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 that exhibit 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 level to the systemic level. Neurodegenerative diseases are considered incurable because 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 and is 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, often limited efficacy, with little or no impact on disease reversal. Currently, two major 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. 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 generically, and are used to treat millions of people worldwide. Memantine, a non-competitive 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 effects 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 garnering increasing attention for their relevance in the treatment of neurodegenerative diseases.

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

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

[0010] WO2021 / 089678A1 describes the use of GLP-1R agonists, in particular semaglutide, to treat dementia or reduce 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 comprising 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 treating 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 conjugates of a GLP1 receptor agonist and an NMDA receptor antagonist, and the use of these conjugates 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 drug 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, wherein said peptide is a glucagon superfamily peptide, said peptide being linked to said NMDAR antagonist directly or via a chemical linker, and wherein said NMDAR antagonist is 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, particularly Alzheimer's disease, in subjects. This novel therapeutic strategy is hypothesized to utilize the benefits 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 these 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 (5xFAD). To the inventors' knowledge, no existing therapeutic strategies for neurodegenerative diseases, either preclinical or clinical, combine NMDA receptor antagonism with glucagon superfamily peptide receptor agonism.

[0021] Without being bound by any particular theory, the inventors speculate that the neuroprotective effect of the conjugate molecule of the present invention is achieved by the 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 glucagon superfamily peptide receptors.

[0022] Peptides have amino and carboxyl termini, which in the context of the present invention are also referred to as the N-terminus and C-terminus, respectively, and corresponding derivatives.

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

[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 the secondary structure, such as a cyclization between glutamic acid at amino acid position 15 and lysine at position 20, counting from the N-terminus of the peptide.

[0026] Peptides can be obtained from any source and can be produced as needed, for example, peptides can be isolated from tissue, or peptides can be recombinantly produced or synthesized by methods well known to those of skill in the art.

[0027] The peptides of the conjugates of the present invention belong to the glucagon superfamily of peptides, 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 co-agonists (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 affects 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 analog thereof that interacts with the GLP-1 and / or GIP receptor as an agonist.

[0031] A peptide 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. Alternatively, a peptide of a composite molecule may have at least 70% amino acid sequence identity with SEQ ID NO: 6 or SEQ ID NO: 7. In certain embodiments, a peptide of a composite molecule has 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, a peptide of a composite molecule has 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 set forth in SEQ ID NO: 5. In one embodiment, the GLP-1 / GIP receptor co-agonist is 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 set forth in SEQ ID NO: 7.

[0033] In one embodiment, the peptide of the conjugate molecule of the present invention has at least 70% amino acid sequence identity with SEQ ID NO: 1. For example, the peptide can 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 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, naturally occurring or synthetic amino acids at amino acid positions 1, 10, 12, 15, 16, 23, 24, 27, 28, 30, and 31, counting from the N-terminus of the peptide of SEQ ID NO: 8. Such a peptide is exemplified by the GLP-1 / GIP co-agonist peptide of the amino acid sequence set forth in SEQ ID NO: 5, see Figure 6. In a further embodiment, the peptide is 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) set forth in SEQ ID NO: 4, and this peptide has GIP activity at the GIP receptor.

[0036] In one embodiment, a 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 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 lacking an exendin-4 extension at the C-terminus of the peptide. In the context of the present invention, an exendin-4 extension has the amino acid sequence GPSSGAPPPS (SEQ ID NO: 9).

[0037] When the peptide of the present invention is complexed with an NMDAR antagonist, it can accumulate at a high rate at the glucagon superfamily peptide receptor site, resulting in improved efficacy of the NMDAR antagonist. An example of a peptide of the present invention having at least 80% amino acid sequence identity with SEQ ID NO: 1 is shown in Figure 6. An example of a peptide of a complex having at least 70% amino acid sequence identity with SEQ ID NO: 1 is shown in Figure 7. The alignment of GLP-1 / GIP (peptide of SEQ ID NO: 5) and GLP-1 (peptide of SEQ ID NO: 1) shown in Figure 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 a native glucagon superfamily peptide at a glucagon superfamily peptide receptor. Generally, this is observed for peptides containing at least 10 amino acids; 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, e.g., 20 to 50 amino acids. The amino acid sequences of the present invention should have a certain percentage of identity with other peptide sequences and sufficiently contain the amino acid sequence of the peptide, e.g., 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 conjugation agents, and / or PEG conjugation. Peptides modified by conjugation to carrier proteins, such as antibodies, are also contemplated. Modifications are preferably made at positions 16, 17, 20, 21, 24, 29, and 40 (counting from the N-terminus), within the C-terminal region, or at the C-terminal amino acid of the peptide. Conjugation can be achieved using any suitable linker, such as disulfide, maleimide, alpha ketone, or click chemistry-based conjugation. Those skilled in the art will know 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. It is generally preferable to add a spacer between the modification (PEG / fatty acid / diacid) and the peptide, and the linker is preferably a gamma-Glu linker, a short PEG chain.

