GLP1R agonist-NMDAR antagonist conjugate

Abstract

The present invention relates to a conjugate molecule comprising a peptide that exhibits at least 0.1% of the activity of native glucagon-like peptide 1 (GLP-1) at the GLP-1 receptor and an N-methyl-D-aspartate receptor (NMDAR) antagonist, wherein the peptide is covalently attached to the NMDAR antagonist directly or via a chemical linker, the conjugate molecule for use in therapy, a pharmaceutical composition comprising the conjugate molecule, a method for reducing the weight of a mammal comprising administering the conjugate molecule to the mammal, and a non-therapeutic method for reducing the weight of a mammal comprising orally administering the conjugate molecule to the mammal.

Description

[Technical Field]

[0001] The present invention relates generally to the field of therapeutic conjugates, and more particularly to conjugates having glucagon-like peptide 1 (GLP-1) receptor activity and N-methyl-D-aspartate receptor (NMDAR) antagonists. [Background technology]

[0002] Obesity is the most prevalent nutritional disease in affluent societies in humans and domestic animals such as dogs and cats, far exceeding the number of nutritional deficiencies. As an alternative to bariatric surgery, many attempts have been made to develop weight-loss drugs for the treatment of obesity. This has resulted in drugs that act by preventing fat absorption by acting as lipase inhibitors in the intestine or by inhibiting food intake via selective serotonin receptor 2C agonism in the hypothalamus.

[0003] Glucagon-like peptide 1 (GLP-1) is a 30- or 31-amino acid-long peptide hormone derived from tissue-specific post-translational processing of the proglucagon peptide. Because it acts on the appetite-regulating center in the brain, a recent application of GLP-1 analogs is weight loss. GLP-1 is associated with appetite and weight maintenance because it not only acts on the gastrointestinal tract but also influences the CNS, which is involved in appetite regulation. It also slows gastric emptying and intestinal motility in humans, potentially contributing to the regulation of food intake. GLP-1-based therapies for the treatment of metabolic disorders are known from the prior art. Parlevliet et al. (J Pharmacol Exp Ther. 2009 Jan;328(1):240-8)" and related patent applications EP1968645 A2, EP2125003 A2, and EP1843788 A2 describe the use of human GLP-1 mimetibodies™ comprising GLP-1 peptides to treat obesity and obesity-related disorders. More specifically, Parlevliet et al. (2009) describe a particular GLP-1CNTO 736, which can reduce food intake and body weight in high-fat fed mice by reducing body fat mass.

[0004] NMDAR antagonists act by blocking the action of NMDA receptors, and there is some preclinical evidence supporting the possibility that NMDAR antagonism is related to appetite reduction and weight maintenance. Deng et al. (2019, Frontiers in Psychiatry, 10, Article 15) described the use of the NMDAR antagonist memantine hydrochloride to drive weight loss in diet-induced obese mice induced by a high-fat diet. Smith et al. (Neuropsychopharmacology (2015) 40, 1163-1171) reported that memantine selectively and dose-dependently reduced binge-like eating and completely blocked food-seeking behavior and compulsive eating in rats fed a highly palatable high-sugar diet. Additionally, Popik et al. (Amino Acids (2011) 40:477-485) reported that chronic administration of memantine hydrochloride to rats can selectively reduce the consumption of a highly palatable food without significantly affecting the consumption of standard chow, and that this effect persists even after treatment is discontinued.

[0005] The effectiveness of memantine in treating binge eating disorder in humans has also been reported. Hermanussen and Tresguerres (Economics and Human Biology 3 (2005) 329-337) reported that in a clinical trial of five obese young women, memantine treatment significantly reduced appetite within the first 24 hours, suppressed binge eating disorder, and could result in weight loss within a few days. Brennan et al. (Int J Eat Disord 2008; 41:520-526) described a preliminary study showing that daily administration of memantine for 12 weeks could improve binge eating in human subjects.

[0006] There is an increasing need for novel weight loss treatments that are more effective, have improved safety (fewer toxicological effects), and also offer convenient and safe administration options. Summary of the Invention

[0007] In view of the above, it is therefore an object of the present invention to provide an effective and safe therapeutic agent for reducing food intake and weight loss in obese human subjects.

[0008] Accordingly, a first aspect of the present invention relates to a conjugate molecule comprising a peptide that exhibits at least 0.1% of the activity of native GLP-1 at the GLP-1 receptor and an N-methyl-D-aspartate receptor (NMDAR) antagonist, wherein said peptide is covalently attached to said NMDAR antagonist either directly or via a chemical linker.

[0009] The inventors have surprisingly found that conjugation of peptides with GLP-1 receptor agonism and NMDAR antagonism represents a novel therapeutic strategy for effectively reversing obesity. Conjugates based on this strategy are superior to GLP-1 peptides, memantine, or MK801 alone in suppressing food intake, as shown in Figures 3-13. Furthermore, conjugates based on GLP-1 peptide variants, such as the GLP-1 / gastric inhibitory polypeptide (GIP) peptide (SEQ ID NO: 9), and alternative NMDAR antagonists have been shown to have similar beneficial effects on food intake and weight loss. This is supported by further findings testing the GLP-1 / GIP co-agonist and the NMDAR antagonist neramexane, as shown in Figures 33-34 and 36-38, respectively. Furthermore, these conjugates benefit from the weight loss effects of NMDAR antagonism, while the central nervous system effects of NMDAR antagonism are avoided by this strategy. Without wishing to be bound by any particular theory, the inventors speculate that this effect is achieved by the NMDAR antagonist accumulating at and / or near the site of the GLP-1 receptor in the body due to the affinity of the peptide for the GLP-1 receptor.

[0010] Peptides have an amino terminus and a carboxyl terminus, which in the context of the present invention may also be referred to as the N-terminus and C-terminus, respectively, and corresponding derivatives.

[0011] Peptides may consist of amino acids encoded by the genetic code, or may contain amino acids encoded by the genetic code as well as 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 may incorporate D-alanine and D-leucine, or synthetic amino acids such as α-aminoisobutyric acid (Aib), d-serine (dSer), and N-methyl-serine.

[0012] In a preferred embodiment, the second amino acid (counting from the N-terminus) of the peptide is dSer, dAla, Aib, glycine, N-methyl-Ser, or valine.

[0013] The peptide may also have one or more modifications to stabilize secondary structure, such as a cyclization between glutamic acid at position 15 and lysine at position 20 of the peptide, these positions being counted from the N-terminus.

[0014] The peptides can be obtained from any source, or the peptides can be produced if desired. For example, the peptides can be isolated from tissue, or the peptides can be produced recombinantly or synthesized by methods well known to those of skill in the art.

[0015] The conjugate molecule comprises a peptide, which (in its free form) exhibits at least 0.1% of the activity of native GLP-1 at the GLP-1 receptor. In the context of the present invention, GLP-1 receptor activity, also referred to as GLP-1 activation (GLP-1R activity), can be measured in an in vitro assay by measuring cAMP induction in HEK293 cells overexpressing the GLP-1 receptor. Specifically, HEK293 cells co-transfected with DNA encoding the GLP-1 receptor and a luciferase gene linked to a cAMP response element (reporter assay) can be used. This assay can be performed as described by Bech et al. (J. Med. Chem. 2017, 60, 17, 7434-7446). Using this assay, the GLP-1R activity of each conjugate can be determined and compared to the activity obtained with native GLP-1 (SEQ ID NO: 1) peptide in the same assay. In certain embodiments, the peptide of the conjugate exhibits at least 1% of the activity of native GLP-1, eg, at least 5%, 10%, 15%, 20%, or 30% of the activity.

