WO2023118263A1

WO2023118263A1 - Agonists for treatment of eating disorder

Info

Publication number: WO2023118263A1
Application number: PCT/EP2022/087180
Authority: WO
Inventors: Jeppe Hvidtfeldt EKBERG; Dan Ploug CHRISTENSEN; Jakob Bondo HANSEN
Original assignee: Embark Biotech Aps
Priority date: 2021-12-21
Filing date: 2022-12-21
Publication date: 2023-06-29

Abstract

The present invention relates to neurokinin receptor (NKR) agonists for use in treatment of an eating disorder, such as binge eating disorder.

Description

Agonists for treatment of eating disorder

Technical field

The present invention relates to neurokinin receptor 2 (NK2R) agonists for use in treatment of an eating disorder, such as binge eating disorder.

Background

Feeding and Eating Disorders involve abnormal eating or feeding behaviours that are not explained by another health condition and are not developmentally appropriate or culturally sanctioned. Feeding disorders involve behavioural disturbances that are not related to body weight and shape concerns, such as eating of non-edible substances or voluntary regurgitation of foods. Eating disorders involve abnormal eating behaviour and preoccupation with food as well as prominent body weight and shape concerns.

Binge eating disorder is characterised by frequent, recurrent episodes of binge eating (e.g. once a week or more over a period of several months). A binge eating episode is a distinct period of time during which the individual experiences a subjective loss of control over eating, eating notably more or differently than usual, and feels unable to stop eating or limit the type or amount of food eaten. Binge eating is experienced as very distressing, and is often accompanied by negative emotions such as guilt or disgust. However, unlike in Bulimia Nervosa, binge eating episodes are not regularly followed by inappropriate compensatory behaviours aimed at preventing weight gain (e.g. self-induced vomiting, misuse of laxatives or enemas, strenuous exercise). There is marked distress about the pattern of binge eating or significant impairment in personal, family, social, educational, occupational or other important areas of functioning.

Lisdexamfetamine (Vyvanse) is approved by the FDA to treat binge eating disorder. The drug, which is also used to treat ADHD, helps reduce the number of episodes and is the first FDA-approved medication to treat binge eating disorder. The most common side effects are dry mouth, trouble sleeping, increased heart rate, and jittery feelings. But it also has some risk of serious problems, like psychiatric disturbances, heart attack, and stroke. The anti-seizure drug topiramate (Topamax) may also help, but its side effects can include memory problems, tingling sensations in fingers and toes, trouble speaking, and sedation. Even off-label use of unapproved drugs, such as dasotraline for treatment of binge eating disorder has been reported.

Thus, the current treatment options are sparse and there is a need in the art for better options to treat subjects suffering from an eating disorder, such as binge eating disorder.

Summary

The present inventors have surprisingly found that agonists of the tachykinin receptor display promising effects toward treatment of an eating disorder, such as binge eating disorder. In particular, agonists of the tachykinin 2 receptor are effective in treatment of an eating disorder, such as binge eating disorder.

Thus, in a first aspect a neurokinin receptor (NKR) agonist is provided for use in the treatment of an eating disorder, such as binge eating disorder.

In a second aspect, a NKR agonist is provided for use in the treatment of an eating disorder, such as binge eating disorder, wherein the NKR agonist is of formula (I), (A)-(B) (I), wherein;

(A) is a peptide comprising an amino acid sequence of the general formula X1X2X3X4X5X6X7, wherein

Xi is selected from the group consisting of: aspartic acid (D) and glutamic acid (E) ;

X₂ is selected from the group consisting of: lysine (K), arginine (R), and histidine (H) ;

X₃ is selected from the group consisting of: tyrosine (Y), phenylalanine (F), meta- tyrosine (m-Y), valine (V), tryptophan (W), methionine (M), leucine (L), isoleucine (I), and alanine (A);

X₄ is selected from the group consisting of: valine (V), threonine (T), serine (S), asparagine (N), glutamine (Q), glycine (G), and alanine (A);

X₅ is selected from the group consisting of: glycine (G), 2-aminoisobutyric acid (Aib), serine (S), alanine (A), valine (V), leuicine (L), beta-alanine (bA) and isoleucine (i); X₆ is selected from the group consisting of: leucine (L), isoleucine (I), alanine (A) and N-methyl leucine (Me-Leu); and

X? is selected from the group consisting of: norleucine (Nle), methoxinine (Mox), methionine (M), 4-fluorophenylalanine (4fF), and 4-methoxyphenylalanine (4MeOF); and

(B) is a conjugated moiety of the general formula (II) Fa-Lg (II), wherein;

Fa is a C10-C20 fatty acid, optionally substituted with one or more carboxylic acid groups,

Lg is a linking group, which covalently links (B) to the peptide (A), and wherein (B) is covalently linked to a terminal amino acid or to a non-terminal amino acid.

