region may be up to 50% of the amino acids counting from the C-terminus, for example up to 40%, 30%, 25%, 20% or 10% of the amino acids counting from the C-terminus. For example, the C-terminal region of SEQ ID NO: 1 may be amino acids 21-40, 26-40 or 31-40 (counting from the N-terminus). Thus, the NMDAR antagonist, such as memantine or dizocilpine, may be bound directly or via a linker to any one of the 10 amino acids counting from the C-terminus. For example, the NMDAR antagonist, such as memantine, may be bound directly to an amino acid within 5 amino acids from the C-terminus. This allows the NMDAR antagonist to cause little or no steric hindrance at the N-terminus of the peptide. Since the N-terminus is involved in binding to the glucagon superfamily peptide receptor, no or low steric hindrance at the N-terminus may increase affinity for the glucagon superfamily peptide receptor. It is also contemplated that multiple NMDAR antagonists may be attached to the same peptide molecule.

[0049] In one embodiment, the NMDAR antagonist is linked to the peptide via a chemical linker that contains a disulfide group. The disulfide group allows the NMDAR antagonist to be released from the peptide when chemically reduced. The chemical linker that contains a disulfide group, also known as a disulfide linker, ensures that the peptide and the NMDAR antagonist of the complex remain complexed for a long period of time in the systemic circulation. The disulfide group of the disulfide linker is reduced in a reducing environment, such as an intracellular environment, so that the complex is cleaved and the peptide part of the complex is separated from the NMDAR antagonist part of the complex. Reduction can be performed by disulfide exchange with, for example, a thiol, such as glutathione, or a reducing enzyme, such as an intracellular protein disulfide isomerase enzyme.

[0050] The chemical linker can be selected from chemical linkers having the general formula R'-SS-R'' known in the art, where the R' and R'' groups can be the same or different from each other. Advantageously, due to the affinity of the peptide to the glucagon superfamily peptide receptor, the complex can accumulate at and near the site of the glucagon superfamily peptide receptor in the body, and the NMDAR antagonist can be released at and near the site of the glucagon superfamily peptide receptor. In the absence of the peptide portion of the complex, the NMDAR antagonist can have the appropriate effect as a site-specific NMDAR binding. The inventors speculate that the complex may be cleaved in the extracellular environment adjacent to cells with glucagon superfamily peptide receptors, or the complex may be internalized by cells with glucagon superfamily peptide receptors and cleaved in the reducing environment of the cells.

[0051] In one embodiment, the conjugate molecules are linked via a chemical linker, the chemical linker having the formula R1-R3-SS-R4-R5-O-CO-R2, where R1 is a peptide, R2 is an NMDAR antagonist, R3 is optional and, if present, is selected from C(CH3)2, CH2-CH2 or CH2 and is attached to a carbon atom in the side chain of the peptide or in the backbone of the peptide, and R4 is (CH2) n or C6H4, R5 is optional and, when present, is selected from C(CH3)2, CH2-CH2 or CH2, and n is 1, 2 or 3. When the chemical linker is reduced, the free NMDAR antagonist portion of the conjugate undergoes intramolecular cyclization, thereby releasing the NMDAR antagonist in its free form, see FIG. 2.

[0052] In one embodiment, the chemical linker has the formula R1-R3-SS-(CH2) n It has -O-CO-R2, where R1 is a peptide, R2 is an NMDAR antagonist, R3 is optional and, if present, is selected from C(CH3)2, CH2-CH2 or CH2 and is attached to a carbon atom in the peptide side chain or the peptide backbone, and n is 1, 2 or 3.

[0053] In one embodiment, the chemical linker has the formula R1-R4-R3-SS-(CH2) n has -O-CO-R2, where R1 is a peptide, R2 is an NMDAR antagonist, R3 is optional and, if present, is selected from CH(CH3)2, CH2-CH2 or CH2 and is attached to a carbon atom in the peptide side chain or the peptide backbone, R4 is optional and, if present, is selected from CH(CH3)2, CH2-CH2 or CH2 and is attached to a carbon atom in the peptide side chain or the peptide backbone, and n is 1, 2 or 3.

[0054] In one embodiment, the second radical bond is to the backbone of the peptide of the invention. In another embodiment, the second radical bond is to the side chain of the peptide of the invention.

[0055] In the context of the present invention, when R1 is attached to the peptide backbone, C(CH3)2 (L-penicillamine) may be referred to as Pen, CH2-CH2 (L-homocysteine) may be referred to as hCys, and CH2 (L-cysteine) may be referred to as Cys, see Figure 1.

[0056] As used herein, first and second radical bonds are used to indicate that there are at least two free bonds in the chemical linkers disclosed herein.

[0057] The present invention facilitates the design and synthesis of a library of conjugate molecules that include peptides and NMDAR antagonists attached via chemical linkers. Figure 1 shows the method of designing such conjugate molecules. As shown in Figure 1, conjugates can be prepared by chemically linking NMDAR antagonists (MK801 in Figure 1) and peptides. Those skilled in the art will understand that a vast number of different chemical linkers can be prepared by the methods disclosed herein and other methods reported in the literature, and these chemical linkers can be used to attach peptides and NMDAR antagonists according to the methods disclosed herein and other methods reported in the known art.

[0058] The inventors have found that the peptides of the invention can function as targeting agents to site-selectively deliver non-specific small molecules, such as NMDAR antagonists, to regions of the brain.

[0059] The conjugate molecule disclosed herein provides selectivity and concentrates drug action in target area.This targeting enabled by the conjugate molecule can improve therapeutic index, i.e., reduce the minimum effective concentration.The tissue-selective targeting of NMDAR can be used for targeted treatment of neurological diseases.