[0016] NMDAR antagonists bind to NMDARs and have a dissociation constant, K, between the NMDAR antagonist and a specific NMDA receptor, e.g., in the free form of the NMDAR antagonist. d NMDAR antagonists generally have dissociation constants in the nanomolar range; for example, the dissociation constant of MK801 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 with the NMDA receptor of 1.3 nM. dThe NMDA receptor may be, for example, a human NMDA receptor, and the NMDAR antagonist may have a K d In the context of the present invention, the NMDA receptor antagonist in free form refers to an antagonist that is not bound (especially chemically linked) to any chemical group and is therefore in its natural, unmodified form. Those skilled in the art will understand that only minor species variations between NMDA receptors are to be expected. Thus, the K measured in rodents, such as mice or rats, or in higher mammals, such as pigs, is d Values ​​are 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.

[0017] The peptide of the conjugate molecule can be any peptide that has at least 0.1% of the activity of native GLP-1 at the GLP-1 receptor. In one embodiment, the peptide of the conjugate is from the glucagon superfamily, a group of peptides that are structurally related in their N- and C-terminal regions (see, e.g., Sherwood et al., Endocrine Reviews 21: 619-670 (2000)). Members of this group include all glucagon-related peptides, as well as growth hormone-releasing hormone (SEQ ID NO:2), vasoactive intestinal peptide (SEQ ID NO:3), pituitary adenylate cyclase-activating polypeptide 27 (SEQ ID NO:4), secretin (SEQ ID NO:5), gastric inhibitory polypeptide (GIP) (SEQ ID NO:6), exendin-4 (SEQ ID NO:7), unmodified GLP-1 (SEQ ID NO:8), GLP-1 / GIP co-agonist (SEQ ID NO:9), and analogs, derivatives, or conjugates thereof having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications relative to the native peptide. Such peptides preferably retain the ability to interact (as agonists) with receptors of the glucagon receptor superfamily, preferably the GLP-1 receptor. The peptide of the conjugated molecule may have at least 80% amino acid sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. The peptide of the conjugate molecule may also have at least 80% amino acid sequence identity to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In certain embodiments, the peptide of the conjugate 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, the peptide of the conjugate molecule has the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In certain embodiments, the peptide of the invention is exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, or semaglutide.

[0018] Also contemplated is the peptide with co-agonist activity, which shows the ability to bind to different receptors of glucagon receptor superfamily.In one embodiment, this co-agonist is GLP-1 / GIP receptor co-agonist.The effect of the conjugate molecule based on the co-agonist of SEQ ID NO: 9 and NMDAR antagonist on food intake and body weight is shown in Figure 33-34.

[0019] In some embodiments, the conjugated peptide has at least 80% amino acid sequence identity to SEQ ID NO: 1. For example, the peptide may have at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than about 97% identity to SEQ ID NO: 1. In certain embodiments, the peptide has the amino acid sequence of SEQ ID NO: 1. Such peptides may have significantly higher GLP-1 activity at the GLP-1 receptor compared to native GLP-1 at the GLP-1 receptor. Thus, when conjugated to an NMDAR antagonist, the peptide may accumulate at the GLP-1 receptor site at a higher rate, which in turn may enhance the efficacy of the NMDAR antagonist. An example of a conjugated peptide having at least 80% amino acid sequence identity to SEQ ID NO: 1 is shown in Figure 35, and the effects of this peptide are supported by Figures 33-34. The alignment of GLP-1 / GIP Pen40 / MK801 (peptide according to SEQ ID NO: 9) with GLP-1 Pen40 / MK801 (peptide according to SEQ ID NO: 1) is further shown below: [ka]

[0020] The peptide of the conjugate molecule has a length sufficient to ensure that the peptide (in its free form) exhibits at least 0.1% of the activity of native GLP-1 at the GLP-1 receptor. Generally, this is observed for peptides comprising at least 10 amino acids, but activity may not be exhibited if the peptide comprises more than 60 amino acids. Thus, in one embodiment, the peptide has a length in the range of 10 to 60 amino acids, e.g., 20 to 50 amino acids. An amino acid sequence of the present invention that is a certain percentage identical to the sequence of another peptide should comprise enough of the amino acid sequence of the peptide, e.g., at least 10 amino acids, to provide putative identification of the peptide by manual evaluation of the sequence by one of skill in the art or by automated computer-based 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).

[0021] In the context of the present invention, peptides may vary in % identity by having substitutions, natural or synthetic amino acid insertions and / or amino acid deletions. In one embodiment, the peptide of the conjugate has the amino acid sequence of SEQ ID NO: 1.

[0022] In some embodiments, the peptide is modified by acetylation, fatty acid conjugation, diacid conjugation, albumin conjugation, small molecule albumin binder, and / or PEG conjugation. Peptides modified by linking to a carrier protein, such as an antibody, are also contemplated. The modification is preferably at positions 16, 17, 20, 21, 24, 29, or 40 (counting from the N-terminus) of the peptide, within the C-terminal region, or at the C-terminal amino acid. Conjugation can be achieved by any suitable linker, such as disulfide, maleimide, α-ketone, or click chemistry-based conjugation. Those skilled in the art will know how to prepare such conjugates. Preferably, the PEG molecule is larger than 1 kDa, and the fatty acid and diacid may contain more than 12 carbon atoms. It is generally preferred to add a spacer between the modification (PEG / fatty acid / diacid) and the peptide; the linker is preferably a γ-Glu linker or a short PEG chain.

[0023] The conjugate molecule comprises an NMDAR antagonist. Any NMDAR antagonist can be used with the conjugate. However, it is preferred that the NMDAR antagonist is a small molecule, e.g., up to 900 kDa. For example, in one embodiment, the NMDAR antagonist is selected from MK801, memantine, ketamine, phencyclidine (PCP), neramexane, and amantadine. MK801, neramexane, and memantine are preferred. MK801 and memantine are also preferred. Neramexane is a non-limiting example of a compound related to memantine, and the effects of neramexane are shown in Figures 36-38.

[0024] The peptide of the present invention and the NMDAR antagonist are covalently bonded.In the context of the present invention, said conjugate molecule is also called peptide-drug conjugate (PDC).Said peptide and NMDAR antagonist can be directly bonded.For example, NMDAR antagonist can be covalently bonded through amide bond, and this amide bond is from the amino group of NMDAR antagonist to the carboxylic acid group of peptide.Such amide bond can be made to any residue of peptide that has carboxylic acid group, such as glutamic acid residue, aspartic acid residue, synthetic residue with carboxylic acid group, or C-terminal carboxylic acid.For example, when NMDAR antagonist is MK801, the amine of MK801 can be bonded to the carboxylic acid of the amino acid residue of peptide.Correspondingly, when NMDAR antagonist is memantine, the amine of memantine can be bonded to the carboxylic acid of the amino acid residue of peptide.

[0025] In the context of the present invention, "directly covalently bonded" means that the peptide has a covalent bond with the NMDAR antagonist, for example, there is no additional chemical group, such as a linker group, between the two molecules.The peptide and the NMDAR antagonist can also be bonded via a chemical linker.Any chemical linker can be used.However, it is generally preferred that the chemical linker has a length of up to 30 atoms.A longer chain can have the advantage that when the peptide interacts with the GLP-1 receptor, the NMDAR antagonist is farther away from the peptide, so that there is no or little steric hindrance between the peptide and the NMDAR antagonist.The absence or low steric hindrance of the peptide increases its affinity for the GLP-1 receptor.The higher the affinity of the conjugate for the GLP-1 receptor, the more likely it is to accumulate at the site of the GLP-1 receptor. Chemical linker is preferably cleavable linker, for example, acid cleavable linker, enzyme cleavable linker, peptide cleavable linker or disulfide linker, which are generally known in the art for use in peptide-drug conjugates.The example of such cleavable linker is the compound comprising glucuronide, β-galactoside, disulfide, hydrazone, and / or these compounds can be cleaved by galactosidase, glucuronidase, pyrophosphatase, phosphatase, arylsulfatase, protease or esterase.For example, linker can comprise the peptide, for example, GFLG, that can be cleaved by cathepsin.Linker can further comprise 4-aminobenzoic acid (PAB), which can be covalently bonded to the amino group of NMDAR antagonist via amide bond or carbamate bond. The linker preferably releases the NMDAR antagonist in its free form (i.e., its native form), which can be achieved by many different linker chemistries, such as the disulfide linkers disclosed herein. These and other linker chemistries are well known to those skilled in the art.

