agonists of tacr2

Abstract

The present disclosure relates to agonists of Tacr2, such as peptide agonists of Tacr2, and methods of using the same for treating insulin resistance, obesity, and / or diabetes. The present disclosure also relates to the use of the described agonists of Tacr2 for increasing energy expenditure in an individual.

Description

Invention Field

[0001] This disclosure relates to agonists of Tacr2, such as peptide agonists of Tacr2, and methods of using them to treat obesity and / or diabetes. The disclosure also relates to the use of said Tacr2 agonists to increase an individual's glucose and energy expenditure. Background of the Invention

[0003] There are approximately 2 billion overweight or obese individuals and 350 million individuals with diabetes worldwide (according to the World Health Organization), and despite ongoing research, effective treatments are still needed.

[0004] One of the therapeutic strategies investigated is the activation of brown and beige adipose tissue. The classic activation of brown and beige adipose tissue by norepinephrine (NE) increases energy expenditure in these cells via beta-adrenergic receptors (β3AR) (Cannon and Nedergaard, 2004). One of the main effectors of this brown and beige adipose energy expenditure is uncoupling protein 1 (Ucp1), which utilizes the proton gradient to generate heat rather than for ATP production. In addition to the activation of brown and beige adipose tissue, cold exposure also induces the expansion of brown and beige adipose tissue depots. The expansion and activation of brown and beige adipose tissue is significant for the treatment of obesity and diabetes because it can deplete large amounts of lipids and glucose. NE is currently considered the main neurotransmitter and effector in the expansion and activation of brown and beige adipose tissue. Unfortunately, NE cannot be used to treat obesity and diabetes because it causes increased blood pressure and heart rate.

[0005] Currently, there are no brown and beige adipose tissue activators for the treatment of obesity and / or diabetes. Among adrenergic activators, Mirabegron (Myrbetriq, Astellas Pharma, Inc.), approved for the treatment of overactive bladder, has shown activation of human brown adipose tissue in a proof-of-concept study (Cypess et al., 2015). However, this drug causes increased blood pressure and heart rate, undesirable side effects for potentially obese patients, reducing enthusiasm for this treatment approach.

[0006] Therefore, there is a need for means that can more specifically activate brown and beige fat stores without the need for systemic administration of adrenergic activators. Summary of the Invention

[0007] This disclosure relates to agonists of Tacr2 or pharmaceutically acceptable salts thereof for the treatment of diabetes and / or obesity in individuals with this need. The inventors have discovered that agonists of Tacr2, particularly peptide agonists of Tacr2, can activate brown and beige adipose tissue independently of norepinephrine (NE) / β-adrenergic receptor signaling. This invention discloses that administration of agonists of Tacr2 to an individual has multiple effects on brown and beige adipocytes, which is beneficial for the treatment of obesity and diabetes. Specifically, agonists of Tacr2 lead to an increase in the oxygen consumption rate of brown and beige adipocytes, and preferably also lead to an increase in glucose uptake, oxygen consumption rate, energy expenditure, and heat production of brown and beige adipocytes. Preferably, agonists of Tacr2, particularly peptide agonists of Tacr2, lead to the activation of brown adipose tissue without causing harmful side effects such as increased blood pressure and heart rate.

[0008] One aspect of this disclosure relates to agonists of Tacr2 or pharmaceutically acceptable salts thereof, particularly peptide agonists of Tacr2, for the treatment of diabetes and / or obesity in individuals with this need.

[0009] Another aspect of this disclosure relates to a pharmaceutical composition comprising an agonist of Tacr2 as defined herein or a pharmaceutically acceptable salt thereof, particularly a peptide agonist of Tacr2, for the treatment of obesity and / or diabetes in individuals with such need.

[0010] Another aspect of this disclosure relates to the use of Tacr2 agonists or pharmaceutically acceptable salts thereof, particularly peptide agonists of Tacr2, in the preparation of medicaments for the treatment of diabetes and / or obesity.