In a third aspect, a NKR agonist is provided for use in the treatment of an eating disorder, such as binge eating disorder, wherein the NKR agonist is of formula (I), (A)-(B) (I), wherein;

Xi is selected from the group consisting of: aspartic acid (D) and glutamic acid (E); X₂ is selected from the group consisting of: lysine (K), arginine (R), and histidine (H);

X₅ is selected from the group consisting of: glycine (G), 2-aminoisobutyric acid (Aib), serine (S), alanine (A), valine (V), leuicine (L), beta-alanine (bA) and isoleucine (I);

X₆ is selected from the group consisting of: leucine (L), isoleucine (I), alanine (A) and N-methyl leucine (Me-Leu); and X? is selected from the group consisting of: norleucine (Nle), methoximne (Mox), methionine (M), 4-fluorophenylalanine (4fF), and 4-methoxyphenylalanine (4MeOF); and

(B) is a conjugated moiety of the general formula (II) Fa-Lg (II), wherein;

Fa is of formula (Fa-1 ),

wherein n is from 14 to 17, preferably wherein n is 15; and wherein X is selected from the group consisting of -OH, -OC1-6, -NH₂, -NHC1-6, and N(CI-₆)2, and

Lg is a linking group of formula (Lg-1 ),

wherein Z is a chain comprising from 18 to 23 atoms in the backbone selected from the group consisting of: C, O, and N; and wherein R is selected from the group consisting of H, and C1-6 alkyl; and Lg covalently links (B) to the peptide (A), and wherein (B) is covalently linked to a terminal or non-terminal amino acid, preferably a terminal amino acid.

Description of Drawings

Fig. 1 : Compound 344 dose-dependently reduces binge eating of palatable foods.

Male wild-type mice, trained to binge eat on palatable foods, were treated with various doses of EB-344 or vehicle. Graph show means ±SEMs, one-way ANOVA with Dunnett’s multiple comparisons test, ****p<0.0001 . Detailed description

Definitions

To facilitate the understanding of the following description, a number of definitions are presented in the following paragraphs.

The term "alkyl" whether used alone or as part of a substituent group, refers to straight and branched carbon chains having 1 to 8 carbon atoms, such as 1 to 6 carbon atoms. Therefore, designated numbers of carbon atoms (e.g., Ci-s) refer independently to the number of carbon atoms in an alkyl moiety or to the alkyl portion of a larger alkyl- containing substituent. In substituent groups with multiple alkyl groups such as, (Ci- 6alkyl)₂amino-, the Ci -ealkyl groups of the dialkylamino may be the same or different. Alkyl, as defined herein may be substituted by one or more substituents such as a halogen or one or more halogens. In one embodiment, an alkyl is substituted by 1 ,2 or 3 fluorine atoms. In one embodiment, an alkyl is substituted by a carboxy group (CO2), such as a carboxy methyl (CC^Me).

It is understood that substituents and substitution patterns on the NKR agonists of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth herein.

The term "subject" refers to an animal, preferably a mammal, and most preferably a human.

Proteinogenic “amino acids” (AA) are named herein using either their 1 -letter or 3-letter code according to the recommendations from IIIPAC, see for example http://www.chem.qmul.ac.uk/iupac/AminoAcid/. Capital letter abbreviations indicate L- amino acids, whereas lower case letter abbreviations indicate D-amino acids.

A series of non-proteinogenic amino acids are referred to herein. The names employed should be clear and understandable to the skilled person. meta-Tyrosine (m-Y) is 3- hydroxyphenylalanine. The structure of methoxinine (Mox) is shown below:

The amino acid, beta-alanine (bA) as used herein is also known as 3-aminopropanoic acid. The amino acid, A/-methyl-Leucine is referred to as (NmLeu) herein.

A “terminal fatty acid” is a fatty acid wherein the carboxylic acid group is localized on a terminal carbon atom of the fatty chain. A “terminal C16-C20 fatty acid” is thus a fatty acid chain consisting of 16 to 20 carbon atoms, wherein the acid group is located terminally and the carbon atom of the carboxylic acid group is a terminal chain carbon.

Eating disorders

In one embodiment, NKR agonists are provided for use in treatment of eating disorders.

In one embodiment, the eating disorder is selected from the group consisting of: binge eating disorder (BED), Prader-Willi syndrome (PWS), bulimia nervousa, hyperphagia and night eating syndrome.

In one embodiment, NKR agonists are provided for use in treatment of binge eating disorder.

In one embodiment, the eating disorder is a disease associated with binge-eating.

In some aspects, the disease associated with binge-eating is selected from the group consisting of: binge eating disorder, bulimia nervousa, night eating syndrome, type-1 diabetes, type-2 diabetes, frontotemporal dementia, Kleine-Levin syndrome, and Prader-Willi syndrome.

In one embodiment, NKR agonists are provided for use in treatment of eating disorders in a subject suffering from a comorbidity. In one embodiment, the eating disorder is due to the comorbidity. In one embodiment, the eating disorder is binge eating or hyperphagia. In one embodiment, the eating disorder is binge eating in a subject suffering from binge eating disorder, bulimia nervousa, night eating syndrome, type-1 diabetes, type-2 diabetes, frontotemporal dementia, Kleine-Levin syndrome, and Prader-Willi syndrome.