[0026] In one embodiment, the NMDAR antagonist is covalently bound to the C-terminal region of the peptide.In the context of the present invention, the C-terminal region can 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 can be amino acids 21-40, 26-40, or 31-40 (numbered from the N-terminus).Therefore, the NMDAR antagonist, for example, memantine or MK801, can be directly bound to any one of the 10 amino acids counting from the C-terminus or via a linker.For example, the NMDAR antagonist, for example, memantine or MK801, can be directly bound to an amino acid within 5 amino acids from the C-terminus.Therefore, the NMDAR antagonist causes little or no steric hindrance at the N-terminus of the peptide. Since N-terminus is involved in binding to GLP-1 receptor, if N-terminus has no or low steric hindrance, it can have high affinity to GLP-1 receptor.The higher the affinity of conjugate to GLP-1 receptor, the more likely it is to accumulate at GLP-1 receptor site.It is also contemplated that two or more NMDAR antagonists can be bound to the same peptide molecule.

[0027] In another highly preferred embodiment, the NMDAR antagonist is covalently bonded to the peptide via a chemical linker that comprises disulfide group.When disulfide group is chemically reduced, it releases the NMDAR antagonist from peptide.The chemical linker that comprises disulfide group is also known as disulfide linker, and ensures that the peptide and NMDAR antagonist of the conjugate remain conjugated for a long period of time in systemic circulation.The disulfide group of disulfide linker can be reduced in a reducing environment such as intracellular environment, and the conjugate can be cleaved, so that the peptide part of the conjugate is separated from the NMDAR antagonist part of the conjugate.The reduction can be, for example, by disulfide exchange with thiol such as glutathione or reductase such as intracellular protein disulfide isomerase enzyme. The chemical linker can be selected from chemical linkers known in the art having the general formula R'-SS-R'', where the R' and R' groups can be the same or different. Experiments have shown that a conjugate of a peptide and an NMDAR antagonist conjugated via a chemical linker comprising a disulfide group has a human plasma cleavage half-life of approximately 0.5 to 13 hours, as shown in Figure 3. Advantageously, due to the affinity of the peptide for the GLP-1 receptor, the conjugate can accumulate at and / or near the site of the GLP-1 receptor in the body, and the NMDAR antagonist can be released at and / or near the site of the GLP-1 receptor. Without the peptide portion of the conjugate, the NMDAR antagonist can have a favorable effect as a site-specific NMDAR binding site. It is speculated by the inventors that the conjugate may be cleaved in the extracellular environment directly adjacent to cells bearing GLP-1 receptors, or that the conjugate may be internalized by cells bearing GLP-1 receptors and cleaved in the reducing environment of the cell.

[0028] In one embodiment, the conjugate molecule is conjugated via a chemical linker, the chemical linker having the formula R1-R3-SS-R4-R5-O-CO-R2, where R1 is the peptide, R2 is the NMDAR antagonist, R3 is optional and, if present, is selected from C(CH3)2, CH2-CH2, or CH2 and is attached to a side chain of the peptide or to a carbon atom 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 liberated NMDAR antagonist portion of the conjugate undergoes intramolecular cyclization, and the NMDAR antagonist is released in free form, see Figure 1B.

[0029] In one embodiment, the chemical linker has the formula R1-R3-SS-(CH2) n -O-CO-R2, wherein R1 is the peptide, R2 is the 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 n is 1, 2, or 3.

[0030] In one embodiment, the chemical linker has the formula R1-R4-R3-SS-(CH2) n -O-CO-R2, wherein R1 is the peptide, R2 is the 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 or main chain 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 or main chain of the peptide, and n is 1, 2, or 3.

[0031] In one embodiment, the second radical bond is to the backbone of the peptide of the invention.

[0032] In another embodiment, the second radical bond is to a side chain of a peptide of the invention.

[0033] 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 1A.

[0034] As used herein, first and second radical bonds are used to describe the presence of at least two free bonds in the chemical linkers disclosed herein.

[0035] The present invention facilitates the design and synthesis of a library of conjugate molecules comprising peptides and NMDAR antagonists attached via chemical linkers. Figure 1 shows the design method of such conjugate molecules. As shown in Figure 1A, conjugates can be prepared by chemically linking NMDAR antagonists (MK801 in Figure 1) to peptides. Those skilled in the art will understand that a vast number of different chemical linkers can be prepared by the methods disclosed herein or other methods reported in literature, and these chemical linkers can be used to attach peptides and NMDAR antagonists according to the methods disclosed herein and as reported elsewhere in the art.

[0036] The present inventors have unexpectedly found that the peptide of the present invention can play a dual role as weight loss drug and targeting drug, and can otherwise enable the site-selective delivery of non-specific small molecules, such as NMDAR antagonists, to the brain regions that control feeding (which may be but are not limited to the hypothalamic nucleus, area postrema, nucleus tractus solitarius and ventral tegmental area).Therefore, the conjugate molecule of the present invention provides a means for selectively regulating glutamatergic signal transduction in the brain regions that control food intake, while preventing it from free signal transduction throughout the brain.It is understood that the targeting property of the peptide of the present invention can also facilitate the delivery of NMDAR antagonists to other regions, such as the endocrine pancreas.

[0037] The conjugate molecule disclosed herein provides selectivity and enhances the drug action in the target area.This targeting made possible by this conjugate molecule can improve the therapeutic index, that is, reduce the minimum effective concentration.In addition, coupling can add another metabolic drug effect to the effectiveness of the medicine that targets GLP-1 receptor.Tissue-selective targeting of NMDAR can be used to control eating behavior, and can reduce relapse after treatment cessation as a result of synaptic plasticity re-establishment at a lower body weight set point.

[0038] The present inventors have demonstrated a surprising synergistic effect of the conjugates of the present invention on appetite, food intake, and body weight, which is significantly greater than the effect obtained by administering the peptide or drug alone. See Figures 4-14. The surprising synergistic effect of the conjugates of the present invention is further supported by the findings shown in Figures 21-28, 33-34, and 36-38.

[0039] The present inventors have further demonstrated a surprising synergistic effect of the conjugates of the present invention on food reward and satiety, which is significantly greater than the effect obtained by administration of either the peptide or the drug alone. See Figure 31.

[0040] In addition, the synergistic effect of the conjugates of the present invention has been demonstrated in relation to the treatment of diabetic patients. See Figure 32.

[0041] Thus, administration of the conjugates of the invention results in an unexpected decrease in food intake and body weight in obese animals.

[0042] In one embodiment of the invention, the conjugated molecule is for use in therapy.

[0043] In certain embodiments, the conjugated molecules of the invention are for use in the treatment of obesity, binge eating disorders, insulin resistance, type 2 diabetes, dyslipidemia, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease.

[0044] Another aspect of the present invention relates to a pharmaceutical composition comprising a conjugate molecule according to the present invention and a pharmaceutically acceptable carrier. Any embodiment of the conjugate molecule may be used in the pharmaceutical composition.

[0045] In a further aspect, the present invention relates to the use of a conjugate molecule according to the present invention in the manufacture of a pharmaceutical composition. Specifically, the pharmaceutical composition is for use in the treatment of obesity, binge eating disorder, insulin resistance, type 2 diabetes, dyslipidemia, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease. Any embodiment of the conjugate molecule may be used in the manufacture of the pharmaceutical composition.