[0011] Another aspect of this disclosure relates to a method for treating diabetes and / or obesity in an individual with this need, said method comprising administering to said individual a therapeutically effective amount of a Tacr2 agonist or a pharmaceutically acceptable salt thereof, particularly a peptide agonist of Tacr2.

[0012] Another aspect of this disclosure relates to a method for treating diabetes and / or obesity in an individual with this need, wherein the method comprises enhancing the activity of brown adipose tissue cells in the individual by administering to the individual a therapeutically effective amount of a Tacr2 agonist or a pharmaceutically acceptable salt thereof, particularly a peptide agonist of Tacr2. Attached Figure Description

[0013] Figure 1: Expression and functional characterization of NKA / TACR2 in brown and white adipose tissues. Tacr2 expression profiles in cold-induced (a) interscapular brown adipose tissue (iBAT), (b) inguinal white adipose tissue (iWAT), and (c) epididymal white adipose tissue (eWAT) of C57BI / 6 mice relative to thermoneutrality. Data show the mean and SEM for n=5.

[0014] Figure 2: (a) Tacr2 mRNA expression levels in mature adipocytes and the stromal vascular fraction of iBAT from thermoneutral or cold-provoked mice. (b) Tacr2 mRNA expression in iBAT from 12-week-old C57BI / 6 mice fed a chow diet or a 60% high-fat diet (HFD). Data show the mean and SEM for n=8. (c)

[0015] Figure 3 (a) NKA-mediated IP3 production in primary brown adipocytes transfected with Tacr2 or the vector control. (b) Oxygen consumption rate (OCR) in primary brown adipocytes treated with NKA or the vector for 72 hours, determined using a Seahorse Bioscience Analyzer. Basal respiration was measured followed by NE (5 μM), oligomycin (1 μM), FCCP (1 μM), and rotenone and antimycin A (1 μM each). Data are presented as mean values ​​and SEM images for n=8.

[0016] Figure 4(a) During a 9-day photoperiod, mice were injected twice daily with 1 mg / kg NKA (grey) or a mediator (black), marked with arrows. Oxygen consumption was measured every 5 minutes using PhenoMaster. Data represent all days and are shown as mean ± SEM for n = 6. (b) Body weight was measured daily throughout the experiment. Data are shown as mean ± SEM for n = 6–7, *p < 0.05, **p < 0.01, using two-sided ANOVA and Sidak multiple comparisons. (c) On day 1 post-treatment, mice fasted for 3 hours underwent an insulin tolerance test, receiving 1 UI insulin / kg lean body mass intraperitoneally. Data are shown as mean ± SEM for n = 6–7, *p < 0.05, **p < 0.01, using two-sided ANOVA and Sidak multiple comparisons. (d) At the end of the experiment, the three major adipose tissues were harvested and weighed. Data are shown as mean ± SEM for n = 6–7, *p < 0.05, using an unpaired Student's t-test.

[0017] Figure 5 a) Weight loss; b) Food intake; c) Calorie burning in mice treated with the medium (black) or [Lys2-γ-Glu-C16]NKA (gray). Data are presented as mean + / - SEM for n=6. Invention Details

[0019] definition

[0020] As used herein, the term "agonist" refers to an agonist of Tacr2 unless otherwise specified. An agonist of Tacr2, as defined herein, is a compound comprising or composed of a peptide that binds to Tacr2 and thereby enhances its activity.

[0021] As used herein, the term "activity of brown and / or beige adipocytes" refers to several typical biochemical parameters of brown and beige adipose tissue cells that can change in response to the interaction between Tacr2 and agonists. Some examples of parameters defining brown and / or beige adipocyte activity are basal oxygen consumption (OCR), norepinephrine (NE)-induced OCR, maximal respiration, glucose uptake / absorption, lipid uptake / absorption, and heat production.

[0022] Proteinogenic "amino acids" are named in this paper using their single-letter codes (as suggested by IUPAC, see, for example, http: / / www.chem.qmw.ac.uk / iupac). Unless otherwise specified, amino acids can be D- or L-forms.