[0060] The inventors have demonstrated that the complex of the present invention exerts a surprising synergistic effect against neurodegeneration, which is significantly greater than the effect obtained by administration of the peptide alone, see Figures 3 and 4. Furthermore, long-term administration of the complex molecule of the present invention does not seem to affect the body weight of the mice used in the study, see Figure 5.

[0061] Although the data disclosed in this invention were obtained from mouse studies, the conclusions apply equally to humans, since the major hormonal pathways controlling energy metabolism are similar between mice and humans and show comparable receptor expression profiles.

[0062] In one embodiment, the neurodegenerative disease treated with the conjugate molecule of the present invention is selected from dementia, mild cognitive impairment, Alzheimer's disease, Parkinson's disease, stroke, traumatic brain injury, Huntington's disease, schizophrenia and depression. In one embodiment, the conjugate molecule of the present invention is used to treat and / or prevent Alzheimer's disease. There are five stages of Alzheimer's disease: preclinical Alzheimer's disease, mild cognitive impairment due to Alzheimer's disease, mild dementia due to Alzheimer's disease, moderate dementia due to Alzheimer's disease, and severe dementia due to Alzheimer's disease. It is envisioned that the conjugate molecule of the present invention can be used to treat any stage of Alzheimer's disease. Similarly, the conjugate molecule of the present invention can be used to treat other neurodegenerative diseases, regardless of the stage or progression of the disease.

[0063] In one embodiment, the conjugated molecule is administered in the form of a pharmaceutical composition, which comprises the conjugated molecule or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier. Any embodiment of the conjugated molecule may be used in the pharmaceutical composition.

[0064] Pharmaceutical preparations can be prepared by conventional techniques.Simply put, pharmaceutically acceptable carriers are either solid or liquid.Solid form preparations include powder, tablet, pill, capsule, cachet, suppository and dispersible granule.Solid carriers can be one or more excipients that also function as diluents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents or encapsulating materials.

[0065] The complexes contained in the pharmaceutical preparations may be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization from a solution which is constituted with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0066] In one embodiment, pharmaceutical composition is suitable for subcutaneous administration, intramuscular administration, intraperitoneal administration, intravenous administration or oral administration.Therefore, the composition of the present invention can be provided in unit dosage form in ampoules, pre-filled syringes, small volume injections or multi-dose containers, optionally with the addition of preservatives.Composition can be in the form of suspension, solution or emulsion in oily or aqueous medium, etc.

[0067] In one embodiment, the conjugated molecule or pharmaceutical composition thereof for use in the treatment and / or prevention of a neurodegenerative disease is administered at least once a week for 12 months or more. In another embodiment, the conjugated molecule or pharmaceutical composition thereof is administered 2-3 times a week for 12 months or more. In another embodiment, the conjugated molecule or pharmaceutical composition thereof is administered to a subject once a day for 12 months or more.

[0068] The conjugate molecule of the present invention can be administered as a chronic treatment, administering the conjugate molecule or a pharmaceutical composition comprising the conjugate molecule for at least 12 months, for example at least 16 months or at least 18 months. It is understood that chronic treatment with the conjugate molecule of the present invention or a pharmaceutical composition thereof may involve treatment over several years. In one embodiment, the conjugate molecule of the present invention or a pharmaceutical composition thereof is administered once a day or once a week for one, two, five, ten or more years. In one embodiment, the therapeutic conjugate molecule of the present invention or a pharmaceutical composition thereof is administered 2-3 times a week for five years or more.

[0069] The present invention has been described above mainly with reference to some embodiments. However, as can be easily understood by those skilled in the art, other embodiments than those disclosed above are also possible within the scope of the present invention.

[0070] Other aspects and advantageous features of the present invention are described in detail and illustrated by the following non-limiting examples.

[0071] In general, all terms used herein are to be interpreted according to their ordinary meaning in the technical field of the present invention unless expressly defined or otherwise stated, and apply to all aspects and embodiments of the present invention. All references to "a / an / the [conjugate, molecule, linker, peptide, etc.]" are to be clearly interpreted as referring to at least one example of the conjugate, agent, molecule, linker, peptide, etc. described above, unless expressly stated otherwise.

[0072] In the context of the present invention, the term "peptide" refers to a compound consisting of a sequence of 10-60 amino acids linked by peptide bonds and belonging to the glucagon superfamily of peptides. Such peptides include, but are not limited to, the incretin hormones glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP) and glucagon (GCG). The peptides of the present invention are agonists of the glucagon superfamily of peptide receptors and are believed to be neuroprotective peptides, and may function as agents to actively deliver the conjugate molecules of the present invention to sites in the brain, including the hypothalamus.

[0073] The term "derivative" or "analog" as used herein in connection with a peptide or amino acid means a chemically modified peptide or amino acid having at least one substituent not present in the unmodified peptide or amino acid or its analog, i.e., a covalently modified peptide or amino acid. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, and the like.

[0074] In the context of the present invention, the term "percentage identity" or "% identity" means the % of identical amino acids between two peptides compared, in particular using the BLAST algorithm.