[0046] Although the data disclosed in this invention were obtained from mouse studies, the conclusions are equally relevant to humans as the major hormonal pathways governing energy metabolism are similar between mice and humans, with comparable receptor expression profiles.

[0047] The conjugate of the present invention can be administered in the form of a pharmaceutical composition. Thus, the present invention further provides a pharmaceutical composition comprising the conjugate of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Pharmaceutical formulations can be prepared by conventional techniques. Briefly, pharmaceutically acceptable carriers can be either solid or liquid. Solid preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Solid carriers can be one or more excipients that can also function as diluents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or encapsulating materials.

[0048] The conjugate included in the pharmaceutical formulation may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0049] In one embodiment, said 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 the form of a single dose or a multi-dose container in ampule, pre-filled syringe, small volume infusion, and optionally preservative is added.These compositions can be in the form of suspension, solution or emulsion in oily or aqueous vehicle, etc.

[0050] According to the present disclosure, a pharmaceutical composition is provided in which the agonistic and food intake-reducing effects of a peptide with GLP-1R activity are combined with NMDAR antagonism in a single format. Active delivery of a peptide with GLP-1R activity to the hypothalamic nucleus, area postrema, nucleus tractus solitarius, and ventral tegmental area and / or endocrine pancreas separates the prototypical undesirable NMDAR-mediated neurobiological effects, such as dissociative, psychotic, and behavioral effects, from the positive metabolic effects. Undesirable neurobiological effects caused by NMDAR antagonism may include hallucinations, paranoid delusions, confusion, difficulty concentrating, agitation, mood changes, nightmares, catatonia, ataxia, sensory loss, and learning and memory impairments. The positive metabolic effects of NMDAR antagonists may include improved glucose metabolism, reduced food intake, and suppression of hyperphagia, which may be beneficial for alleviating obesity and obesity-related metabolic disorders in humans or mammals.

[0051] Therefore, the therapeutic utility of the combination of the peptide of the present invention and NMDAR antagonist provides a new approach for the treatment of obesity and its related metabolic disorders.The treatment of obesity can be achieved by administering the conjugate molecule to humans or mammals, thereby reducing food intake and food motivation, and reducing overeating episodes;Therefore, another aspect of the present invention relates to a method for reducing the body weight of mammals, comprising administering the conjugate molecule of the present invention or the pharmaceutical composition of the present invention.

[0052] In certain embodiments, the method for reducing weight involves reducing food intake in a mammal by administering to the mammal a conjugated molecule of the invention or a pharmaceutical composition of the invention.

[0053] The conjugated molecule or the pharmaceutical composition may be administered subcutaneously, orally, intramuscularly, intraperitoneally, or intravenously.

[0054] The conjugate molecule, and therefore the pharmaceutical composition, is superior in suppressing food intake compared to the prior art. Therefore, the conjugate molecule and the pharmaceutical composition can be used in the treatment of obesity at any level. Obesity can be expressed as body mass index (BMI), which is defined as weight divided by height squared, for example, kg / m 2 Without wishing to be bound by theory, the inventors believe that BMI can be used to define the limit between pathological and non-pathological obesity. For example, in the context of the present invention, a BMI of 30 kg / m 2 may be interpreted as the limit between pathological and non-pathological obesity. However, other BMI values ​​can also be considered to define the limit between pathological and non-pathological obesity. Thus, for example, a BMI value of 24 kg / m 2 , 26 kg / m 2 , 27 kg / m 2 , 28 kg / m 2 , 29 kg / m 2 , 30 kg / m 2 , 31 kg / m 2 , 32 kg / m 2 , 33 kg / m 2 , 34 kg / m 2 , and 35 kg / m 2 is believed to define the threshold between pathogenic and non-pathogenic obesity. In a further aspect, the present invention relates to a non-therapeutic treatment of a mammal for weight loss, comprising orally administering a conjugated molecule according to the present invention to said mammal. For example, the mammal may have a non-pathogenic BMI. Specifically, the method may comprise orally administering said conjugated molecule to a subject having a BMI below the threshold that defines non-pathogenic obesity.

[0055] The present invention has been described above primarily with reference to certain embodiments, however, as will be readily apparent to those skilled in the art, embodiments other than those disclosed above are equally possible within the scope of the present invention.

[0056] Other aspects and advantageous features of the present invention are illustrated in detail below by way of non-limiting examples.

[0057] In general, all terms used herein should be interpreted according to their ordinary meaning in the art and are applicable to all aspects and embodiments of the invention unless expressly defined or otherwise noted. All references to "a / an / the [conjugate, molecule, linker, peptide, etc.]" should be openly interpreted as referring to at least one instance of said conjugate, agent, molecule, linker, peptide, etc., unless expressly stated otherwise.

[0058] In the context of the present invention, the term "GLP1", "GLP-1" or "GLP1 peptide" refers to a peptide of the glucagon superfamily, in particular the incretin hormone glucagon-like peptide 1. The peptides of the present invention are considered to be food intake-regulating hormone peptides and may function as active delivery agents of the conjugated molecules of the present invention to the hypothalamus and / or pancreas.

[0059] In the context of the present invention, the term "peptide" means a compound consisting of a stretch of 10 to 60 amino acids linked by peptide bonds.

[0060] In the context of the present invention, a peptide derived from GLP-1 means a peptide having amino acid sequence identity with the natural GLP-1 peptide from which it originates, ie SEQ ID NO:1.

[0061] The term "derivative," as used herein with respect 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.

[0062] In the context of the present invention, the term "percentage identity" or "% identity" means the percentage of identical amino acids between two peptides compared, in particular using the BLAST algorithm.

[0063] As used herein, the term " NMDAR antagonist " refers to the compound that is the antagonist of NMDA receptor (NMDAR).Examples of NMDAR antagonist include but are not limited to memantine, memantine hydrochloride, amantadine, ketamine or MK801.Further examples of NMDAR antagonist include but are not limited to norketamine and neramexane. [Brief explanation of the drawings]

[0064] The above and additional 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, with reference to the accompanying drawings.