[0023] As used in this article, the term "C" min "C" refers to the lowest blood concentration of the compound. Therefore, C is related to blood glucose levels after insulin administration. min This is the lowest blood glucose concentration observed after the administration. Typically, C involves blood glucose levels. min It is the lowest blood glucose concentration observed within the insulin dosing interval.

[0024] As used herein, the term "functional analog" refers to a compound that functions in a manner similar to that of a reference compound. Therefore, a functional analog of a Tacr2 peptide agonist functions in the same way as a Tacr2 peptide agonist. A functional analog can be, for example, a compound containing a peptide, wherein the peptide may be modified with a portion that is not necessarily composed of amino acid residues, which may bind to Tacr2 to enhance its activity.

[0025] "Non-standard amino acids" are amino acid residues that are not encoded by the genetic code of any organism. Non-standard amino acid residues can exist naturally in organisms. Non-limiting examples of non-standard amino acids are: naturally occurring amino acids with the D conformation, β-amino acids, γ-amino acids, and non-proteinogenic amino acids.

[0026] The term "PEG" or polyethylene glycol refers to a polymer of ethylene glycol, with the chemical formula C6H2O. 2n H 4n+2 O n+1 And it has the following repeating structure:

[0027]

[0028] As used herein, the term “peptide” is a sequence of at least two amino acid residues linked by an amide bond.

[0029] Unless otherwise specified, the term "peptide agonist" refers to a compound agonist of Tacr2. As defined herein, a Tacr2 peptide agonist is a compound comprising or composed of peptides capable of binding to Tacr2 and thus enhancing its activity. Tacr2 peptide agonists may consist of a peptide and a conjugated moiety. The conjugated moiety may be, for example, a sugar, a lipid, a secondary peptide, or any other chemical group that a person skilled in the art would consider beneficial.

[0030] As used herein, the term "sequence identity" refers to the percentage of identical amino acids or nucleotides between a candidate sequence and a reference sequence based on alignment. Therefore, a candidate sequence with 80% amino acid identity to a reference sequence requires that, based on alignment, 80% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. The identity of the present invention was determined by computer analysis, including, but not limited to, the Clustal Omega computer alignment program (Sievers et al. (2011 October 11) Molecular Systems Biology 7:539, PMID:21988835; Li et al. (2015 April 06) Nucleic Acids Research 43(W1):W580-4 PMID:25845596; McWilliam et al., (2013 May 13) Nucleic Acids Research 41(Web Serverissue):W597-600 PMID:23671338), and the suggested default parameters therein. The Clustal Omega software is available from EMBL-EBI at https: / / www.ebi.ac.uk / Tools / msa / clustalo / . Using the program and its default settings, the mature (bioactive) portions of the query and reference peptides are aligned. The number of completely conserved residues is counted and divided by the length of the reference polypeptide. The MUSCLE or MAFFT algorithm can be used for nucleotide sequence alignment. Sequence identity can be calculated in a similar manner to that described for amino acid sequences. Therefore, the sequence identity presented herein is calculated over the entire length of the reference sequence.

[0031] As used herein, the term "treatment" refers to the management and care of a patient for the purpose of combating a condition, disease, or symptom. The term is intended to encompass comprehensive treatment for a given condition suffered by a patient, such as the administration of active compounds for the purpose of: reducing or alleviating symptoms or complications; delaying the progression of the condition, disease, or symptom; improving, curing, or eliminating the condition, disease, or symptom; and / or preventing the condition, disease, or symptom. Patients to be treated are preferably mammals, especially humans. Patients to be treated can be of various ages.

[0032] Tacr2 agonists

[0033] One aspect of this disclosure relates to agonists of Tacr2 or pharmaceutically acceptable salts thereof, particularly peptide agonists of Tacr2, for the treatment of obesity and / or diabetes. The Tacr2 agonist can be any compound capable of binding to Tacr2 and enhancing its activity. In some embodiments, the Tacr2 agonist is a peptide agonist. The Tacr2 peptide agonist may comprise or consist of a peptide and an optional conjugated moiety, as described in detail in the “Peptide Agonists” and “Conjugated Moiety” sections below.