In one embodiment, the eating disorder is hyperphagia in a subject suffering from binge eating disorder, bulimia nervousa, night eating syndrome, type-1 diabetes, type-2 diabetes, frontotemporal dementia, Kleine-Levin syndrome, and Prader-Willi syndrome.

In one embodiment, the comorbidity is Prader-Willi syndrome. Hyperphagia is a hallmark pf Prader-Willi syndrome. In one specific embodiment, the eating disorder is binge eating in a subject suffering from Prader-Willi syndrome. In one specific embodiment, the eating disorder is hyperphagia in a subject suffering from Prader-Willi syndrome.

In one embodiment, the eating disorder is binge eating in a subject diagnosed with a disease selected from the group consisting of binge eating disorder, bulimia nervousa, night eating syndrome, type-1 diabetes, type-2 diabetes, frontotemporal dementia, Kleine-Levin syndrome, and Prader-Willi syndrome.

In one embodiment, the eating disorder is hyperphagia in a subject diagnosed with a disease selected from the group consisting of binge eating disorder, bulimia nervousa, night eating syndrome, type-1 diabetes, type-2 diabetes, frontotemporal dementia, Kleine-Levin syndrome, and Prader-Willi syndrome.

In one embodiment, the eating disorder is binge eating in a subject with Prader-Willi syndrome. In one embodiment, the eating disorder is hyperphagia in a subject with Prader-Willi syndrome.

Binoe eatino disorder

As demonstrated in example 3 and 4 , NKR agonists, such as compound 344, 369 and 383, can be used to effectively treat eating disorders, such as binge eating disorder.

In one embodiment, a neurokinin receptor (NKR) agonist is provided for use in the treatment of an eating disorder. In some embodiments, the eating disorder is binge eating disorder. Preferably, the NKR agonist provided is a neurokinin receptor 2 (NK2R) agonist used in the treatment of binge eating disorder.

Binge eating disorder (BED) is a life-threatening psychologic disorder with frequent defined periods of overeating without regularly using unhealthy compensatory measures (e.g., purging) to counter the binge eating. The prevalence of BED is approximately 2% of the adult population with even distribution between men and women, and is associated with depression, abuse, obesity, diabetes etc. Overeating occurs in defined periods, on average of 2 hours at least once weekly for more than three months, by rapid consumption of large amounts of food until feeling uncomfortably full. Bingeing often occurs through consumption of palatable foods, but is not restricted to, and is commonly associated with a feeling of loss-of-control and often shame.

According to the ICD-11 , Binge eating is an episode in which an individual eats notably more than usual and feels that she or he is unable to stop or limit the amount or type of food eaten.

Recurrent episodes of binge eating: An episode of binge eating is characterized by either or both of the following:

• Eating, in a discrete period of time (e.g., within any 2-hour period), an amount of food that is definitely larger than what most people would eat in a similar period of time under similar circumstances, such as an amount of food equivalent to about 2000 kcal or more.

• A sense of lack of control over eating during the episode (e.g., a feeling that one cannot stop eating or control what or how much one is eating).

The binge eating episodes are typically associated with three (or more) of the following:

• Eating much more rapidly than normal.

• Eating until feeling uncomfortably full.

• Eating large amounts of food when not feeling physically hungry.

• Eating alone because of feeling embarrassed by how much one is eating.

• Feeling disgusted with oneself, depressed, or very guilty afterward.

In one embodiment, a neurokinin receptor (NKR) agonist is provided for use in the treatment of hyperphagia.

Hyperphagia is defined in ICD-1 1 as the intermittent or persistent increased motivation or desire to eat food as compared to what is typical for the individual. Hyperphagia is also referred to as Increased Appetite. It is an abnormal condition of intense hunger and excessive eating.

The family of Tachykinin neuropeptides and receptors is a highly conserved family consisting of three G protein-coupled receptors: Tachykinin receptor (Tacr) 1 , Tacr2 and Tacr3, also known as Neurokinin receptor 1 -3, (NK1 -3R). The endogenous ligands for tachykinin receptors are the neuropeptides Substance P (SP) being the preferred ligand for NK1 R, Neurokinin A (NKA) the preferred ligand for NK2R, both transcribed from the Tac1 gene, and Neurokinin B, expressed from the Tac2 gene, is the preferred ligand for NK3R. Neither of the endogenous ligands are specific for their receptor and each peptide can, thus, cross-activate all members of the Tachykinin receptor family with a potency close to its preferred receptor. Upon activation, the Tachykinin receptors preferentially couples to Gq, generating an intracellular IP3 signaling response. All receptors can, however, also couple to Gs and induce cAMP accumulation, although with lower potency than Gq-activation (PMID: 9357319). Tachykinins have been proposed to control various aspects of metabolism and behaviour, predominantly investigated in lower organisms and rodents. In drosophila, the NK2R has previously been shown to regulate fat oxidation through a central mechanism, while Tac1 controls circadian rhythm feeding behaviour and associates with stress-induced anhedonia behaviour in mice.