[0065] [Figure 1] FIG. 1 shows an example of a conjugate of a peptide and an NMDAR antagonist. [Figure 2] FIG. 2 shows the mechanism by which MK801 is released from the conjugate of FIG. [Figure 3] FIG. 3 shows the in vitro human plasma stability of three forms of the conjugates of FIGS. [Figure 4] FIG. 4 shows the weight-reducing effect of a conjugate of the peptide of SEQ ID NO: 1 and memantine (GLP-1 Cys40 / memantine). [Figure 5] FIG. 5 shows the effect of GLP-1 Cys40 / memantine on cumulative food intake in mice. [Figure 6] FIG. 6 shows the effect of GLP-1 Cys40 / memantine on daily food intake in mice. [Figure 7] FIG. 7 shows the effect of GLP-1 Cys40 / memantine on body composition in mice. [Figure 8] FIG. 8 shows the weight-reducing effect of a conjugate of the peptide of SEQ ID NO: 1 and MK801 (GLP-1 Cys40 / MK801). [Figure 9] FIG. 9 shows the effect of the GLP-1 Cys40 / MK801 conjugate on cumulative food intake in mice. [Figure 10] FIG. 10 shows the effect of the GLP-1 Cys40 / MK801 conjugate on daily food intake in mice. [Figure 11] FIG. 11 shows the effect of the GLP-1 Cys40 / MK801 conjugate on body composition in mice. [Figure 12] FIG. 12 shows the weight loss effect of a conjugate of the peptide of SEQ ID NO: 1 and MK801 (GLP-1 Pen40 / MK801), in which the cysteine ​​residue of SEQ ID NO: 1 is replaced with L-penicillamine. [Figure 13] FIG. 13 shows the effect of the GLP-1 Pen40 / MK801 conjugate on daily food intake in mice. [Figure 14] FIG. 14 shows the effect of the GLP-1 Pen40 / MK801 conjugate on mouse body weight. [Figure 15] FIG. 15 shows a synthetic route to chemical linker-derivatized memantine. [Figure 16] FIG. 16 shows an example of a synthetic route for the conjugation of a peptide with a small molecule bearing an amino group. [Figure 17] FIG. 17 shows a synthetic route for synthesizing chemical linker-derivatized MK801. [Figure 18] FIG. 18 shows the conjugation reaction of linker-derivatized MK801 with a peptide (a peptide having the amino acid sequence given by SEQ ID NO:1). [Figure 19] FIG. 19 shows a synthetic route for the chemical synthesis of linker-derivatized MK801. [Figure 20]FIG. 20 shows the reaction for conjugation of a peptide having the amino acid sequence of SEQ ID NO: 1 and having a Pen40 modification with linker-derivatized MK801. [Figure 21] FIG. 21 shows the effect of different doses of GLP-1 Pen40 / MK801 conjugate on the body weight of mice. [Figure 22] FIG. 22 shows the effect of different doses of GLP-1 Pen40 / MK801 conjugate on daily food intake in mice. [Figure 23] FIG. 23 shows the effect of different doses of GLP-1 Pen40 / MK801 conjugate on blood glucose in mice after compound challenge. [Figure 24] FIG. 24 shows the effect of active and inactive MK801 in the GLP-1 Pen40 / MK801 conjugate on mouse body weight. [Figure 25] FIG. 25 shows the effect of active and inactive MK801 in the GLP-1 Pen40 / MK801 conjugate on cumulative food intake in mice. [Figure 26] FIG. 26 shows the in vitro human plasma stability of active and inactive MK801 used in the conjugate GLP-1 Pen40 / MK801. [Figure 27] FIG. 27 shows the effect of GLP-1 / MK801 conjugates with different linkers on mouse body weight. [Figure 28] FIG. 28 shows the effect of GLP-1 / MK801 conjugates with different linkers on cumulative food intake in mice. [Figure 29] Figure 29 shows GLP-1 / MK801 conjugates with one type of linker. [Figure 30] Figure 30 shows GLP-1 / MK801 conjugates with one type of linker. [Figure 31] FIG. 31 shows the effect of the GLP-1 Pen40 / MK801 conjugate on sucrose intake in mice. [Figure 32]FIG. 32 shows the effect of the GLP-1 Pen40 / MK801 conjugate on blood glucose in db / db mice after a compound challenge. [Figure 33] FIG. 33 shows the effect of the co-agonist GIP / GLP-1 / MK801 conjugate on mouse body weight. [Figure 34] FIG. 34 shows the effect of the co-agonist GIP / GLP-1 / MK801 conjugate on cumulative food intake in mice. [Figure 35] Figure 35 shows the amino acid sequence alignment of the co-agonist GLP-1 / GIP of SEQ ID NO: 9 used in the drug conjugate with the GLP-1 peptide of SEQ ID NO: 1, where X1 is D-alanine, D-serine, α-aminoisobutyric acid, N-methyl-serine, glycine, or valine, and X2 is cysteine ​​(hCys40 / Cys40) or L-penicillamine (Pen40). [Figure 36] FIG. 36 shows the effect of different NDMAR antagonists conjugated to GLP-1 Pen40 on mouse body weight. [Figure 37] FIG. 37 shows the effect of different NDMAR antagonists conjugated to GLP-1 Pen40 on daily food intake in mice. [Figure 38] FIG. 38 shows the effect of different NDMAR antagonists conjugated to GLP-1 Pen40 on cumulative food intake in mice. Specific Description of the Invention

[0066] 1 shows an example of a peptide-NMDAR antagonist conjugate 100 consisting of MK801 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 comprises a disulfide group 105. The side chain 106 of the C-terminal cysteine ​​102 may optionally be 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, has a length of n=2 carbon atoms and R=hydrogen. A modification of the side chain 106, designated hCys40, has a length of n=1 carbon atom and R=methyl. Normal cysteine ​​is designated Cys40.

[0067] Figure 2 shows the mechanism by which MK801 is released from the conjugate 100 of Figure 1. The chemical linker 104, comprising a disulfide group 105, is self-immolative and can be reduced in a reducing environment (not shown), such as an intracellular environment, to generate a thiol group, separating the peptide portion of the conjugate 107 from the MK801 portion 108 of the molecule, where the liberated nucleophilic thiol 109 undergoes spontaneous intramolecular cyclization, releasing MK801 as the native, unmodified MK801 drug (the free form of MK801).

[0068] Figure 3 shows the in vitro human plasma stability of three forms of conjugate 100 from Figures 1 and 2, each with a different cysteine ​​derivative or residue. The first form, GLP-1 Pen40 / MK801, has the cysteine ​​derivative Pen40; the second form, GLP-1 hCys40 / MK801, has the cysteine ​​derivative hCys40; and the third form, GLP-1 Cys40 / MK801, has an unmodified cysteine ​​Cys40. The plasma stability of each form is shown as the percentage recovery over time. LCMS analysis (not shown) revealed that the major contribution to the degradation of the conjugate was likely due to deconjugation of MK801 via disulfide exchange in the linker. As a result, a single substitution of the C-terminal cysteine ​​102 (hCys40 / Cys40) with L-penicillamine (Pen40) dramatically increased plasma stability due to decreased accessibility of the disulfide bond due to increased steric hindrance.

[0069] 4-14 show the results of the in vivo mouse study disclosed in Example 8.

[0070] Figure 4 shows the weight-reducing effect of conjugates of memantine with the peptide of SEQ ID NO: 1 chemically attached via the linker shown in Figures 1 and 2, where the cysteine ​​residue is an unmodified cysteine ​​(GLP-1-Cys40 / memantine) (40 nmol / kg), and equimolar doses of the peptide of SEQ ID NO: 1 (GLP-1 Cys40) or memantine were administered for 8 days as measured by body weight percentage (BW%) in diet-induced idiopathic (DIO) mice. Data are presented as mean ± SEM, with an N of 8 for each group. Both GLP-1 Cys40 and GLP-1 Cys40 / memantine resulted in a reduction in BW% in DIO mice, with the latter conjugate resulting in a reduction in BW% of approximately 7% after 8 days of treatment.

[0071] Figure 5 shows the effect of GLP-1 Cys40 / memantine and equimolar doses of GLP-1 Cys40 or memantine on cumulative food intake (FI cumulative, grams per day) in DIO mice treated for 8 days. Data are expressed as mean ± SEM, N = 8 per group. Over the course of treatment, a decrease in cumulative food intake was observed in mice treated with GLP-1 Cys40 and GLP-1 Cys40 / memantine compared to control (vehicle) and memantine.

[0072] Figure 6 shows the effect of GLP-1 Cys40 / memantine (40 nmol / kg) or equimolar doses of GLP-1 Cys40 or memantine on daily food intake (FI per day, grams per day) in DIO mice treated for 8 days. Data are expressed as mean ± SEM, N = 8 per group. During the 8-day treatment, GLP-1 Cys40 and GLP Cys40 / memantine showed an overall reduction in daily food intake compared to memantine-treated mice and the control group (vehicle, i.e., saline). At the end of the study, mice treated with GLP-1 showed only a slight decrease in food intake compared to the control group (vehicle).