[0034] Preferably, the agonist of Tacr2 enhances the activity of Tacr2, as determined by any suitable assay, especially by assays that can measure Tacr2 activity (e.g., Ca). 2+ Mobilization assay (Ca 2+ The cellular function assay for Tacr2 activity can be either a mobilization assay or an inositol 3-phosphate (IP3) production assay, both described by Ablas J. et al. (1995). A person skilled in the art may choose a radioligand assay or other assays such as a functional monoreceptorial bioassay as a suitable means of determining Tacr2 activity. Therefore, the agonist of Tacr2 can be any compound, such as a peptide agonist, that, as determined by any of the above assays, enhances Tacr2 activity, preferably by at least 20%, for example at least 30%, for example at least 40%.

[0035] In some embodiments, agonist activity is determined by measuring IP3 production in cells expressing Tacr2. Preferably, the Tacr2 agonist is a compound that causes an increase in IP3 production upon contact with such cells. Therefore, whether a compound is an agonist of Tacr2 can be determined by a method including the following steps:

[0036] • Provide cells expressing Tacr2,

[0037] • Contact the cells with myo-inositol and a compound that is a presumed agonist of Tacr2.

[0038] • The production of IP3 in the cells was measured in both the absence and presence of the assumed Tacr2 agonist.

[0039] The increase in IP3 in the presence of the compound indicates that the compound is an agonist of Tacr2.

[0040] Cells may endogenously express Tacr2 or they may be recombinant cells that express Tacr2, for example, the cells may be COS-7 or HEK 293 cells containing a heterologous nucleic acid encoding Tacr2. IP3 production can be determined using a dose-response profile.

[0041] The cells can also be primary brown adipocytes, such as primary brown adipocytes containing a heterologous nucleic acid encoding Tacr2. Therefore, it is preferable that the Tacr2 agonist is a compound that induces IP3 production in the assay performed as described in Example 2. Preferably, the agonist is a compound that induces IP3 production in a manner similar to NKA or induces at least 80% of IP3 production compared to NKA (in the assay performed as described in Example 2).

[0042] Therefore, a Tacr2 agonist can be a compound that enhances the activity of Tacr2 by at least 20%, as determined by the assay for monitoring IP3 production described above. A Tacr2 agonist can also be a compound that enhances the activity of Tacr2 by at least 30%, as determined by the assay for monitoring IP3 production described above.

[0043] A Tacr2 agonist can also be a compound that enhances the activity of Tacr2 by at least 40%, as determined by the assay described above for monitoring IP3 production.

[0044] Tacr2 agonists preferably have a high affinity for Tacr2. The affinity of a compound for its receptor, i.e., its affinity for Tacr2, can be determined by different assays, resulting in different affinity units as shown in Table 1, as described below:

[0045] -pIC 50 It is an agonist's IC 50 The negative logarithm to base 10, in this IC 50 "IC" is a term used in a variety of ways, and in particular it can refer to the molar concentration of an unlabeled agonist that inhibits the binding of a radioligand by 50%; the concentration of the radioligand should be given; IC in this disclosure 50 The unit is M (moles), unless otherwise specified;

[0046] -pEC 50 EC is an agonist 50 The negative logarithm to base 10, where EC 50 This is the molar concentration at which the agonist produces its maximum possible effect, determined using the appropriate assay as defined above in this section. Other percentage values ​​(EC) may be cited as examples. 20 EC 40(etc.); EC in this disclosure 50 The unit is M (moles), unless otherwise specified;

[0047] -pK d It is K d The negative logarithm to the base 10, where K d It refers to the equilibrium dissociation constant of an agonist, which is directly measured in a binding assay using labeled agonists;

[0048] -pK i It is K i The negative logarithm to the base 10, where K i This refers to the equilibrium dissociation constant of the ligand determined in inhibition studies. Given the ligand's K... i Typically (but not always), in competitive radioligand binding studies, this is determined by measuring the inhibition of the binding of the competitive ligand of interest to the reference radioligand under equilibrium conditions.