Agonist-induced G-protein coupled receptor (GPCR) activation can be measured by an lnositol-1 ,4,5-Trisphosphate [³H] Radioreceptor Assay (IP₃ Assay) as described in Example 1 . The IP₃ assay takes advantage of the tachykinin receptors’ ability to induce production of the inositol trisphosphate (IP3) second messenger upon agonist (ligand) binding on receptor expressing cells following an initial ³H- inositol labelling period. In effect this means that production of the second messenger IP3 as a measure of receptor activity can be assessed by counting ³H-activity. An “NK2R agonist” may possess varying degrees of selectivity relative to activity at the NK1 receptor and/or NK3 receptor as measured in biological assays, such as the IP3 assay presented herein. A “selective NK2R agonist” is herein defined as a ligand that binds to or activates the NK2 receptor with at least about 10 times or greater potency than it binds to or activates the NK1 and/or NK3 receptors. It is not necessary that a molecule be considered selective in both binding and functional (activation) assays to be a selective NK2R agonist. Binding potency is routinely reported as the EC50, with a lower EC50 value equating with greater potency. Thus, a selective NK2R agonist possesses an NK2R binding EC50 that is at least about 10 times or more lower than its NK1 and/or NK3 binding EC50. Potency to activate a receptor is also routinely reported as the Ki, with the lower Ki value equating with a greater potency.

In some embodiments, use of a NKR agonist, such as a NK2R agonist is provided for the manufacture of a medicament for the treatment of an eating disorder, such as binge eating disorder.

Selective NK2R agonists

In a preferred embodiment, the NKR agonist provided herein is a neurokinin receptor 2 (NK2R) agonist. In one embodiment, the NKR agonist is a selective neurokinin receptor 2 (NK2R) agonist.

In one embodiment, the NKR agonist has an EC50 towards human NK2R of 300 nM or less, such as 250 nm or less, such as 200 nm or less, such as 150 nM or less, such as 100 nM or less, such as 90 nM or less, such as 80 nM or less, such as 70 nM or less, such as 60 nM or less, such as 50 nM or less.

In one embodiment, the NKR agonist has an EC50 towards human NK2R of 50 nM or less, such as 40 nm or less, such as 30 nm or less, such as 20 nM or less, such as 15 nM or less, such as 14 nM or less, such as 13 nM or less, such as 12 nM or less, such as 11 nM or less, such as 10 nM or less. In one embodiment, the NKR agonist has an EC50 towards human NK1 R of at least 100 nM, such as at least 200 nM, such as at least 300 nM, such as at least 400 nM, such as at least 500 nM.

In one embodiment, the NKR agonist has an EC50 towards human NK3R of at least 100 nM, such as at least 200 nM, such as at least 300 nM, such as at least 400 nM, such as at least 500 nM.

In one embodiment, the NKR agonist has an EC50 towards human NK2R of 50 nM or less; and an EC50 towards human NK1 R of at least 100 nM. In one embodiment, the NKR agonist has an EC50 towards human NK2R of 50 nM or less; and an EC50 towards human NK3R of at least 100 nM. In one embodiment, the NKR agonist has an EC50 towards human NK2R of 50 nM or less; an EC50 towards human NK1 R of at least 100 nM; and an EC50 towards human NK3R of at least 100 nM.

In one embodiment, the NKR agonist is a selective neurokinin receptor 2 (NK2R) agonist which is used for treatment of binge eating disorder. In one embodiment, the NK2R agonist used for treatment of binge eating disorder has an EC50 towards human NK2R of 50 nM or less; an EC50 towards human NK1 R of at least 100 nM; and an EC50 towards human NK3R of at least 100 nM.

Structural aspects of NKR agonists

In one embodiment, a NKR agonist, such as a NK2R agonist is provided according to formula (I),

(A)-(B) (I), wherein;

Xi is selected from the group consisting of: aspartic acid (D) and glutamic acid (E);

X₂ is selected from the group consisting of: lysine (K), arginine (R), and histidine (H);

X₃ is selected from the group consisting of: tyrosine (Y), phenylalanine (F), meta- tyrosine (m-Y), valine (V), tryptophan (W), methionine (M), leucine (L), isoleucine (I), and alanine (A); X₄ is selected from the group consisting of: valine (V), threonine (T), serine (S), asparagine (N), glutamine (Q), glycine (G), and alanine (A);

X₆ is selected from the group consisting of: leucine (L), isoleucine (I), alanine (A) and N-methyl leucine (Me-Leu); and

(B) is a conjugated moiety of the general formula (II)

Fa-Lg (II), wherein;

Fa is a C10-C20 fatty acid, optionally substituted with one or more carboxylic acid groups, and

In some embodiments, the NKR agonist is provided wherein the peptide (A) is of the general formula X1X2X3X4X5X6X7, wherein

X₂ is selected from the group consisting of: lysine (K), and arginine (R);

X₃ is selected from the group consisting of: tyrosine (Y), and phenylalanine (F), and meta-tyrosine (m-Y),

X₄ is selected from the group consisting of: valine (V), and threonine (T);

X₅ is selected from the group consisting of: glycine (G), 2-aminoisobutyric acid (Aib), beta-alanine (bA) and serine (S);

X₆ is selected from the group consisting of: leucine (L), and N-methyl leucine (Me-Leu); and

X7 is selected from the group consisting of: norleucine (Nle), methoxinine (Mox), methionine (M), 4-fluorophenylalanine (4fF), and 4-methoxyphenylalanine (4MeOF). In some embodiments, X2 is arginine (R).