[0073] Figure 7 shows the effect of GLP-1 Cys40 / memantine (40 nmol / kg) or equimolar doses of GLP-1 Cys40 or memantine on body composition (in terms of change in fat and lean body mass) (Δchange, g) in DIO mice treated for 8 days. Data are expressed as mean ± SEM, N = 8 per group. After 8 days, mice treated with memantine, GLP-1 Cys40, and GLP-1 Cys40 / memantine all showed a decrease in fat mass, while lean body mass was largely unchanged. GLP-1 Cys40 / memantine produced the greatest change in fat mass, with a loss of approximately 4 g of fat mass observed in mice treated with this conjugate.

[0074] Figure 8 shows the weight loss effect (% BW) of GLP-1 Cys40 / MK801 (100 nmol / kg) or equimolar doses of GLP-1 Cys40 or MK801 in DIO mice treated for 10 days. Data are expressed as mean ± SEM, N = 8 per group. MK801 showed little change in body weight (BW), whereas both GLP-1 Cys40 and GLP-1 Cys40 / MK801 reduced BW by approximately 8 and 12%, respectively, after 10 days of treatment.

[0075] Figure 9 shows the effect of GLP-1 Cys40 / MK801 (100 nmol / kg) or equimolar doses of GLP-1 Cys40 or MK801 on cumulative food intake (FI cumulative value) in DIO mice treated for 10 days. Data are expressed as mean ± SEM, with N = 8 per group. Over the course of 10 days of treatment, a decrease in cumulative food intake was observed in mice treated with GLP-1 Cys40 and GLP-1 Cys40 / MK801 compared to control (vehicle) and MK801. The best results were observed in GLP-1 Cys40 / MK801-treated mice, with cumulative food intake of approximately 13 g / day, approximately 10 g / day less than vehicle-treated mice (approximately 23 g / day).

[0076] Figure 10 shows the effect of GLP-1 Cys40 / MK801 (100 nmol / kg) or equimolar doses of GLP-1 Cys40 or MK801 on daily food intake (daily FI) in DIO mice treated for 10 days. Data are expressed as mean ± SEM, N = 8 per group. Overall, daily food intake fluctuated to various degrees over the 10 days of treatment, but a decrease in food intake was observed in mice treated with GLP-1 Cys40 / MK801 compared to the control group (vehicle) for all 10 days.

[0077] Figure 11 shows the effect of GLP-1 Cys40 / MK801 (100 nmol / kg) or equimolar doses of GLP-1 Cys40 or MK801 on body composition (in terms of change in fat and lean body mass) (Δchange, g) in DIO mice treated for 10 days. Data are expressed as mean ± SEM, N = 8 per group. After 10 days of treatment, the GLP-1 Cys40 / MK801-treated group of mice showed a decrease in both fat and lean body mass, with the change in body fat mass (a decrease of approximately 5 g) being the most significant.

[0078] Figure 12 shows the effect of GLP-1 Pen40 / MK801 (100 nmol / kg) or equimolar doses of GLP-1 Cys40 or MK801 on body weight percent in DIO mice treated for 5 days. Data are expressed as mean ± SEM, N = 8 per group. After 5 days of treatment, GLP-1 Pen40 / MK801-treated mice showed approximately 15% body weight loss. In comparison, GLP-1 Cys40-treated mice showed approximately 4% body weight loss.

[0079] Figure 13 shows the effect of GLP-1 Pen40 / MK801 (100 nmol / kg) or equimolar doses of GLP-1 Cys40 or MK801 on food intake (g / day) in DIO mice treated for 5 days. Data are expressed as mean ± SEM, N = 8 per group. Mice treated with GLP-1 Pen40 / MK801 showed an immediate decrease in food intake compared to the control group (vehicle-treated mice). Furthermore, the decrease in food intake was maintained at approximately 0.2-0.7 g / day throughout the 5-day treatment period.

[0080] Figure 14 shows the effect of GLP-1 Pen40 / MK801 (100 nmol / kg) or equimolar doses of GLP-1 Pen40 or GLP-1 Cys40 on body weight percent in DIO mice treated for 5 days. Data are expressed as mean ± SEM, N = 7 per group. Mice treated with GLP-1 Pen40 or GLP-1 Cys40 showed a similar decrease in body weight percent (approximately 6%), with GLP-1 Pen40 / MK801 showing an approximately 12% decrease in body weight. In addition, if treatment were extended, further weight loss would appear to be expected with GLP-1 Pen40 / MK801 based on the slope of the curve.

[0081] Figures 21 and 22 show the effects of different doses of the GLP-1 Pen40 / MK801 conjugate (50 nmol / kg and 100 nmol / kg) on ​​body weight (BW%, Figure 21) and daily food intake (FI in grams per day, Figure 22) of DIO mice treated for 5 days, compared with the control group (vehicle, i.e., saline). Data are expressed as mean ± SEM, with N = 5–6 per group. Over the course of treatment, a decrease in body weight and daily food intake was observed in mice treated with both doses (50 nmol / kg and 100 nmol / kg) compared with the control group, with the most significant decrease observed in mice receiving daily subcutaneous injections of 100 nmol / kg of the conjugate.

[0082] Figure 23 shows the effect of different doses of the GLP-1 Pen40 / MK801 conjugate (50 nmol / kg and 100 nmol / kg) on ​​blood glucose levels (mmol / L) in DIO mice undergoing an ipGTT on day 7 of the treatment course, compared to the control group (vehicle, i.e., saline). Blood glucose levels were measured over a 120-minute period. Data are presented as mean ± SEM, with N = 5–6 per group. Overall, both doses of the conjugate, i.e., 50 nmol / kg and 100 nmol / kg, result in a significantly lower initial rise and overall lower blood glucose levels compared to the control group.

[0083] Figures 24 and 25 show the effects of active and inactive MK801 conjugated to GLP-1 Pen40 on body weight (ΔBW in %), Figure 24) and cumulative food intake (cumulative FI in grams, Figure 25) of DIO mice treated for 7 days compared with the control group (vehicle, i.e., saline). Data are expressed as mean ± SEM, with N = 8 per group. Over the course of treatment, a decrease in body weight and cumulative food intake was observed in mice treated with GLP-1 Pen40 conjugated to active MK801. Conjugates with inactive MK801 showed similar results to unconjugated GLP-1 Pen40. It is concluded that MK801 and GLP-1 have a synergistic effect in reducing body weight and cumulative food intake in mice.

[0084] Figure 26 shows the in vitro human plasma stability of the active and inactive MK801 forms of the conjugate GLP-1 Pen40 / MK801 compared to a PBS control. The plasma stability of inactive and active MK801 is shown as percentage recovery (%) (hours) over time. The two conjugates show nearly identical plasma stability, regardless of whether the MK801 is active or inactive.

[0085] Figures 27 and 28 show the effects of GLP-1 / MK801 conjugates (100 nmol / kg) with different linkers compared to the control group (vehicle, i.e., saline) on body weight (BW in %) and cumulative food intake (cumulative FI in grams) of DIO mice over 7 days. Data are presented as mean ± SEM, with N = 5–6 per group. The structures of GLP-1 / MK801 conjugates with different linkers are shown in Figure 20 (GLP-1 Pen40 / MK801), Figure 29 (GLP-1 Lys40-triazole-PEG4-Val-Cit-PAB-MK801), and Figure 30 (GLP-1 Cys40-mc-Val-Cit-PAB-MK801). Over the course of treatment, mice treated with GLP-1 / MK801 conjugates with different linkers showed similar decreases in cumulative food intake. The most significant weight loss (approximately 20% decrease) was observed in the group of mice treated with the GLP-1 Pen40 / MK801 conjugate at 7 days of treatment.

[0086] Figure 31 shows the effects of GLP-1 Pen40 / MK801 (100 nmol / kg) and equimolar doses of GLP-1 Pen40, MK801, or semaglutide on sucrose intake (% %) in DIO mice treated for 8 days, compared with the control group (vehicle, saline injection). Data are expressed as mean ± SEM, N = 8 per group. The most significant reduction in sucrose intake, expressed compared with the control group (vehicle), was observed in mice treated with semaglutide and the GLP-1 / MK801 conjugate. It was concluded that the conjugate molecules of the present invention are effective in inducing food reward and satiety effects in treated mice.