[0049] In particular, Tacr2 agonists, such as peptide agonists, can be pEC. 50 Compounds of 8 to 10 are determined, such as in assays described in Bellucci F. et al. (2002) for radioligand binding assays or “single-receptor” bioassays.

[0050] In some embodiments, the peptide agonist may be the pEC of the mouse receptor as determined as described in Example 7. 50 Compounds in the range of 6.7 to 9.5.

[0051] For example, peptide agonists can be pECs of mouse receptors. 50 For example, pEC, it should be at least 6.8. 50 For at least 7, pEC is preferred. 50 For at least 7.5, such as pEC 50 For at least 8, for example, pEC 50 The value should be at least 8.5, preferably pEC. 50 Compounds with at least 9.

[0052] In some embodiments, the peptide agonist may be the pEC of the human receptor as determined as described in Example 7. 50 Compounds in the range of 7.2 to 9.5.

[0053] For example, peptide agonists can be pECs of human receptors. 50 For at least 7.5, such as pEC 50 The value should be at least 7.8, preferably pEC. 50 For at least 8, for example, pEC 50 For example, pEC. 50 Compounds with at least 9.

[0054] In particular, Tacr2 agonists, such as peptide agonists, can be pK d Compounds of 6 to 10 are determined, for example, in assays such as radioligand binding assays or “single-receptor” bioassays described in Bellucci F. et al. (2002).

[0055] In particular, Tacr2 agonists, such as peptide agonists, can be pK i Compounds of 5 to 10 are determined, for example, in assays such as radioligand binding assays or “single-receptor” bioassays described in Bellucci F. et al. (2002).

[0056] The assay for Tacr2 activity described at the beginning of this section can also be used to determine whether a potential agonist has a high affinity for Tacr2. Agonists are those that increase Tacr2 activity, such as Ca2+. 2+ Compounds that mobilize or increase inositol accumulation are preferred, especially peptide agonists. Preferably, the peptide agonists of this disclosure are further characterized by a higher affinity for Tacr2 than for Tacr1. Their affinity for Tacr1 may be very low or negligible. They may also be able to bind Tacr1, but their affinity for Tacr2 is higher than their affinity for Tacr1. Therefore, preferably, Tacr2 agonists, such as Tacr2 peptide agonists, have an affinity for Tacr2 at least twice that for Tacr1, preferably at least five times.

[0057] Tacr2 agonists can be either naturally occurring or artificial compounds. A non-limiting list of natural Tacr2 agonists found in humans (Homo sapiens, Hs), rats (Rattus norvegicus, Rn), and guinea pigs (Cavia porcellus, Cp) and their affinity for Tacr2 is presented in Table 1 (Douglas SD, et al. 2015).

[0058] Table 1: Tacr2 agonists and their affinity for receptors.

[0059]

[0060]

[0061] Tacr2 agonists, such as peptide agonists, can be highly potent compounds that can be measured in assays such as the radioligand binding assay or the “single receptor” bioassay described by Bellucci F. et al. (2002), where the highest value indicates the efficacy of the agonist being measured, and the difference between the highest and lowest values ​​represents the efficacy span of the measured value.

[0062] In one embodiment of this disclosure, the Tacr2 agonist, such as a peptide agonist, can be a compound with the highest possible efficacy. This efficacy can be obtained from the highest values ​​obtained in assays such as the radioligand binding assay or the "single receptor" bioassay described by Bellucci F. et al. (2002).

[0063] In one embodiment of this disclosure, the Tacr2 agonist, such as a peptide agonist, may be a compound having an efficacy of at least 1260 relative units, for example, at least 1300 relative units, for example, at least 1400 relative units, for example, at least 1500 relative units, for example, at least 1600 relative units, for example, at least 1700 relative units, for example, at least 1800 relative units, for example, at least 1900 relative units, for example, at least 2000 relative units, for example, at least 2400 relative units, for example, at least 2700 relative units, against the mouse receptor as determined as described in Example 7.