[0041] NMDA receptor antagonists bind to NMDA receptors, and NMDAR antagonists, for example, in their free form, have 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 in rat brain membranes. d = 6.3 nM, and K in mouse brain homogenate d = 10 nM, K in pig brain d The dissociation constant K is 1.3 nM. The determination of dissociation constants 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 NMDA receptor is, for example, a human NMDA receptor, and the NMDAR antagonist has a K dIn the context of the present invention, a free NMDA receptor antagonist refers to an antagonist that is not bound to any chemical group, particularly not chemically linked, and is therefore in its natural, unmodified form. Those skilled in the art will appreciate that interspecies differences between NMDA receptors are expected to be slight. Thus, the K measured in rodents such as mice and rats, or higher mammals such as pigs, is d Values ​​are based on K values ​​measured at human NDMA receptors or other relevant animal or mammalian NMDA receptors. d It is expected to be similar to the value.

[0042] Any NMDAR antagonist can be used. As used herein, "antagonist" refers to a type of receptor ligand or drug that binds to and blocks receptors, thereby blocking or attenuating biological responses, rather than activating receptors like agonists. Channel blockers are antagonists of each ion channel. The NMDAR antagonist in the present invention is selected from the group of NMDA receptor open channel blockers. NMDA receptor open channel blockers 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 NMDA receptor open channel blockers 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, for example, a small molecule of up to 900 kDa.In one embodiment, NMDAR antagonist is selected from dizocilpine (also known as MK801), memantine, ketamine, phencyclidine, argiotoxin (such as argiotoxin-636), dextrorphan, aptiganel, alkaline, polyamines (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 NMDAR antagonist in the present invention are bonded.In the context of the present invention, this conjugated molecule is also called peptide-drug conjugate (PDC).The peptide and NMDAR antagonist can be linked or directly bonded to each other.For example, the NMDAR antagonist can be linked via an amide bond, and this amide bond is from the amino group of the NMDAR antagonist to the carboxylic acid group of the peptide.This amide bond can be formed in any residue on the peptide that has a carboxylic acid group, such as glutamic acid residue, aspartic acid residue, synthetic residue with a carboxylic acid group, or C-terminal carboxylic acid.In one embodiment, the NMDAR is memantine.For example, when the NMDAR antagonist is memantine, the amine of memantine can be bonded to the carboxylic acid of the amino acid residue of the peptide.

[0046] In one embodiment, the conjugated molecule is for use in treating and / or preventing a neurodegenerative disease in a subject in need thereof, the conjugated molecule comprising a peptide having the amino acid sequence set forth in SEQ ID NO: 1, wherein 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 set forth 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 for example, 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, it is usually preferred that the length of the chemical linker is at most 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 interacts with the glucagon superfamily peptide receptor.When the peptide has no or low steric hindrance, it has a high affinity for the glucagon superfamily peptide receptor.A complex with high affinity for the glucagon superfamily peptide receptor may accumulate 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 containing glucuronide, β-galactoside, disulfide, hydrazone, and / or compounds that can be cleaved by galactosidase, glucuronidase, pyrophosphatase, phosphatase, arylsulfatase, protease, or esterase.For example, the linker can include a peptide that can be cleaved by cathepsin, such as GFLG.The linker can also include 4-aminobenzoic acid (PAB), which can be bonded to the amino group of NMDAR antagonist via amide bond or carbamate bond. The linker preferably releases the NMDAR antagonist in a free form (i.e., 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, an 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, an NMDAR antagonist, such as memantine, may be directly bound 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. Because the N-terminus is involved in binding to glucagon superfamily peptide receptors, low or no steric hindrance at the N-terminus may increase affinity for glucagon superfamily peptide receptors. It is also contemplated that multiple NMDAR antagonists may be attached to the same peptide molecule.