[0087] Figure 32 shows the effect of the GLP-1 Pen40 / MK801 conjugate (100 nmol / kg) and equimolar doses of MK801 or semaglutide on blood glucose (mmol / L) in db / db (diabetic) mice undergoing an ipGTT on day 7 of the treatment course. Blood glucose levels were measured over 24 hours. Data are expressed as mean ± SEM, N=8 per group. Mice treated with either semaglutide or conjugated GLP-1 Pen40 / MK801 showed overall lower blood glucose levels compared to the control group (vehicle), and it was concluded that the conjugated molecules of the invention are suitable for treating diabetic mice.

[0088] Figures 33 and 34 show the effects of the co-agonist GIP / GLP-1 Pen40 / MK801 conjugate (SEQ ID NO: 9) (50 nmol / kg) and an equimolar dose of GLP-1 / GIP on body weight (in %, Figure 33) and cumulative food intake (cumulative FI in grams, Figure 34) of DIO mice treated for 7 days, compared with the control group (vehicle, i.e., saline injection). Data are expressed as mean ± SEM, N = 8 per group. The most significant effect was observed in mice treated with the GIP / GLP-1 / MK801 conjugate, which showed an overall decrease in body weight of approximately 25% compared to the control group, and a cumulative food intake of approximately 3 g compared to the 15 g observed in the control group.

[0089] Figures 36-38 show the effects of different NDMAR antagonists conjugated with GLP-1 Pen40 (100 nmol / kg), i.e., MK-801, memantine, and neramexane, on body weight (in %), daily food intake (food intake, grams per day, Figure 37), and cumulative food intake (cumulative FI in grams, Figure 38) of DIO mice treated for 5 days, compared with the control group (vehicle, i.e., saline). Data are expressed as mean ± SEM, with N = 8 per group. Over the course of treatment, all mice treated with GLP-1 Pen40 conjugated with either MK801, memantine, or neramexane showed significant decreases in body weight, daily food intake, and cumulative food intake compared with the control group. We conclude that different NMDAR antagonists can be conjugated to the peptides of the present invention to achieve the same beneficial effects on mouse body weight and food intake.

[0090] conclusion The data presented demonstrate that chemical conjugation of a GLP-1 analogue with an NMDAR antagonist represents a novel therapeutic strategy for effectively reversing obesity. Conjugates based on this strategy are superior to GLP-1 peptide controls in suppressing food intake and reducing body weight, without the negative central effects of NMDAR antagonism. [Example]

[0091] Example 1: Preparation of peptides and peptide-NMDAR antagonist conjugates materialAll solvents and reagents were purchased from commercial sources 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 The -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-pentamethylcoman-6-sulfonyl, O t Bu = tert-butyl ester, Trt = trityl, Boc = tert-butyloxycarbonyl, and t Bu = tert-butyl ether).

[0092] All peptides and peptide-NMDAR antagonist conjugates were characterized by analytical reversed-phase ultra-performance liquid chromatography (RP-UPLC) (Waters) coupled to an Agilent 6410 triple quadrupole mass filter using a C18 column (Zorbax Eclipse, XBD-C18, 4.6 × 50 mm) and electrospray ionization liquid chromatography-mass spectrometry (ESI-LCMS). ESI-LCMS 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 determined by RP-UPLC equipped with a C18 column (Acquity UPLC BEH C18, 1.7 μm, 2.1 × 50 mm) and 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.

[0093] Automated Peptide Synthesis Protocol for Fmoc Protection Schemes Peptides were prepared as C-terminally 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 procedures using the following protocol: deprotection (2 × 2 min, room temperature, 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 AA / HCTU / DIPEA (ratio 1:1.25:2.5) in a 5-fold excess relative to the resin.

[0094] Peptide cleavage The synthesized 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, followed by 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.

[0095] purificationCrude peptides or peptide-NMDAR antagonist conjugates were analyzed by RP-UPLC and ESI-LCMS or MALDI-TOF mass spectrometry before purification. Purification was performed on a reversed-phase high-performance liquid chromatography (RP-HPLC) system (Waters) equipped with a reversed-phase C18 column (Zorbax, 300 SB-C18, 21.2 x 250 mm) 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 determined by RP-UPLC at 214 nm. Fractions with purity >95% were pooled and lyophilized. The final lyophilized product was used for further experiments.

[0096] Conjugation Protocol for Assembly of Peptide and NMDAR Antagonist Conjugates The pure peptide and pure thiopyridyl-activated NMDAR antagonist conjugate were dissolved in a binary solvent system (A:DMF in HO; 6 M guanidine, 1.5 M imidazole, 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.

[0097] desalination All peptides were desalted before biological experiments. Desalting was performed by sequentially redissolving the peptide or peptide-NMDAR antagonist conjugate in 0.01 M dilute aqueous HCl followed by lyophilization, which was repeated three times. The purity of the peptide or conjugate was monitored by RP-UPLC and ESI-LCMS until use in in vivo or in vitro experiments.

[0098] Preparation of GLP-1 Cys40 / memantine (cysteine-linked) 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 via the synthetic pathway shown in Figure 15. The first step of the synthetic pathway was carried out in MeOH 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 a buffer of 6 M guanidine, 1.5 M imidazole at room temperature for 2 hours.

[0099] 2'-pyridyldithioethanol In a dry round-bottom flask equipped with a magnetic stir bar and under a N2 atmosphere, 2'-aldrithiol (4.71 g, 21.3 mmol, 3 equiv.) was dissolved in dry MeOH (20 mL), followed by the dropwise addition of 2-mercaptoethanol (0.56 g, 7.1 mmol, 0.5 mL, 1 equiv.) via syringe. The reaction was left at ambient temperature for 2 h and then concentrated in vacuo. The crude yellow oil was purified by silica gel flash chromatography (EtOAc:CHCl, 2:8) to give 2'-pyridyldithioethanol as a clear oil (1.33 g, 100%). 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.

[0100] 4-Nitrophenyl(2-(pyridin-2-yldisulfanyl)ethyl)carbonateIn a dry round-bottom flask equipped with a magnetic stir bar and 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 ambient 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: EtOAc, 2:1) to give 4-nitrophenyl (2-(pyridin-2-yldisulfanyl) ethyl) carbonate as a clear viscous oil (2.21 g, 89%). f =0.34; Purity >95% (HPLC), R t= 15.99 minutes; UPLC / MS(ESI):C 14 H 12 N2O5S2[M+H] + Calculated m / z for = 353.0, measured m / z 353.3; 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.

[0101] 2-(Pyridin-2-yldisulfanyl)ethyl (3,5-dimethyladamantan-1-yl)carbamateIn a dry round-bottom flask equipped with a magnetic stir bar under N2, 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 subsequently heated to 80 °C. The reaction was then transferred to a separatory funnel with EtOAc (50 mL) and washed thoroughly with 5× half-saturated brine (50 mL) and brine (50 mL) to remove the 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 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):C 20 H 28 N2O2S2[M+H] + Calculated m / z for = 393.2, measured m / z 393.4; 1 H 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); 13C 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.

[0102] GLP-1 Cys40 and GLP-1 Cys40 / memantine were prepared using the protocols described above, and purity was determined to be >95% by RP-UPLC and ESI-LCMS analysis.

[0103] Preparation of GLP-1 Pen40 / Memantine (Penicillamine Linked) . 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 a buffer of 6 M guanidine, 1.5 M imidazole at room temperature for 2 hours.