[0064] In one embodiment of this disclosure, the Tacr2 agonist, such as a peptide agonist, may be a compound having an efficacy of at least 2000 relative units, for example, at least 2200 relative units, for example, at least 2400 relative units, for example, at least 2600 relative units, for example, at least 2800 relative units, for example, at least 3000 relative units, against a human receptor as determined as described in Example 7.

[0065] In one embodiment of this disclosure, the Tacr2 agonist, such as a peptide agonist, can be a compound with the widest possible efficacy span. This efficacy span can be determined by subtracting the lowest value from the highest value obtained in assays such as radioligand binding assays or “single-receptor” bioassays as described in, for example, Bellucci F. et al. (2002).

[0066] In one embodiment of this disclosure, the Tacr2 agonist, such as a peptide agonist, may be a compound having a mouse receptor-related efficacy span of at least 1100 relative units, for example, at least 1300 relative units, for example, at least 1400 relative units, for example, at least 1500 relative units, for example, at least 1600 relative units, for example, at least 1700 relative units, for example, at least 1800 relative units, for example, at least 1900 relative units, for example, at least 2000 relative units, for example, at least 2400 relative units, for example, at least 2700 relative units, when measured as described in Example 7.

[0067] In one embodiment of this disclosure, the Tacr2 agonist, such as a peptide agonist, may be a compound having a human receptor-related efficacy span of at least 1700 relative units, for example, at least 2000 relative units, for example, at least 2200 relative units, for example, at least 2400 relative units, for example, at least 2600 relative units, for example, at least 2800 relative units, for example, at least 3000 relative units, as determined as described in Example 7.

[0068] peptide agonists

[0069] One aspect of this disclosure relates to a peptide agonist of Tacr2 or a pharmaceutically acceptable salt thereof for treating diabetes and / or obesity in individuals with this need, wherein said peptide agonist comprises or is composed of a peptide. As described in the “Conjugation Part” section below, when the peptide agonist of Tacr2 comprises a peptide, said peptide may be covalently linked to the conjugation part. Preferably, the peptide agonist is capable of binding Tacr2 in cells expressing Tacr2 and causing Ca2+. 2+ Increased mobilization or inositol accumulation. As described in the "Tacr2 Agonists" section above, peptide agonists are characterized by a high affinity for Tacr2. Some examples of Tacr2 peptide agonists and their affinities are listed in Table 1.

[0070] In one embodiment, the peptide agonist comprises or consists of neurokinin A or a functional analogue thereof. Neurokinin A, also known as substance K, is a peptide translated from the pre-protachykinin gene. Neurokinin A can be translated from different isoforms of the pre-protachykinin gene via alternative splicing. In humans, the gene encoding neurokinin A has two isoforms, and therefore two precursor proteins, namely, the protachykinin-1 isoform β precursor (SEQ ID NO:12) and the protachykinin-1 isoform γ precursor (SEQ ID NO:13). Similarly, in mice, the gene encoding neurokinin A also has two isoforms, and therefore two precursor proteins, called the protachykinin-1 isoform 2 precursor (SEQ ID NO:14) and the protachykinin-1 isoform 1 precursor (SEQ ID NO:15).

[0071] In some embodiments, the peptide agonist of Tacr2 comprises a precursor protein of neurokinin A or a fragment thereof, or is composed of a precursor protein of neurokinin A or a fragment thereof. Therefore, in some embodiments, the peptide agonist comprises a precursor protein of neurokinin A of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or a functional analog thereof having at least 75%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% sequence identity with it. Preferably, the peptide agonist comprises a precursor of human tachykinin-1 isoform β or a fragment thereof, or is composed of a precursor of human tachykinin-1 isoform β or a fragment thereof. More preferably, the peptide agonist comprises a precursor of human tachykinin-1 isoform γ or a fragment thereof, or is composed of a precursor of human tachykinin-1 isoform γ or a fragment thereof. More preferably, the peptide agonist comprises a precursor of murine tachykinin-1 isoform 2 or a fragment thereof, or is composed of a precursor of murine tachykinin-1 isoform 2 or a fragment thereof. More preferably, the peptide agonist comprises a precursor of murine protakinin-1 isoform 1 or a fragment thereof, or is composed of a precursor of murine protakinin-1 isoform 1 or a fragment thereof.