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

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

[0051] In one embodiment, the conjugated molecules are joined 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 a side chain of the peptide or in the backbone of the peptide, and R4 is (CH2) n or C6H4, R5 is optional and, if present, is selected from C(CH3)2, CH2-CH2, or CH2, and n is 1, 2, or 3. Upon reduction of the chemical linker, the free NMDAR antagonist portion of the conjugate undergoes intramolecular cyclization, thereby releasing the NMDAR antagonist in its free form, see Figure 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 a side chain of the peptide or in the backbone of the peptide, 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, wherein 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 side chain of the peptide or the backbone of the peptide, 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 side chain of the peptide or the backbone of the peptide, and n is 1, 2 or 3.

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

[0055] In the context of the present invention, when R1 is attached to the backbone of the peptide, 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 comprising a peptide and an NMDAR antagonist attached via a chemical linker. Figure 1 shows a method for designing such conjugate molecules. As shown in Figure 1, conjugates can be prepared by chemically linking an NMDAR antagonist (MK801 in Figure 1) with a peptide. 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 present inventors have found that the peptides of the present 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 that conjugate molecule enables can improve therapeutic index, that is, reduce minimum effective concentration.The tissue-selective targeting of NMDAR can be used for targeted treatment of neurological diseases.

[0060] The present inventors have demonstrated that the complex of the present invention exerts a surprising synergistic effect on neurodegeneration, which is significantly greater than the effect obtained by administering the peptide alone, see Figures 3 and 4. Furthermore, long-term administration of the complex molecule of the present invention does not appear 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, as the major hormonal pathways regulating energy metabolism are similar between mice and humans and show comparable receptor expression profiles.

[0062] In one embodiment, the neurodegenerative disease treated with the conjugated 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 conjugated molecule of the present invention is used to treat and / or prevent Alzheimer's disease. Alzheimer's disease has five stages: 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 contemplated that the conjugated molecule of the present invention can be used to treat any stage of Alzheimer's disease. Similarly, the conjugated 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 pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Any embodiment of the conjugated molecule may be used in the pharmaceutical composition.

[0064] Pharmaceutical preparations can be prepared by conventional technology.Simply put, pharmaceutically acceptable carriers are either solid or liquid.Solid form preparations include powder, tablets, pills, capsules, cachets, suppositories and dispersible granules.Solid carriers can also be one or more excipients that function as diluents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents or encapsulating materials.

[0065] The conjugates contained in the pharmaceutical preparations may be in powder form, obtained by aseptic isolation of the 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 optionally added with preservative and provided in the form of unit dosage in ampule, pre-filled syringe, small volume injection or multi-dose container.Composition can be in the form of suspension, solution or emulsion in oily medium or aqueous medium etc.

[0067] In one embodiment, the conjugated molecule or pharmaceutical composition thereof for use in the treatment and / or prevention of neurodegenerative diseases 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 conjugated molecules of the present invention can be administered as a chronic treatment by administering the conjugated molecules or pharmaceutical compositions containing the conjugated molecules for at least 12 months, e.g., at least 16 months or at least 18 months. It is understood that chronic treatment with the conjugated molecules of the present invention or pharmaceutical compositions thereof can involve treatment over several years. In one embodiment, the conjugated molecules of the present invention or pharmaceutical compositions thereof are administered once daily or once weekly for 1 year, 2 years, 5 years, 10 years, or more. In one embodiment, the therapeutic conjugated molecules of the present invention or pharmaceutical compositions thereof are administered 2-3 times weekly for 5 years or more.

[0069] Although the present invention has been described above primarily with reference to certain embodiments, it will be readily apparent to those skilled in the art that other embodiments than those disclosed above are equally 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, unless expressly defined or otherwise stated, are to be construed in accordance with their ordinary meaning in the art of the present invention 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 construed as referring to at least one example of said conjugate, agent, molecule, linker, peptide, etc., 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 to 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 glucagon superfamily peptide receptors and are thought to be neuroprotective peptides, and may function as active delivery agents for 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 reference to a peptide or amino acid, refers to a chemically modified peptide or amino acid in which at least one substituent is 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, etc.