[0075] The term "NMDAR antagonist" as used herein refers to a compound that is an antagonist of NMDA receptor (NMDAR). Examples of NMDAR antagonists that belong to the group of NMDA receptor open channel blockers include, but are not limited to, 3,5-dimethyladamantan-1-amine (memantine), 3,5-dimethyladamantan-1-amine hydrochloride (memantine hydrochloride), adamantane-1-amine (amantadine), 2-(2-chlorophenyl)-2-(methylamino)cyclohexan-1-one (ketamine), 1-(1-phenylcyclohexyl) Piperidine (phencyclidine), argiotoxins, such as (2S)-N-[5-[3-[3-[[(2S)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]propylamino]propylamino]pentyl]-2-[[2-(2,4-dihydroxyphenyl)acetyl]amino]butanediamide (argiotoxin-636), (1S,9S,10S)-17-methyl-17-azatetracyclo[7.5.3.0 1,10 .0 2,7 ]heptadeca-2(7),3,5-trien-4-ol (dextrorphan), 1-(3-ethylphenyl)-1-methyl-2-naphthalen-1-ylguanidine (aptiganel), polyamines such as 2-[4-(diaminomethylideneamino)butyl]guanidine (alkaline), N'-(2-aminoethyl)ethane-1,2-diamine (diethylenetriamine) and butane-1,4-diamine (putrescine), 1,3,3,5,5-pentamethylcyclohexan-1-amine (neramexane), 2-amino-2-(2-chlorophenyl)cyclohexan-1-one (norketamine) and (1S,9R)-1-methyl-16-azatetracyclo[7.6.1.0] 2,7 .0 10,15 ]Hexadeca-2,4,6,10,12,14-hexaene (dizocilpine or MK801).

[0076] In the context of the present invention, the term "treatment and / or prevention" as used herein encompasses the prevention, delay, risk reduction, amelioration or curative treatment of the neurodegenerative disease referred to herein. Treatment may be symptomatic or disease-modifying treatment. In some embodiments, prevention as used herein refers to the prevention of the neurodegenerative disease referred to herein. Prevention as used in the context of the prevention of neurodegenerative disease may be the prevention or reduction of disease progression. Individuals already diagnosed with a neurodegenerative disease may experience a reduction in disease progression, i.e., a delay in further deterioration, by treating with the conjugate molecule of the present invention, compared to subjects not suffering from a neurodegenerative disease.

[0077] In the context of the present invention, the term "subject in need thereof" refers to a subject, particularly a human, diagnosed with a neurodegenerative disease. The diagnosis of a neurodegenerative disease and the selection of an appropriate course of treatment are performed by a general practitioner (GP), who may collaborate with specialists such as psychiatrists, elderly care physicians (especially in the case of age-related neurodegenerative diseases such as Alzheimer's disease and dementia), and / or neurologists. Following the initial diagnosis, subjects diagnosed with a neurodegenerative disease may undergo regular check-ups by their GP and / or specialists to follow the progression of the neurodegenerative disease. [Brief description of the drawings]

[0078] The above and further objects, features and advantages of the present invention will be better understood from the following illustrative and non-limiting detailed description of embodiments of the invention, taken in conjunction with the accompanying drawings. [Figure 1] FIG. 1 shows examples of complexes of peptides and NMDAR antagonists. [Diagram 2] FIG. 2 shows the mechanism by which an NMDAR antagonist, here MK801 (dizocilpine), is released from the complex of FIG. [Diagram 3] FIG. 3 shows the neuroprotective effects of GLP-1 Pen40 / memantine in primary mouse hippocampal neurons. [Figure 4]FIG. 4 shows the effect of GLP-1 Pen40 / memantine on gene expression of proinflammatory markers IL-1b, IL-6 and TNF-α in the hippocampus of mice. [Diagram 5] FIG. 5 shows the effect of GLP-1 Pen40 / memantine on weight gain in 5xFAD mice. [Figure 6] FIG. 6 shows an amino acid sequence alignment between the co-agonist GLP-1 / GIP of SEQ ID NO:5 and the modified GLP-1 peptide of SEQ ID NO:1, where X1 is D-alanine, D-serine, α-aminoisobutyric acid, N-methyl-serine, glycine or valine, X2 is cysteine ​​(hCys40 / Cys40) or L-penicillamine (Pen40) and Cex is the exendin-4 extension (SEQ ID NO:9) at the C-terminus of the peptide. [Figure 7] FIG. 7 shows an amino acid sequence alignment between the triagonist GLP-1 / GIP / GCG of SEQ ID NO: 7 and the modified GLP-1 peptide of SEQ ID NO: 1, where X1 is D-alanine, D-serine, α-aminoisobutyric acid, N-methyl-serine, glycine or valine, X2 is cysteine ​​(hCys40 / Cys40) or L-penicillamine (Pen40) and Cex is the exendin-4 extension (SEQ ID NO: 9) at the C-terminus of the peptide. [Figure 8] FIG. 8 shows the number of amyloid plaques in the frontal cortex (left graph) and hippocampus (right graph) after 49 days of treatment with saline (control) or GLP-1 / memantine in 5xFAD mice. Detailed Description of the Invention

[0079] FIG. 1 shows an example of a peptide-NMDAR antagonist complex 100, which consists of MK801 (dizocilpine) 101 chemically attached to the C-terminal cysteine ​​102 of peptide 103 of SEQ ID NO: 1 via a chemical linker 104, which contains a disulfide group 105. The side chain 106 of the C-terminal cysteine ​​102 may be optionally derivatized such that the length n of the side chain 106 is 1 or 2 carbon atoms and / or R is hydrogen or methyl. A modification of the side chain 106, designated hCys40, is of length n=2 carbon atoms and R=hydrogen. A modification of the side chain 106, designated hCys40, is of length n=1 carbon atom and R=methyl. A normal cysteine ​​is designated Cys40.

[0080] Figure 2 illustrates the mechanism by which an NMDAR antagonist, here MK801 (dizocilpine), is released from the conjugate 100 of Figure 1. The chemical linker 104, which contains a disulfide group 105, is self-destructive and is reduced in a reducing environment (not shown), such as an intracellular environment, to generate a thiol group, separating the peptide portion 107 of the conjugate from the MK801 portion 108 of the conjugate. In the MK801 portion 108 of the molecule, the liberated nucleophilic thiol 109 spontaneously undergoes intramolecular cyclization, releasing MK801 as the native, unmodified MK801 drug (the free form of MK801).