[0104] Preparation of GLP-1 Cys40 / MK801 (cysteine-linked) . A peptide having the sequence of SEQ ID NO: 1 was synthesized using the Fmoc protocol disclosed above and conjugated with a chemical linker-derivatized MK801 analog. The synthesis of chemical linker-derivatized MK801 was carried out 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.

[0105] Linker-derivatized MK801 was conjugated to GLP-1 Cys40 using the chemistry shown in Figure 18. The reaction was carried out in a buffer of 6 M guanidine, 1.5 M imidazole at room temperature for 2 hours.

[0106] 2-(Pyridin-3-yldisulfanyl)ethyl 5-methyl-10,11-dihydro-5H-5,10-epiminodibenzo[a,d][7]annulene-12-carboxylateIn a flame-dried round-bottom flask equipped with a magnetic stir bar and under a N 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 showed complete consumption of the starting material. The reaction was diluted with EtOAc (50 mL) and washed thoroughly with half-saturated brine (5 × 60 mL), 0.5 M aqueous NaOH (5 × 60 mL), and brine. The organic layer was collected, dried over MgSO, filtered, and concentrated in vacuo. Purification by preparative HPLC (isocratic elution at 60% B over 17 mL / min) followed by lyophilization afforded 11 as a clear solid (250.2 mg, 80.1%); purity >95% (HPLC), R t= 18.17 minutes; UPLC / MS(ESI):C 24 H 22 N2O2S2[M+H] + m / z calculated for = 435.1, measured 435.4; 1 H 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); 13C 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.

[0107] 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-12-carboxylate and GLP-1 Cys40 using the protocol disclosed above. RP-UPLC and ESI-LCMS analyses confirmed the product, and the purity was determined to be >95%.

[0108] Preparation of GLP-1 hCys40 / MK801 (homocysteine-linked) A peptide having the amino acid sequence of SEQ ID NO:1 and containing an hCys40 modification was synthesized using the Fmoc protocol disclosed above and conjugated to a chemical linker-derivatized MK801 analog. Chemical synthesis of the linker-derivatized MK801 was performed via the synthetic pathway shown in Figure 19. The chemical reaction was carried out in a buffer of 6 M guanidine, 1.5 M imidazole at room temperature for 2 hours.

[0109] GLP-1 hCys40: A peptide having the amino acid sequence of SEQ ID NO: 1 and having 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.

[0110] Preparation of GLP-1 Pen40 / MK801 (penicillamine-linked) 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 via the pathway disclosed in Figure 16.

[0111] GLP-1 Pen40 / MK801: This conjugate was prepared using the protocol disclosed above according to the chemistry shown in Figure 20, which was carried out in a buffer of 6 M guanidine, 1.5 M imidazole for 2 hours at room temperature. Purity was determined to be >95% by RP-UPLC and ESI-LCMS analysis.

[0112] Example 2: In vitro human plasma stability study In vitro human plasma stability assay: Peptide stability was determined using normal human plasma containing citrate phosphate dextrose (3H Biomedical, lot P22). Human plasma was prewarmed at 37°C for 15 minutes. Subsequently, 40 μL of GLP-1 Pen40 / MK801, GLP-1 hCys40 / MK801, or GLP-1 Cys40 / MK801 conjugate stock solution (1 mM, prepared by diluting 10 mM peptide in DMSO stock with PBS buffer) was added to 360 μL of human plasma and incubated at 37°C with gentle shaking. Aliquots of 45 μL were collected at t = 0 and five additional time points (depending on the conjugate stability) and pretreated with urea buffer (50 μL, 30 min) at 0°C, followed by treatment with 20% trichloroacetic acid in acetone and incubation at -20°C overnight. After centrifugation (13400 rpm, 30 min), the supernatant was filtered and analyzed by RP-UPLC and ESI-LCMS at 214 nm. The area under the curve (AUC) was determined and plotted using prism 8.0. The half-life (T 1 / 2 ) was determined by fitting the data to a one-phase decay equation. Data are presented as the mean of three independent experiments.

[0113] Example 3: In vivo pharmacological studies in diet-induced obese (DIO) mice C57BL6J male mice (hereafter referred to as diet-induced obese (DIO) mice) were maintained on a high-fat diet (58% energy from fat) and had an average body weight of over 45 grams before the start of each study. Mice were housed individually or double-housed. Mice were maintained at 21-23°C with a 12-h light / dark cycle. Compounds were administered subcutaneously once daily (2:00 PM-5:00 PM), and food intake (FI) and body weight (BW) were measured at the corresponding times. Body composition was measured using an MRI scanner (EchoMRI) before the study (1-3 days before the start of the study) and on the final day of the study. A group of mice injected with vehicle (saline) served as the control group.

[0114] Example 4: Sucrose preference test in normal-fed mice C57BL6J male mice were housed singly in cages and maintained on a normal diet. All compounds were administered subcutaneously at a dose of 100 nmol / kg once daily, except for semaglutide, which was administered at a dose of 10 nmol / kg. A group of mice injected with vehicle (saline) served as the control group. Eight mice were included in each treatment group. Two drinking bottles were provided in all cages, and mice were allowed to acclimate for a minimum of 5 days before the start of the study. At the start of the study, the water bottles were replaced with one bottle containing water and one bottle containing a 10% (w / v) sucrose solution in water. To correct for laterality, the sucrose bottles were distributed equally between the left and right bottles. Sucrose water intake and water intake were measured by weighing the bottles after 24 hours.

Claims

1. A conjugate molecule comprising a peptide having at least 90% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 9, and exhibiting at least 0.1% of the activity of native glucagon-like peptide 1 (GLP-1) at the GLP-1 receptor, and an N-methyl-D-aspartate receptor (NMDAR) antagonist, wherein the peptide is covalently bonded to the NMDAR antagonist directly or via a chemical linker.

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

3. 3. The conjugate molecule of claim 1 or 2, wherein the NMDAR antagonist is selected from memantine, memantine hydrochloride, amantadine, ketamine, phencyclidine (PCP), MK801, norketamine, and neramexane.

4. The conjugate molecule of any one of claims 1 to 3, wherein the peptide has SEQ ID NO: 1 or SEQ ID NO:

9.

5. The conjugate molecule of any one of claims 1 to 4, wherein the NMDAR antagonist is covalently attached at the C-terminal region of the peptide.

6. 6. The conjugate molecule of any one of claims 1 to 5, wherein the NMDAR antagonist is covalently attached to the peptide via a cleavable chemical linker, the cleavable chemical linker being selected from an acid-cleavable linker, an enzyme-cleavable linker, a peptide-cleavable linker, and a linker comprising a disulfide group.

7. The chemical linker has the formula R １ -R ３ -S-S-R ４ -R ５ -O-CO-R ２ wherein R １ is the peptide, and R ２ is the NMDAR antagonist, and R ３ is optional and, if present, is C(CH ３ ) ２ , C.H. ２ -CH ２ , or C.H. ２ and attached to a side chain of the peptide or to a carbon atom in the backbone of the peptide; ４ (CH ２ ) ｎ or C ６ H ４ and R ５ is optional and, if present, is C(CH ３ ) ２ , C.H. ２ -CH ２ , or C.H. ２ and n is 1, 2, 3 or 4.

8. The conjugate molecule of any one of claims 1 to 7, wherein the NMDAR antagonist is MK801, neramexane, or memantine.

9. A pharmaceutical composition comprising the conjugate molecule of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

10. 10. The pharmaceutical composition of claim 9 for treating obesity, binge eating disorder, insulin resistance, type 2 diabetes, dyslipidemia, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease.

11. The pharmaceutical composition according to claim 9 for reducing body weight.

12. A composition for reducing the body weight of a mammal, comprising a conjugated molecule according to any one of claims 1 to 8, for oral administration.

13. 13. The composition of claim 12, wherein the mammal has a non-pathogenic body mass index (BMI).

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