In some embodiments, X3 is tyrosine (Y).

In some embodiments, X4 is threonine (T).

In some embodiments, X5 is selected from the group consisting of: 2-aminoisobutyric acid (Aib) and serine (S).

In some embodiments, Xe is N-methyl-leucine (Me-Leu).

In some embodiments, X7 is selected from the group consisting of: methoxinine (Mox) and 4-fluorophenylalanine.

In some embodiments, the NKR agonist is of formula (I):

(A)-(B) (I), wherein;

(B) is a conjugated moiety of the general formula (II)

Fa-Lg (II), wherein;

Fa is of formula (Fa-1 ),

Lg is a linking group of formula (Lg-1 ),

wherein Z is a chain comprising from 18 to 23 atoms in the backbone selected from the group consisting of: C, O, and N; and wherein R is selected from the group consisting of H, and C1-6 alkyl; and Lg covalently links (B) to the peptide (A), and wherein (B) is covalently linked to a terminal or non-terminal amino acid.

A person of skill in the art knows that C, O, and N may be substituted with hydrogen according to the valence of the particular atom. The backbone may comprise one or more carbonyl groups, such as 1 , 2, 3, or 4 carbonyl groups. The backbone may also comprise one or more carboxylic acid groups, such as 1 or 2. In some embodiments, Z comprises fragments of ethylene glycol interrupted by one or more amide functionalities. An example is shown in formula (B1 ), wherein the fatty acid Fa is of formula (Fa-1 ), R of Lg-1 is H, and the backbone of Z comprises 21 atoms selected from the group consisting of C, O, and N.

In some embodiments, the NKR agonist of formula (I) is provided for use in the treatment of an eating disorder, such as binge eating disorder. Preferably, the NKR agonist of formula (I) is a NK2R agonist, such as a selective NK2R agonist, for use in the treatment of binge eating disorder.

In some embodiments, the conjugated moiety is of formula (B1 );

In some embodiments, the conjugated moiety (B) is covalently attached to the N- terminus of (A), optionally via an amide bond.

In some embodiments, the conjugated moiety (B) is covalently attached to the N- terminus of (A) via an amide bond with the N-terminal a-NH2 group.

In some embodiments, the peptide (A) is amidated on the C-terminus.

In some embodiments, the peptide (A) comprises from 7 to 15 amino acids, such as from 7 to 14 amino acids, such as from 7 to 13 amino acids, such as from 7 to 12 amino acids, such as from 7 to 11 amino acids, such as from 7 to 1 1 amino acids, such as from 7 to 10 amino acids, such as from 7 to 9 amino acids, such as from 7 to 8 amino acids, preferably wherein the peptide comprises 7 amino acids.

In some embodiments, the peptide (A) comprises no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids. In some embodiments, the peptide (A) consists of 7 ammo acids of the general formula X1X2X3X4X5X6X7.

In some embodiments, the NKR agonist is provided, wherein

(A) is: Asp;Lys;Phe;Val;Gly;NmLeu;Nle;NH2 (compound 305), and

(B) is of formula (B1) covalently attached to the N-terminal aspartate of (A).

In some embodiments, the NKR agonist is provided, wherein

(A) is: Asp;Lys;Tyr;Val;Gly;NmLeu;Metox;NH2 (compound 344), and

(B) is of formula (B1) covalently attached to the N-terminal aspartate of (A).

In some embodiments, the NKR agonist is a NK2R agonist that consists of the sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 57.

In some embodiments, the NKR agonist is a NK2R agonist selected from the group consisting of:

(ID 344);

(ID 394);

(ID 398).

Pharmaceutical compositions

In one embodiment, a pharmaceutical composition is provided comprising the NKR agonist as defined herein, and one or more pharmaceutically acceptable adjuvants, excipients, carriers, buffers and/or diluents.

Administration

The NKR agonists disclosed herein, such as the NK2R agonists, are preferably administered to subjects, such as human subjects presenting with an eating disorder. In some embodiments, the NKR agonists are administered parenterally, such as by subcutaneous administration. Examples

and efficacies bv Inositol

measurement

Compound selectivity toward NK2R can be determined using the methods disclosed in the present example.