[0081] Figure 3 shows the effect of GLP-1 Pen40 / memantine on neuroprotection of primary hippocampal neurons. Primary rat hippocampal neurons were cultured for 24 h with either buffer, NMDA (to induce neurotoxicity), NDMA+GLP-1, or NMDA+GLP-1 / memantine complex. The medium was collected and lactate dehydrogenase (LDH) activity was analyzed as an indirect measure of cytotoxicity. The data show that the GLP-1 / memantine complex prevents NMDA-induced neurotoxicity, but not the GLP-1 peptide alone. The data in Figure 3 further show that the synergistic effect of GLP-1 and memantine is only obtained when these two compounds are complexed, as evidenced by the significant reduction in cytotoxicity only with the GLP-1 / memantine complexed molecule.

[0082] Figure 4 shows the effect of GLP-1 Pen40 / memantine on gene expression of pro-inflammatory markers IL-1b, IL-6 and TNF-α in the hippocampus after 49 days of compound treatment. Expression of pro-inflammatory markers IL-1b, IL-6 and TNF-α (measured by mRNA) was globally increased in the 5xFAD AD mouse model. Notably, treatment with the GLP-1 / memantine complex completely eliminated pro-inflammatory events in the 5xFAD mouse model.

[0083] Figure 5 shows the effect of GLP-1 Pen40 / memantine on weight gain in 5xFAD mice. Female wild-type (WT) control mice + vehicle treatment (n=12), 5xFAD AD mice + vehicle treatment, and 5xFAD AD mice + GLP-1 / memantine (100 nmol / kg / day) treatment were subcutaneously injected daily for 49 days, after which mouse brain regions were dissected and preserved for ex vivo analysis. It was shown that treatment with the GLP-1 / memantine complex caused only a temporary weight loss, and no long-term weight loss was observed in this in vivo Alzheimer's disease model.

[0084] FIG. 8 shows the number of amyloid plaques in the frontal cortex (left graph) and hippocampus (right graph) after 49 days of saline or GLP-1 / memantine administration in 5xFAD mice. Amyloid plaques are commonly found in Alzheimer's disease patients and are caused by abnormal levels of the natural protein beta amyloid 42, which aggregates to form plaques, collects between neurons, and inhibits cell function. Thus, patients with Alzheimer's disease have elevated levels of amyloid plaques. Treatment with the complexes of the present invention, exemplified by GLP-1 complexed with memantine, has been shown to reduce the formation of amyloid plaques. Thus, without being bound by any particular theory, the inventors hypothesize that the complex molecules of the present invention play a neuroprotective role.

[0085] Overall, the data presented indicate that chemical conjugation of glucagon superfamily peptide analogues, such as GLP-1 analogues, with NMDAR antagonists represents a novel therapeutic strategy for the treatment of neurodegenerative diseases. Conjugates based on this strategy provide superior neuroprotection in primary hippocampal neurons compared to peptide controls, without the central adverse effects of NMDAR antagonism.

[0086] In addition to the experiments and findings reported above, those skilled in the art will understand that other tests can be carried out to evaluate the efficacy of the conjugate molecule of the present invention in treating neurodegenerative diseases.Such experiments or tests include, but are not limited to, the behavioral tests commonly used to evaluate cognitive impairment in rodents.Examples of such behavioral tests include, but are not limited to, the Y-maze test for evaluating short-term memory, the step-through passive avoidance test for testing long-term memory, as well as the open field test, the rotarod performance test, the Morris water maze, the novel object recognition test, the radial maze, fear conditioning, and passive avoidance learning. EXAMPLES

[0087] Example 1: Preparation of peptides and peptide-NMDAR antagonist complexes material: All solvents and reagents were purchased commercially and used without purification. H-Rink amide ChemMatrix® resin was used for peptide elongation. Unless otherwise stated, Fmoc-protected (9-fluorenylmethylcarbamate) amino acids were purchased from Iris-Biotech or Gyros Protein Technologies, and H-Rink amide ChemMatrix® resin, 35-100 mesh; loading 0.40-0.60 mmol / g, was purchased from Sigma Aldrich. Commercially available N α -Fmoc amino acid building blocks were purchased as the following side-chain protected analogs: Arg, Pmc; Asp, O t Bu;Cys, Trt;Gln, Trt;His, Trt;Lys, Trt;Ser,t Bu; and Trp, Boc (Pmc = 2,2,5,7,8-pentamethylchloromane-6-sulfonyl, O t Bu = tert-butyl ester, Trt = trityl, Boc = tert-butyloxycarbonyl, and t Bu=tert-butyl ether).

[0088] All peptides and peptide-NMDAR antagonist complexes were characterized by analytical reversed-phase ultra-performance liquid chromatography (RP-UPLC) (Waters) and electrospray ionization liquid chromatography mass spectrometry (ESI-LCMS) coupled to an Agilent 6410 Triple Quadrupole Massfilter equipped with a C18 column (Zorbax Eclipse, XBD-C18, 4.6 × 50 mm) with a binary buffer system consisting of HO:MeCN:TFA (A: 95:5:0.1, B: 5:95:0.1) and eluted at a flow rate of 0.75 mL / min. Purity was measured by RP-UPLC equipped with a C18 column (Acquity UPLC BEH C18, 1.7 μm, 2.1 × 50 mm) eluted with a binary buffer system consisting of HO:MeCN:TFA (A: 95:5:0.1, B: 5:95:0.1) at a flow rate of 0.45 mL / min.