[0072] In some embodiments, the peptide agonist comprises a fragment of the precursor protein of neurokinin A of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or a functional analog thereof having at least 75%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% sequence identity with it. Thus, in some embodiments, the peptide agonist consists of a peptide optionally linked to a conjugation motif, wherein the peptide consists of a continuous sequence of up to 130 amino acid residues of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or any of the foregoing functional analogs having at least 75%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% sequence identity with it. For example, in some embodiments, the peptide agonist may consist of a peptide optionally linked to a conjugation moiety, wherein the peptide consists of a continuous sequence of up to 130 amino acid residues of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or any of the foregoing functional analogs having at least 75%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% sequence identity therewith, wherein the peptide comprises the sequence of SEQ ID NO:16 and / or SEQ ID NO:30.

[0073] Preferably, the peptide agonist of Tacr2 is a functional analog of neurokinin A. In some embodiments, the peptide agonist of Tacr2 comprises or consists of the concordant sequence X6X1FX2X3X4X5 [SEQ ID NO:30], and wherein...

[0074] X6 = aspartic acid (E) or glutamic acid (D);

[0075] X1 = serine (S) or lysine (K);

[0076] X2 = valine (V) or tryptophan (W);

[0077] X3 = glycine (G), β-alanine (β-ala);

[0078] X4 = methyl-leucine (Me-Leu), L, γ-lactam-leucine (γ-lactam-Leu);

[0079] X5 = ortholeucine (Nle), methionine (M).

[0080] Even more preferably, the peptide agonist of Tacr2 consists of a peptide optionally linked to a conjugation portion, wherein the peptide consists of a sequence of at least 7 amino acids and at most 130 amino acids, wherein the sequence comprises SEQ ID NO:30.

[0081] In another embodiment, the peptide agonist of Tacr2 consists of a peptide optionally linked to a conjugation portion, wherein the peptide comprises at least 7 and at most 130 amino acid residues comprising the sequence of SEQ ID NO: 30. Preferably, the peptide comprises at least 7 and at most 100 amino acid residues comprising the sequence of SEQ ID NO: 30, for example, at least 7 and at most 70 amino acid residues comprising the sequence of SEQ ID NO: 30, for example, at least 7 and at most 50 amino acid residues comprising the sequence of SEQ ID NO: 30, for example, at least 7 and at most 30 amino acid residues comprising the sequence of SEQ ID NO: 30, for example, at least 7 and at most 20 amino acid residues comprising the sequence of SEQ ID NO: 30, for example, at least 7 and at most 15 amino acid residues comprising the sequence of SEQ ID NO: 30. In a further embodiment, the peptide agonist comprises 7 amino acid residues of SEQ ID NO: 30.

[0082] Preferably, the peptide agonist of Tacr2 is a functional analog of neurokinin A. In some embodiments, the peptide agonist of Tacr2 comprises or consists of the concordant sequence DX1FX2X3X4X5 [SEQ ID NO:16], and wherein...

[0083] X1 = serine (S) or lysine (K);

[0084] X2 = valine (V) or tryptophan (W);

[0085] X3 = glycine (G), β-alanine (β-ala);

[0086] X4 = methyl-leucine (Me-Leu), L, γ-lactam-leucine (γ-lactam-Leu);

[0087] X5 = ortholeucine (Nle), methionine (M).

[0088] Even more preferably, the peptide agonist of Tacr2 consists of a peptide optionally linked to a conjugation portion, wherein the peptide consists of a sequence of at least 7 amino acids and at most 130 amino acids, wherein the sequence comprises SEQ ID NO:16.