Materials:

Formic acid, LiCI, CaCl2, Tris-HCI, EDTA, HEPES, NaCI, Chloroquine, and ovalbumin (albumin from chicken eggs) (Sigma Aldrich). COS-7 monkey kidney cell line was obtained from ATCC. DMEM 1885, FBS, Penicillin/Streptomycin (P/S) and HBSS were from Thermo Scientific / Gibco. Clear Costar 96 wells Tissue Culture-treated plates and solid white 96-well plates from Corning. Polylysine Coated Yttrium Silicate SPA Beads (#RPNQ0010) and Myo-[2-³H(N)]-inositol - (#NET114A[005MC]) from Perkin Elmer. lnositol-1 ,4,5-Trisphosphate [³H] Radioreceptor Assay (IP₃ Assay) from Perkin Elmer. pcDNA3.1 (+) containing coding sequences of human and mouse tachykinin receptor 1 , 2, 3 mRNA were obtained from Genscript (custom order). Synthesized peptides diluted in saline + 0.2% (w/v) ovalbumin. mRNA IDs Human Tacrl : NM 001058.4

Human Tacr2: NM 001057.3

Human Tacr3: NM 001059.2

Methods:

Agonist-induced G-protein coupled receptor (GPCR) activation was measured by an lnositol-1 ,4,5-Trisphosphate [³H] Radioreceptor Assay (IP₃ Assay). Assays were carried out using COS-7 cells transiently transfected by calcium phosphate transfection with a vector pcDNA3.1 (+) encoding one of the indicated receptors (Genscript). Briefly, DNA mixed with CaCh (2 M) and TE-buffer (10 mM Tris-HCI, 1 mM EDTA, pH 7.5) was dropwise added to 2xHBS (50 mM HEPES, 280 mM NaCI, 1 .5 mM NaH2PO4, pH 7.2) and incubated for 45 min at room temperature (22±2 °C). The mixture and a final concentration of 100 pM Chloroquine were added to the cells and left to incubate for 5 hours at 37 °C under standard cell culture conditions (10% CO2) before changing medium to fresh medium containing 5 pl/mL Myo-[2-³H(N)]-inositol (labelling medium). The IP3 assay takes advantage of the tachykinin receptors ability to induce production of the inositol trisphosphate (IP3) second messenger upon agonist (ligand) binding on receptor expressing cells following an initial ³H-inositol labelling period. In effect this means that production of the second messenger I P₃ as a measure of receptor activity can be assessed by counting ³H-activity.

Assay solutions used: Wash buffer (HBSS), Assay buffer (HBSS + 10 mM LiCI and 0,2% w/v ovalbumin), Lysis buffer (10 mM formic acid), and SPA YSI beads (12.5mg/ml in H₂O). The assay was performed the day after transfection. Briefly, labelling medium was aspirated, and plates were washed x1 in wash buffer before adding 100 pl assay buffer, pre-incubated for 30 min followed by 120 min incubation with agonist, both at 37 °C. After incubation, plates were immediately placed on ice and the incubation medium was aspirated and 40 pl of 10 mM formic acid per well was added. Plates were incubated for at least 30 min on ice. 60 pl (1 mg/well) SPA YSI beads/well was pipetted into a solid white 96 wells plate and 35 pl of the lysis solution was transferred to the plate before covering plates with seal cover and shaking for 10 min. (max speed). Centrifuge plates and leave for 8 hours at room temperature before counting the plates in a MicroBeta plate counter (Perkin Elmer).

Example 2: Materials and methods for in vivo studies

Materials

NaH₂PO4-H₂O (Sigma), Na₂HPO4-2H₂O (Sigma), propylene glycol (Sigma), maintenance diet for rats and mice (regular chow, #1320, Altromin), C57BL/6NR] mice (Janvier Labs), high fat diet (HFD) (TD.95217, Envigo), milk chocolate (Remal OOO milk chocolate with nuts), peptide analogues (Novo Nordisk).

Methods

Animals were housed with ad libitum access to maintenance diet and water with a 12- hour light-dark cycle and 22-24 degree Celsius temperature. All animal experiments were performed according to Danish Animal Inspectorate regulations. C57BL6/J mice were used for experimentation.

Binge eating model was established using modified protocols as described in: Hildebrandt and Ahmari, Frontiers in Psychiatry 2021 ; Halpern et al. J. Neuro Sci. 2013, Vickers et al. J. Psychopharmacol 2015. The mice were trained to binge by exposure to HFD for one hour during the first hours of the dark period for at least eight days. Binge eating was defined by consumption of more than 25% (in grams) of 24- hour food intake during a one-hour binge. Upon establishment of binge eating, mice were subcutaneously injected with peptide analogue in a volume of 2 ml/kg at least two hours prior to binge eating assay. HFD was weighed manually before and after binge to estimate amount binged. To determine efficacy of NK2R agonists, mice were trained to binge on milk chocolate. Binge criteria and treatment regimen were the same as for HFD-binge.

Example 3: NK2R aqonists reduce HFD binqe eatinq in mice

Materials & Methods

In the present example, the effect of protracted NK2R agonists on HFD binge eating wild-type mice was examined. HFD binge eating was measured in wild-type mice as described in Example 2.

Results

NK2R-selective NKA(4-10) analogue (compound 344) dose-dependently decreased binge eating in wild-type mice trained to binge on palatable diet as shown in Fig 1 .

Conclusions

The present example demonstrates that NK2R selective compounds, such as 344, can be used to treat eating disorders, such as binge eating disorder.

Example 4: NK2R aqonists reduce chocolate binqe eatinq in mice

Materials & Methods

In the present example, the effect of protracted NK2R agonists on chocolate binge eating wild-type mice was examined. Chocolate binge eating was measured in wildtype mice trained to binge as described in Example 2. Results

Table 1 shows the efficacy of selective long-lived NK2R agonists in reducing chocolate binge eating in mice.