[0089] Automated Peptide Synthesis Protocol for Fmoc Protection Scheme:Peptides were prepared as C-terminal amidated derivatives using a Prelude X, induction heating assisted peptide synthesizer (Gyros Protein Technologies, Tucson, AZ, USA) equipped with 10 mL glass vessels. All reagents were freshly prepared as stock solutions in DMF: Fmoc-protected amino acids (0.2 M), HCTU (0.5 M), DIPEA (1.0 M) and piperidine (20% v / v). Peptide elongation was achieved by successive synthetic operations using the following protocol: deprotection (2 × 2 min, RT, 300 rpm shaking) and coupling (2 × 5 min, 75 °C, 300 rpm shaking, for Arg and His, 2 × 5 min, 50 °C, 300 rpm shaking). Peptides were prepared using double and triple couplings consisting of a 5-fold excess of AA / HCTU / DIPEA (ratio 1:1.25:2.5) over resin.

[0090] Peptide cleavage: The synthetic peptides were released from the peptidyl resin by adding 1.5 mL of cleavage cocktail (2.5% EDT, 2.5% HO, 2.5% TIPS, 2.5% thioanisole in TFA) per 100 mg of peptidyl resin and stirring for 2 h. The crude peptides were precipitated with cold diethyl ether, centrifuged at 2500 × g for 10 min at 4 °C, redissolved in MeCN:HO:TFA (ratio 1:1:0.01), filtered, and lyophilized.

[0091] purification:Crude peptides or peptide-NMDAR antagonist complexes were analyzed by RP-UPLC and ESI-LCMS or MALDI-TOF mass spectrometry before purification. Purification was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system (Waters) equipped with a reversed-phase C18 column (Zorbax, 300SB-C18, 21.2 × 250 mm) and eluted with a linear gradient (flow rate 20 mL / min) using a binary buffer system of HO:MeCN:TFA (A: 95:5:0.1, B: 5:95:0.1). Fractions were collected at 0.3 min intervals and characterized by ESI-LCMS. Purity was measured by RP-UPLC at 214 nm, and fractions with purity >95% were pooled and lyophilized. The final lyophilized product was used for further experiments.

[0092] Conjugation protocol for assembling peptide-NMDAR antagonist conjugates: The pure peptide and the pure thiopyridyl-activated NMDAR antagonist complex were dissolved in a binary solvent system (A:DMF, 6 M guanidine, 1.5 M imidazole in HO at pH=8) (ratio 7:1) and stirred for at least 2 h. The crude reaction mixture was monitored by analytical RP-UPLC and ESI-LCMS. Upon completion, the reaction mixture was diluted with buffer A and buffer B and directly purified using RP-HPLC eluting with a linear gradient.

[0093] Desalting: All peptides were desalted before biological experiments. Desalting was performed by successively redissolving the peptides or peptide-NMDAR antagonist complexes in dilute aqueous solutions of 0.01 M HCl followed by lyophilization three times. The purity of the peptides or complexes was monitored by RP-UPLC and ESI-LCMS before use in in vivo or in vitro experiments.

[0094] Preparation of GLP-1 Cys40 / Memantine (Cysteine ​​Linkage) A GLP-1 peptide having the amino acid sequence of SEQ ID NO:1 was synthesized using the Fmoc protocol described above and conjugated to a chemical linker-derivatized memantine analog. The synthesis of the chemical linker-derivatized memantine was performed according to the synthetic pathway shown in FIG. 15. The first step of the synthetic pathway was performed in methanol at room temperature for 2 hours. The second step was performed in CHCl in the presence of pyridine at 0° C. for 2 hours. The third step was performed in DMF in the presence of N,N-diisopropylethylamine (DIPEA) at 55° C. for 5 days. The final step (conjugation) was performed in 6M guanidine, 1.5M imidazole buffer at room temperature for 2 hours.

[0095] 2'-Pyridyldithioethanol In a dry round-bottom flask equipped with a magnetic stirrer and under a N2 atmosphere, 2'-aldrithiol (4.71 g, 21.3 mmol, 3 equiv) was dissolved in dry MeOH (20 mL) and then 2-mercaptoethanol (0.56 g, 7.1 mmol, 0.5 mL, 1 equiv) was added dropwise via syringe. The reaction was left at room temperature for 2 h and then concentrated in vacuo. The crude yellow oil was purified by silica gel flash chromatography (ethyl acetate:CH2Cl2, 2:8) to give 2'-pyridyldithioethanol (1.33 g, 100%) as a clear oil. R f = 0.48; 1 H NMR (600 MHz, Chloroform-d) δ 8.49 (d, J = 5.0 Hz, 1H), 7.57 (td, J = 7.7, 1.8 Hz, 1H), 7.44 - 7.36 (m, 1H), 7.16 - 7.11 (m, 1H), 5.32 (s, 1H), 3.88 - 3.73 (m, 2H), 3.01 - 2.89 (m, 2H); 13 C NMR (151 MHz, CDCl3) δ 159.31, 149.86, 137.00, 122.12, 121.57, 58.37, 42.83.