[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] As used herein, the term " NMDAR antagonist " refers to the compound that is the antagonist of NMDA receptor (NMDAR).The example of NMDAR antagonist that belongs to the group of NMDA receptor open channel blockers includes but is not limited to: 3,5-dimethyladamantan-1-amine (memantine), 3,5-dimethyladamantan-1-amine hydrochloride (memantine hydrochloride), adamantan-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 (alkaine), 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 diseases referred to herein. Treatment may be symptomatic or disease-modifying. In some embodiments, prevention as used herein refers to the prevention of the neurodegenerative diseases referred to herein. Prevention as used in the context of the prevention of neurodegenerative diseases may also refer to 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 treatment with the conjugate molecules 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, who has been 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 (particularly in the case of age-related neurodegenerative diseases such as Alzheimer's disease and dementia), and / or neurologists. Following the initial diagnosis, a subject diagnosed with a neurodegenerative disease may undergo regular check-ups with their GP and / or specialists to track the progression of the neurodegenerative disease. [Brief explanation 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. [Figure 2] FIG. 2 shows the mechanism by which an NMDAR antagonist, here MK801 (dizocilpine), is released from the complex of FIG. [Figure 3] FIG. 3 shows the neuroprotective effect of GLP-1 Pen40 / memantine in primary hippocampal neurons in mice. [Figure 4]FIG. 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 of mice. [Figure 5] FIG. 5 shows the effect of GLP-1 Pen40 / memantine on weight gain in 5xFAD mice. [Figure 6] Figure 6 shows the amino acid sequence alignment between the coagonist 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] Figure 7 shows the 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 conjugate 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, where the chemical linker 104 contains a disulfide group 105. The side chain 106 of the C-terminal cysteine ​​102 may be optionally derivatized so 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, has a length n=2 carbon atoms and R=hydrogen. A modification of the side chain 106, designated hCys40, has a 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, containing 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 (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 hours with either buffer, NMDA (to induce neurotoxicity), NDMA + GLP-1, or NMDA + GLP-1 / memantine complex. Culture medium was collected and analyzed for lactate dehydrogenase (LDH) activity as an indirect measure of cytotoxicity. The data indicate that the GLP-1 / memantine complex prevents NMDA-induced neurotoxicity, but the GLP-1 peptide alone does not. The data in Figure 3 further demonstrate that the synergistic effect of GLP-1 and memantine is only achieved when these two compounds are complexed, as evidenced by the significant reduction in cytotoxicity observed only with the GLP-1 / memantine complex.

[0082] Figure 4 shows the effect of GLP-1 Pen40 / memantine on the gene expression of pro-inflammatory markers IL-1b, IL-6, and TNF-α in the hippocampus after 49 days of compound treatment. Expression of the pro-inflammatory markers IL-1b, IL-6, and TNF-α (measured by mRNA) was generally increased in the 5xFAD AD mouse model. Notably, treatment with the GLP-1 / memantine complex completely eliminated pro-inflammatory responses 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. Treatment with the GLP-1 / memantine complex induced only transient weight loss, indicating that long-term weight loss is not observed in this in vivo Alzheimer's disease model.

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

[0085] Overall, the presented data demonstrate that chemical conjugation of glucagon superfamily peptide analogs, such as GLP-1 analogs, with NMDAR antagonists represents a novel therapeutic strategy for the treatment of neurodegenerative diseases. Conjugates based on this strategy exhibited superior neuroprotection in primary hippocampal neurons compared with peptide controls, without the adverse central 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 also be carried out to evaluate the efficacy of the conjugate molecules 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, rotarod performance test, Morris water maze, novel object recognition test, radial maze, fear conditioning, and passive avoidance learning. [Example]

[0087] Example 1: Preparation of peptides and peptide-NMDAR antagonist conjugates material: All solvents and reagents were purchased commercially and used without further 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-pentamethylchloroman-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) coupled to an Agilent 6410 Triple Quadrupole Mass Filter equipped with a C18 column (Zorbax Eclipse, XBD-C18, 4.6 x 50 mm) and electrospray ionization liquid chromatography-mass spectrometry (ESI-LCMS). Elution was performed using a binary buffer system consisting of HO:MeCN:TFA (A: 95:5:0.1, B: 5:95:0.1) at a flow rate of 0.75 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 with Fmoc Protection Scheme:Peptides were prepared as C-terminal amidated derivatives using a Prelude X induction-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 sequential 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 over resin (ratio 1:1.25:2.5).

[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 (1:1:0.01 ratio), filtered, and lyophilized.