Table 1 : reduction in chocolate binge eating. 0-25 % reduction from control (+); 26-50 % reduction from control (++); 51 -100 % reduction from control (+++).

Conclusions

The present example demonstrates that NK2R selective compounds such as 344, 369, and 383, can be used to treat eating disorders, such as binge eating disorder.

Example 5: Structures of conjugated moieties

Semaglutide protractor: 2xOEG-gammaGlu-C18 diacid. Neutral linker termed “Conj- Neu-C18DA”.

3xgamma-Glu-C18 diacid. Negative charged linker termed “Conj-Neg”.

Glu-C18 diacid. Positive charged linker termed “Conj-Pos1

Glu-C18 diacid. Positive charged linker termed “Conj-Pos2”. Sequence overview

Unless stated otherwise, “*” is “Conj-Neu-C18DA” the structure of which is illustrated in

Example 5. The conjugated moiety is attached to the N-terminal a-amino group, unless stated otherwise.

References

Hildebrandt and Ahmari, Frontiers in Psychiatry 2021

Halpern et al. J. Neuro Sci. 2013

Vickers et al. J. Psychopharmacol 2015

Claims

1 . A neurokinin receptor (NKR) agonist for use in the treatment of an eating disorder.

2. The NKR agonist for use according to claim 1 , wherein the eating disorder is selected from the group consisting to binge eating disorder, bulimia nervousa, hyperphagia and night eating syndrome.

3. The NKR agonist for use according to any one of the preceding claims, wherein the eating disorder is binge eating disorder (BED).

4. The NKR agonist for use according to any one of the preceding claims, wherein the eating disorder is hyperphagia.

5. The NKR agonist for use according to any one of the preceding claims, wherein the eating disorder is binge-eating in a disease associated with binge-eating.

6. The NKR agonist for use according to any one of the preceding claims, wherein the eating disorder is binge-eating in a disease selected from the group consisting of: binge eating disorder, bulimia nervousa, night eating syndrome, type-1 diabetes, type-2 diabetes, frontotemporal dementia, Kleine-Levin syndrome, and Prader-Willi syndrome.

7. The NKR agonist for use in according to any one of the preceding claims, for use in the treatment of binge eating or hyperphagia in a subject suffering from a comorbidity.

8. The NKR agonist for use according to claim 7, wherein the comorbidity is selected from the group consisting of binge eating disorder, bulimia nervousa, night eating syndrome, type-1 diabetes, type-2 diabetes, frontotemporal dementia, Kleine-Levin syndrome, and Prader-Willi syndrome.

9. The neurokinin receptor agonist for use according to any one of the preceding claims, wherein the NKR agonist is a neurokinin receptor 2 (NK2R) agonist. The NKR agonist for use according to any one of the preceding claims, wherein the NKR agonist is of formula (I):

(A)-(B) (I), wherein;

X₂ is selected from the group consisting of: lysine (K), arginine (R), and histidine

(H);

X₃ is selected from the group consisting of: tyrosine (Y), phenylalanine (F), meta- tyrosine (m-Y), valine (V), tryptophan (W), methionine (M), leucine (L), isoleucine

(I), and alanine (A);

X7 is selected from the group consisting of: norleucine (Nle), methoxinine (Mox), methionine (M), 4-fluorophenylalanine (4fF), and 4-methoxyphenylalanine (4MeOF); and

(B) is a conjugated moiety of the general formula (II)

Fa-Lg (II), wherein;

Lg is a linking group, which covalently links (B) to the peptide (A), and wherein (B) is covalently linked to a terminal amino acid or to a non-terminal amino acid. The NKR agonist for use according to claim 10, wherein the peptide (A) is of the general formula X1X2X3X4X5X6X7, wherein

X₂ is selected from the group consisting of: lysine (K), and arginine (R);

X₄ is selected from the group consisting of: valine (V), and threonine (T);

X7 is selected from the group consisting of: norleucine (Nle), methoxinine (Mox), methionine (M), 4-fluorophenylalanine (4fF), and 4-methoxyphenylalanine (4MeOF). The NKR agonist for use according to any one of claims 10-11 , wherein X₂ is arginine (R). The NKR agonist for use according to any one of claims 10-12, wherein X3 is tyrosine (Y). The NKR agonist for use according to any one of claims 10-13, wherein X4 is threonine (T). The NKR agonist for use according to any one of claims 10-14, wherein X5 is selected from the group consisting of: 2-aminoisobutyric acid (Aib) and serine (S). The NKR agonist for use according to any one of claims 10-15, wherein Xe is N- methyl-leucine (Me-Leu). The NKR agonist for use according to any one of claims 10-16, wherein X7 is selected from the group consisting of: methoxinine (Mox) and 4- fluorophenylalanine. The NKR agonist for use according to any one of claims 10-17, wherein the NKR agonist is of formula (I):

(A)-(B) (I), wherein;

(H);

(I), and alanine (A);

(B) is a conjugated moiety of the general formula (II)