[0096] 4-Nitrophenyl(2-(pyridin-2-yldisulfanyl)ethyl)carbonateIn a dry round bottom flask equipped with a magnetic stirrer and under a N2 atmosphere, 2'-pyridyldithioethanol (1.33 g, 7.1 mmol, 1 equiv) and dry pyridine (0.56 g, 8.5 mmol, 0.575 mL, 1.2 equiv) were diluted with anhydrous CHCl (15 mL). The reaction mixture was cooled to 0°C and nitrophenyl chloroformate (1.72 g, 8.5 mmol, 1.2 equiv) was added in one portion. The reaction was stirred for 10 min, allowed to reach room temperature and left with stirring for 2 h. The reaction was diluted to 50 mL and extracted with 3xH0 (30 mL) and brine (30 mL), dried over MgSO, filtered and concentrated in vacuo. The crude oil was purified by silica gel flash chromatography (heptane:ethyl acetate, 2:1) to give 4-nitrophenyl(2-(pyridin-2-yldisulfanyl)ethyl)carbonate (2.21 g, 89%) as a clear viscous oil. f = 0.34; Purity >95 % (HPLC), R t = 15.99 min; UPLC / MS (ESI): m / z calcd. for C 14 H 12 N2O5S2[M+H] + = 353.0, found 353.3 m / z; 1 H NMR (600 MHz, DMSO-d6) δ 8.47 (ddd, J = 4.8, 1.9, 0.9 Hz, 1H), 8.35 - 8.26 (m, 2H), 7.84 (td, J = 7.8, 1.8 Hz, 1H), 7.78 (dt, J = 8.1, 1.1 Hz, 1H), 7.58 - 7.48 (m, 2H), 7.26 (ddd, J = 7.3, 4.8, 1.1 Hz, 1H), 4.48 (t, J = 6.0 Hz, 2H), 3.24 (t, J = 6.1 Hz, 2H); 13 C NMR (151 MHz, DMSO) δ 158.65, 155.17, 151.75, 149.66, 145.18, 137.80, 125.40, 122.53, 121.40, 119.52, 66.54, 36.42.

[0097] 2-(Pyridin-2-yldisulfanyl)ethyl (3,5-dimethyladamantan-1-yl)carbamate In a dry round bottom flask equipped with a magnetic stirrer under a N2 atmosphere, 4-nitrophenyl(2-(pyridin-2-yldisulfanyl)ethyl)carbonate (707 mg, 2.00 mmol, 1 equiv.) and memantine hydrochloride (650 mg, 3.00 mmol, 1.5 equiv.) were dissolved in dry DMF (20 mL) and dry DIPEA (260 mg, 6.00 mmol, 0.35 mL, 3 equiv.) was added via syringe. Memantine did not completely dissolve and the reaction immediately turned yellow upon addition of DIPEA. The reaction was left for 5 days and then heated to 80 °C. The reaction was then transferred to a separatory funnel containing ethyl acetate (50 mL) and washed thoroughly with five portions of saturated brine (50 mL) and brine (50 mL) to remove DMF. The organic layer was then extracted five times with 1M aqueous NaOH (50 mL) (until the yellow color of the aqueous layer disappeared), dried over MgSO4, filtered, and concentrated in vacuo. The crude oil was purified by flash chromatography on silica gel eluting with a gradient (heptane:EtOAc, 9:1 to 3:1) to give 2-(pyridin-2-yldisulfanyl)ethyl (3,5-dimethyladamantan-1-yl)carbamate as a glassy viscous oil (540 mg, 54%). f = 0.26; Purity >95 % (HPLC), R t = 19.36 min; UPLC / MS (ESI): m / z calcd. for C 20 H 28 N2O2S2[M+H] + = 393.2, found 393.4 m / z; 1H NMR (600 MHz, DMSO-d6) δ 8.46 (ddd, J = 4.8, 1.9, 0.9 Hz, 1H), 7.85 - 7.75 (m, 2H), 7.25 (ddd, J = 7.2, 4.8, 1.2 Hz, 1H), 6.89 (s, 1H), 4.10 (t, J = 6.4 Hz, 2H), 3.05 (t, J = 6.3 Hz, 2H), 1.69 - 1.63 (m, 2H), 1.54 - 1.43 (m, 4H), 1.31 - 1.20 (m, 5H), 1.07 (s, 2H), 0.80 (s, 6H); 13 C NMR (151 MHz, DMSO) δ 159.04, 153.78, 149.55, 137.79, 121.21, 119.23, 60.80, 51.40, 50.18, 47.07, 42.22, 37.46, 31.84, 30.05, 29.46.

[0098] GLP-1 Cys40 and GLP-1 Cys40 / memantine were prepared using the protocol described above and were determined to be >95% pure by RP-UPLC and ESI-LCMS analysis.

[0099] Preparation of GLP-1 Pen40 / Memantine (Penicillamine Conjugate) The synthesis of chemical linker-derivatized memantine was carried out using the synthetic route disclosed in Figure 15. GLP-1 Pen40 and memantine were conjugated via the chemical reaction shown in Figure 16, which was carried out in 6M guanidine, 1.5M imidazole buffer at room temperature for 2 hours.

[0100] Preparation of GLP-1 Cys40 / MK801 (cysteine ​​bond)A peptide having the sequence of SEQ ID NO:1 was synthesized using the Fmoc protocol disclosed above and conjugated to a chemical linker-derivatized MK801 analog. The synthesis of chemical linker-derivatized MK801 was performed via the second synthetic route disclosed in FIG. 17. The chemical reaction was carried out in DMF in the presence of DIPEA at 55° C. for 5 days. The linker-derivatized MK801 was conjugated to GLP-1 Cys40 via the chemical reaction shown in FIG. 18. The reaction was carried out in 6M guanidine, 1.5M imidazole buffer at room temperature for 2 hours.