[0091] purification:Crude peptides or peptide-NMDAR antagonist conjugates 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 x 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 conjugates of peptides and NMDAR antagonists: The pure peptide and pure thiopyridyl-activated NMDAR antagonist conjugate were dissolved in a binary solvent system (A: 6 M guanidine, 1.5 M imidazole in DMF, pH 8 HO) (7:1 ratio) 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 peptide or peptide-NMDAR antagonist complex with dilute aqueous solutions of 0.01 M HCl, followed by lyophilization three times. The purity of the peptide or complex 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 ​​bond) 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 carried out according to the synthetic pathway shown in Figure 15. The first step of the synthetic pathway was carried out in methanol at room temperature for 2 hours. The second step was carried out in CHCl in the presence of pyridine at 0°C for 2 hours. The third step was carried out in DMF in the presence of N,N-diisopropylethylamine (DIPEA) at 55°C for 5 days. The final step (conjugation) was carried out in 6 M guanidine, 1.5 M 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:CHCl, 2:8) to give 2'-pyridyldithioethanol (1.33 g, 100%) as a clear oil. 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 under a N 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, extracted 3x with HO (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 N 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 allowed to stand 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 the DMF. The organic layer was then extracted five times with 1 M 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 silica gel flash chromatography, eluting with a gradient (heptane:EtOAc, 9:1 to 3:1) to afford 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 purity was determined to be >95% 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 by the chemical reaction shown in Figure 16, which was carried out in 6 M guanidine, 1.5 M 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 Figure 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 Figure 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, under a N2 atmosphere, MK801 hydrochloride (191 mg, 0.86 mmol, 1.2 equiv.) was dissolved in dry DMF (10 mL), followed by the addition of 4-nitrophenyl(2-(pyridin-2-yldisulfanyl)ethyl)carbonate (253 mg, 0.72 mmol, 1.0 equiv.). 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 indicated complete consumption of the 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 ​​conjugate) A peptide having the amino acid sequence of SEQ ID NO:1 and an hCys40 modification was synthesized using the Fmoc protocol disclosed above and conjugated 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 a 6 M guanidine, 1.5 M 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 conjugated to chemical linker-derivatized MK801 analogs, the chemical synthesis of which was carried out according to the pathway disclosed in Figure 16.

[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. 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). Cells were grown in 24-well cell culture plates precoated with poly-L-lysine, supplemented with B27 supplement (5 × 10 5 Neurobasal medium containing 1000kJ / mL of NMDA was diluted with 1000kJ / mL of NMDA. 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 with either vehicle, NMDA (100 μM), NMDA + GLP-1 (1 μM), or NMDA + GLP-1 / memantine for 24 hours. 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 progression in the 5xFAD Alzheimer's disease mouse model (Figure 5) Experiments were conducted using female 5xFAD (Jackson Laboratory, 34840-JAX) mice fed a chow diet (Altromin #1310, Lage, Germany). Pharmacological studies began at 8 weeks of age. Mice were treated with subcutaneous injections of GLP-1 / memantine or saline (control) once daily for 49 days, and weighed weekly. Specifically, compounds were administered at a volume of 5 μL per gram. Mice were group-housed (n = 2–5 per cage) and maintained at a temperature of 21–23 °C under 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 reassigned to the same groups as in weeks 0–4. Finally, mice were euthanized on day 49, and tissues were harvested, snap-frozen using liquid nitrogen, and stored at −80°C before analysis.

[0109] Example 4: Gene expression in the hippocampus after 49 days of compound treatment (Figure 4). Hippocampal tissue was homogenized in Trizol Lysis Reagent (QIAzol Lysis Reagent, Qiagen) using stainless steel beads (Qiagen) and a Tissue Lyser LT (Qiagen) at 20 Hz for 3 minutes. 200 μl of chloroform (Sigma-Aldrich) was then added, and the tube was vigorously shaken for 15 seconds, left at room temperature for 2 minutes, and then centrifuged at 12,000 × g for 15 minutes 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 500 ng of RNA was converted to cDNA by mixing FS buffer and DTT (Thermo Fisher) with Random Primers (Sigma-Aldrich), and then incubated at 70°C for 3 minutes. After that, dNTPs, RNase out, and Superscript III (Thermo Fisher) were added, and the mixture was placed in a thermal cycler and treated at 25°C for 5 minutes, 50°C for 60 minutes, and 70°C for 15 minutes. The mixture was then stored at -20°C until further processing.

[0110] qPCR: SYBR green qPCR was performed using Precision plus qPCR Mastermix containing SYBR green (Primer Design, #PrecisionPLUS). qPCR was performed in 384-well plates using a Light Cycler 480 Real-Time PCR machine. The PCR was performed with a 2-minute preincubation at 95°C, followed by 45 cycles at 60°C for 60 seconds. A melting curve was performed with a temperature stepwise increase from 60°C to 95°C. Quantification of mRNA expression was performed according to the delta-delta Ct 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 conducted using female 5xFAD (Jackson Laboratory, 34840-JAX) mice fed a chow diet (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 daily for 49 days. Specifically, compounds were administered at a volume of 5 μL per g. Mice were group-housed (n = 2–5 per cage) and maintained at a temperature of 21–23 °C under 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 regrouped into the same groups as those from 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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