Fa-Lg (II), wherein;

Fa is of formula (Fa-1 ),

wherein n is from 14 to 17, preferably wherein n is 15; and wherein X is selected from the group consisting of -OH, -OC1-6, -NH2, -NHC1-

6, and N(CI-6)2, and

Lg is a linking group of formula (Lg-1 ),

wherein Z is a chain comprising from 18 to 23 atoms in the backbone selected from the group consisting of: C, O, and N; and wherein R is selected from the group consisting of H, and C1-6 alkyl; and Lg covalently links (B) to the peptide (A), and wherein (B) is covalently linked to a terminal or non-terminal amino acid. The NKR agonist for use according to any one of claims 10-18, wherein the conjugated moiety is of formula (B1 );

The NKR agonist for use according to any one of claims 10-19, wherein the conjugated moiety (B) is covalently attached to the N-terminus of (A), optionally via an amide bond. The NKR agonist for use according to any one of claims 10-20, wherein the conjugated moiety (B) is covalently attached to the N-terminus of (A) via an amide bond with the N-terminal a-NH2 group. The NKR agonist for use according to any one of claims 10-21 , wherein the peptide (A) is amidated on the C-terminus. The NKR agonist for use according to any one of claims 10-22, wherein the peptide (A) comprises from 7 to 15 amino acids, such as from 7 to 14 amino acids, such as from 7 to 13 amino acids, such as from 7 to 12 amino acids, such as from 7 to 1 1 amino acids, such as from 7 to 11 ammo acids, such as from 7 to 10 amino acids, such as from 7 to 9 amino acids, such as from 7 to 8 amino acids, preferably wherein the peptide comprises 7 amino acids.

24. The NKR agonist for use according to any one claims 10-23, wherein the peptide (A) comprises no more than 15 amino acids, such as no more than 14 amino acids, such as no more than 13 amino acids, such as no more than 12 amino acids, such as no more than 11 amino acids, such as no more than 10 amino acids, such as no more than 9 amino acids, such as no more than 8 amino acids, such as no more than 7 amino acids.

25. The NKR agonist for use according to any one of claims 10-24, wherein the peptide (A) consists of 7 amino acids of the general formula X1X2X3X4X5X6X7.

26. The NKR agonist for use according to any one of claims 10-25, wherein

(A) is: Asp;Lys;Phe;Val;Gly;NmLeu;Nle;NH2 (compound 305), and

(B) is of formula (B1 ) covalently attached to the N-terminal aspartate of (A).

27. The NKR agonist for use according to any one of claims 10-25, wherein

(A) is: Asp;Lys;Tyr;Val;Gly;NmLeu;Metox;NH2 (compound 344), and

(B) is of formula (B1 ) covalently attached to the N-terminal aspartate of (A).

28. The NKR agonist for use according to any one of claims 10-25, wherein

(A) is: Asp;Lys;Tyr;Val;Gly;NmLeu;4-F-Phe;NH2 (compound 369), and

(B) is of formula (B1 ) covalently attached to the N-terminal aspartate of (A).

29. The NKR agonist for use according to any one of claims 10-25, wherein

(A) is: Asp;Lys;Phe;Thr;Gly;NmLeu;Metox;NH2 (compound 383), and

(B) is of formula (B1 ) covalently attached to the N-terminal aspartate of (A).

30. The NKR agonist for use according to any one of the preceding claims wherein the NKR agonist is an NK2R agonist that consists of the sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 57.

31 . The NKR agonist for use according to any one of the preceding claims, wherein the NKR agonist is an NK2R agonist selected from the group consisting of:

10 (ID 369);

(ID 398).

32. The NKR agonist for use according to any one of the preceding claims, wherein the NKR agonist is a selective neurokinin receptor 2 (NK2R) agonist.

33. The NKR agonist for use according to any one of the preceding claims, wherein the NKR agonist has an EC50 towards human NK2R of 300 nM or less, such as 250 nm or less, such as 200 nm or less, such as 150 nM or less, such as 100 nM or less, such as 90 nM or less, such as 80 nM or less, such as 70 nM or less, such as 60 nM or less, such as 50 nM or less.

34. The NKR agonist for use according to any one of the preceding claims, wherein the NKR agonist has an EC50 towards human NK2R of 50 nM or less, such as 40 nm or less, such as 30 nm or less, such as 20 nM or less, such as 15 nM or less, such as 14 nM or less, such as 13 nM or less, such as 12 nM or less, such as 11 nM or less, such as 10 nM or less.

35. The NKR agonist for use according to any one of the preceding claims, wherein the NKR agonist has an EC50 towards human NK1 R of at least 100 nM, such as at least 200 nM, such as at least 300 nM, such as at least 400 nM, such as at least 500 nM.

36. The NKR agonist for use according to any one of the preceding claims, wherein the NKR agonist has an EC50 towards human NK3R of at least 100 nM, such as at least 200 nM, such as at least 300 nM, such as at least 400 nM, such as at least 500 nM.

37. Use of a NKR agonist as defined in any one of the preceding claims, for the manufacture of a medicament for the treatment of an eating disorder, such as binge eating disorder.