[0101] 2-(Pyridin-3-yldisulfanyl)ethyl 5-methyl-10,11-dihydro-5H-5,10-epiminodibenzo[a,d][7]annulene e-12-carboxylate In a flame-dried Schlenk round-bottom flask equipped with a magnetic stirrer and under a N2 atmosphere, MK801 hydrochloride (191 mg, 0.86 mmol, 1.2 equiv) was dissolved in dry DMF (10 mL) and then 4-nitrophenyl(2-(pyridin-2-yldisulfanyl)ethyl)carbonate (253 mg, 0.72 mmol, 1.0 equiv) was added. Dry DIPEA (375 μL, 2.14 mmol, 3.0 equiv) was then added, turning the solution yellow. The reaction was heated to 55 °C in an oil bath and stirred for 4 days until UPLC-MS showed complete consumption of starting material. The reaction was diluted with ethyl acetate (50 mL) and washed thoroughly with half-saturated brine (5 × 60 mL), 0.5 M aqueous sodium hydroxide (5 × 60 mL), and brine. The organic phase was collected, dried over MgSO4, filtered, and concentrated in vacuo. Purification by preparative HPLC (elution over 17 mL / min with isocratic 60% B) followed by lyophilization afforded 11 as a clear solid (250.2 mg, 80.1%); purity >95% (HPLC), R t = 18.17 min; UPLC / MS (ESI): m / z calcd. for C 24 H 22 N2O2S2[M+H] + = 435.1, found 435.4; 1H NMR (600 MHz, DMSO-d6) δ 8.41 (dt, J = 4.8, 1.4 Hz, 1H), 7.68 (dt, J = 7.9, 4.1 Hz, 2H), 7.45 (d, J = 7.1 Hz, 1H), 7.38 - 7.31 (m, 1H), 7.25 - 7.15 (m, 4H), 7.15 - 7.06 (m, 2H), 7.01 - 6.87 (m, 1H), 5.38 (d, J = 5.5 Hz, 1H), 4.27 - 4.13 (m, 2H), 3.59 (dd, J = 17.3, 5.7 Hz, 1H), 3.10 (s, 2H), 2.67 - 2.58 (m, 1H), 2.20 (s, 3H); 13 C NMR (151 MHz, DMSO) δ 158.92, 149.56, 143.37, 139.04, 137.70, 131.78, 130.25, 127.42, 127.34, 127.31, 125.88, 122.12, 121.66, 121.20, 119.19, 65.33, 62.21, 59.20, 37.55.

[0102] GLP-1 Cys40 / MK801 was prepared from 2-(pyridin-3-yldisulfanyl)ethyl 5-methyl-10,11-dihydro-5H-5,10-epiminodibenzo[a,d][7]annulene e-12-carboxylate and GLP-1 Cys40 using the protocol described above. The product was confirmed by RP-UPLC and ESI-LCMS analysis and the purity was determined to be >95%.

[0103] Preparation of GLP-1 hCys40 / MK801 (homocysteine ​​binding) A peptide having the amino acid sequence of SEQ ID NO:1 and hCys40 modification was synthesized using the Fmoc protocol disclosed above and coupled to a chemical linker-derivatized MK801 analog. The chemical synthesis of the linker-derivatized MK801 was carried out according to the synthetic route shown in Figure 19, and the chemical reaction was carried out in 6M guanidine, 1.5M imidazole buffer at room temperature for 2 hours.

[0104] GLP-1 hCys40: A peptide having the amino acid sequence of SEQ ID NO:1 and an hCys40 modification was prepared using the protocol disclosed above. Purity was determined to be >95% by RP-UPLC and ESI-LCMS analysis. GLP-1 hCys40 / MK801 was prepared using the protocol disclosed above. Purity was determined to be >95% by RP-UPLC and ESI-LCMS analysis.

[0105] Preparation of GLP-1 Pen40 / MK801 (penicillamine conjugate) GLP-1 peptide derivatives were synthesized using the Fmoc protocol disclosed above and coupled to chemical linker-derivatized MK801 analogs. Chemical synthesis of chemical linker-derivatized MK801 was carried out according to the pathway disclosed in FIG.

[0106] GLP-1 Pen40 / MK801: The conjugate was prepared using the protocol disclosed above and the chemistry shown in Figure 20, which was carried out in 6 M guanidine, 1.5 M imidazole buffer at room temperature for 2 hours, and purity was determined to be >95% by RP-UPLC and ESI-LCMS analysis.

[0107] Example 2: Neuroprotection in primary hippocampal neurons (Figure 3) Primary cultures of hippocampal neurons were prepared from Sprague-Dawley rat fetuses (E18), and cells were cultured in 24-well cell culture plates precoated with poly-L-lysine with B27 supplement (5 × 10 5 Neurobasal medium containing 100 μM of NMDA (100 μM), NMDA+GLP-1 (1 μM) or NMDA+GLP-1 / memantine was diluted in 100 μM of NMDA-1000 cells / mL. Neurons were kept at 37 °C in a humidified incubator with a 5% CO2 atmosphere. After 8 days of in vitro culture, cultures were treated for 24 h with either vehicle, NMDA (100 μM), NMDA+GLP-1 (1 μM) or NMDA+GLP-1 / memantine. Cell culture medium (50 μL) was transferred to a 96-well ELISA plate and analyzed for lactate dehydrogenase (LDH) activity according to the manufacturer's protocol (CytoTox-96, Nonradioactive Cytotoxicity assay, Promega).

[0108] Example 3: Body weight change in the 5xFAD Alzheimer's disease mouse model (Figure 5) Experiments were performed with female 5xFAD (Jackson Laboratory, 34840-JAX) mice fed on chow (Altromin#1310, Lage, Germany). Pharmacological studies began at 8 weeks of age, and mice were treated with subcutaneous injections of GLP-1 / memantine or saline (control) once a day for 49 days and weighed weekly. Specifically, compounds were administered in a volume of 5 μL per g, mice were housed in groups (n=2–5 per cage) and kept at a temperature of 21–23 °C with a 12-h light / dark cycle (light: 6 am–6 pm, dark: 6 pm–6 am). During weeks 5 and 6, mice were singly housed and transferred to a digitally ventilated cage system. During week 7, mice were reclassified into the same groups as in weeks 0–4. Finally, mice were euthanized on day 49, and tissues were harvested, flash frozen using liquid nitrogen, and stored at −80°C prior to analysis.

[0109] Example 4: Gene expression in the hippocampus after 49 days of compound treatment (Figure 4). Hippocampal tissue was homogenized in Trizol reagent (QIAzol Lysis Reagent, Qiagen) using stainless steel beads (Qiagen) and Tissue LyserLT (Qiagen) at 20 Hz for 3 min. Then, 200 μl of chloroform (Sigma-Aldrich) was added, the tube was shaken vigorously for 15 s, left at room temperature for 2 min, and then centrifuged at 12,000 × g for 15 min at 4 °C. The aqueous phase was mixed 1:1 with 70% ethanol and further processed using the RNeasy Lipid Mini Kit according to the manufacturer's instructions. For muscle tissue, the lysis procedure described in the protocol included with the Fibrous Tissue Mini Kit (Qiagen) was followed. After RNA extraction, the RNA content was measured using NanoDrop 2000 (Thermo Fisher), and 500ng of RNA was converted to cDNA by mixing FS buffer and DTT (Thermo Fisher) with Random Primers (Sigma-Aldrich), and incubated at 70°C for 3 min, after which dNTPs, RNase out, and Superscript III (Thermo Fisher) were added, and the mixture was placed in a thermal cycler for 5 min at 25°C, 60 min at 50°C, and 15 min at 70°C, and then stored at -20°C until further processing.

[0110] qPCR: SYBR green qPCR was performed using Precision plus qPCR Mastermix with SYBR green (Primer Design, #PrecisionPLUS). qPCR was performed in 384-well plates using a Light Cycler 480 Real-Time PCR machine with a preincubation at 95°C for 2 min followed by 45 cycles at 60°C for 60 s, and melting curves were performed with a stepwise increase in temperature from 60°C to 95°C. Quantification of mRNA expression was performed according to the Δ-ΔCt method.

[0111] Example 5: Number of amyloid plaques after 49 days of saline or GLP-1 / memantine treatment in 5xFAD mice (Figure 8) Experiments were performed with female 5xFAD (Jackson Laboratory, 34840-JAX) mice fed on chow (Altromin#1310, Lage, Germany). Pharmacological studies began at 8 weeks of age, and mice were treated with subcutaneous injections of GLP-1 / memantine or saline (control) once a day for 49 days. Specifically, compounds were administered in a volume of 5 μL per g, mice were group-housed (n=2–5 per cage) and kept at a temperature of 21–23 °C with a 12-h light / dark cycle (light: 6 am–6 pm, dark: 6 pm–6 am). During weeks 5 and 6, mice were singly housed and transferred to a digitally ventilated cage system. During week 7, mice were reclassified into the same groups as in weeks 0–4. On day 49, mice were perfusion-fixed with paraformaldehyde, and brains were dissected and sliced ​​into 30-micron sections. Every sixth slide was collected from the hippocampus (preskull: -2.80 mm to -0.94 mm) and frontal cortex (preskull: 0.14 mm to 2.96 mm) and stained with Thioflavine S to quantify amyloid plaque deposition.

Claims

1. A conjugate molecule for treating and / or preventing a neurodegenerative disease in a subject, comprising a peptide and an N-methyl-D-aspartate receptor (NMDAR) antagonist, wherein the peptide has at least 85% amino acid sequence identity with SEQ ID NO: 1, or the peptide is set forth in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and the peptide is linked to the NMDAR antagonist directly or via a chemical linker, and the NMDAR antagonist is selected from memantine, neramexane, and dizocilpine.

2. The conjugated molecule of claim 1 , wherein the peptide is an incretin hormone.

3. The conjugated molecule of claim 1, wherein the peptide is glucagon-like peptide 1 (GLP-1), gastric inhibitory peptide (GIP), a combination thereof, and / or a derivative or analog thereof.

4. The composite molecule of claim 1, wherein the peptide has an amino acid sequence identity of more than 90% with SEQ ID NO:

1.

5. 2. The conjugated molecule according to claim 1, wherein the NMDAR antagonist in its free form has a dissociation constant Kd with the NMDA receptor in the range of about 0.5 nM to 1000 nM.

6. 2. The conjugated molecule of claim 1, wherein the NMDAR antagonist is linked to the peptide via a cleavable chemical linker, and the cleavable chemical linker is selected from an acid-cleavable linker, an enzyme-cleavable linker, a peptide-cleavable linker, and a linker containing a disulfide group.

7. The conjugated molecule of claim 6 , wherein the cleavable chemical linker is selected from linkers containing disulfide groups.

8. The conjugated molecule according to any one of claims 1 to 6, wherein the neurodegenerative disease is selected from dementia, mild cognitive impairment, Alzheimer's disease, Parkinson's disease, stroke, traumatic brain injury, Huntington's disease, schizophrenia and depression.

9. The conjugated molecule of claim 8, wherein the neurodegenerative disease is Alzheimer's disease.

10. The conjugated molecule according to any one of claims 1 to 6, wherein the conjugated molecule is administered in the form of a pharmaceutical composition, the pharmaceutical composition comprising the conjugated molecule or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

11. The conjugated molecule according to any one of claims 1 to 6, wherein said conjugated molecule is administered at least once a week for 12 months or more.

12. The conjugate molecule described in claim 6, wherein the peptide is shown in SEQ ID NO: 1, the amino acid at the second position from the N-terminus of the peptide is α-aminoisobutyric acid, and the NMDAR antagonist is memantine.

13. A pharmaceutical composition comprising a conjugated molecule described in any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutically acceptable carrier is a pharmaceutically acceptable solid carrier selected from excipients, diluents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents and encapsulating materials.

15. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is in the form of a powder, tablet, pill, capsule, cachet, suppository or dispersible granules.

16. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is adapted for subcutaneous, intramuscular, intraperitoneal, intravenous or oral administration.

17. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is provided in unit dosage form in an ampoule, prefilled syringe, small volume injection or multi-dose container.

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