[0089] In some embodiments, the peptide agonist may contain non-standard amino acid residues, such as D-γ-lactam, β-alanine, γ-lactam-leucine, methyl-leucine, ororoleucine, or other amino acids that may enhance the affinity of the peptide agonist for Tacr2 or its agonistic behavior.

[0090] In another embodiment, the peptide agonist of Tacr2 consists of a peptide optionally linked to a conjugation portion, wherein the peptide comprises at least 7 and at most 130 amino acid residues comprising the sequence of SEQ ID NO:16. Preferably, the peptide comprises at least 7 and at most 100 amino acid residues comprising the sequence of SEQ ID NO:16, for example, at least 7 and at most 70 amino acid residues comprising the sequence of SEQ ID NO:16, for example, at least 7 and at most 50 amino acid residues comprising the sequence of SEQ ID NO:16, for example, at least 7 and at most 30 amino acid residues comprising the sequence of SEQ ID NO:16, for example, at least 7 and at most 20 amino acid residues comprising the sequence of SEQ ID NO:16, for example, at least 7 and at most 15 amino acid residues comprising the sequence of SEQ ID NO:16. In a further embodiment, the peptide agonist comprises 7 amino acid residues of SEQ ID NO:16.

[0091] In another embodiment, the peptide agonist of Tacr2 comprises or consists of sequences selected from or composed of sequences selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and any of the aforementioned sequences in which up to two amino acids have been replaced with standard or non-standard amino acids.

[0092] Specifically, the peptide agonist of Tacr2 may comprise or consist of sequences selected from or composed of sequences selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and any of the aforementioned sequences in which at most one amino acid has been substituted. More preferably, the peptide agonist of Tacr2 comprises or consists of sequences selected from or composed of sequences selected from the following: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

[0093] In another embodiment, the peptide agonist of Tacr2 consists of a peptide optionally linked to a conjugation portion, wherein the peptide comprises at least 7 and at most 130 amino acid residues selected from the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and any of the aforementioned sequences wherein at most 2, for example, at most 1 amino acid has been replaced with a standard or non-standard amino acid. Specifically, the peptide agonist of Tacr2 may consist of a peptide optionally linked to a conjugated portion, wherein the peptide comprises at least 7 and at most 100 amino acid residues comprising a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, for example comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. The sequences of NO:26 and SEQ ID NO:27 contain at least 7 and at most 70 amino acid residues.For example, it may contain at least 7 and at most 50 amino acid residues selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; or, for example, it may contain at least 7 and at most 30 amino acid residues selected from SEQ ID NO:1, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; or, for example, it may contain at least 7 and at most 30 amino acid residues selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; or, for example, it may contain at least 7 and at most 30 amino acid residues selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; or, for example, it may contain at least 7 and at most 5 ... The sequences NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27 contain at least 7 and at most 15 amino acid residues.

[0094] Therefore, in some embodiments, the peptide agonist comprises a peptide optionally linked to a conjugation portion, wherein the peptide comprises a continuous sequence of 7 to 130 amino acids, for example, 7 to 100 consecutive amino acids, for example, 7 to 50 consecutive amino acids, for example, 7 to 15 consecutive amino acids, or any of the aforementioned fragments, or a functional analog having at least 75%, for example, at least 80%, for example, at least 85%, for example, at least 90%, for example, at least 95% sequence identity with it, wherein the peptide comprises a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0095] In a preferred embodiment, the peptide agonist comprises a peptide optionally linked to a conjugation portion, wherein the peptide comprises a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0096] In another embodiment, the peptide agonist of Tacr2 consists of a peptide optionally linked to a conjugation portion, wherein the peptide consists of at least 7 and at most 130 amino acid residues comprising the sequence SEQ ID NO:2, wherein at most 2, for example, at most 1 amino acid has been replaced with a standard or non-standard amino acid. Specifically, the peptide agonist of Tacr2 may consist of a peptide optionally linked to a conjugation portion, wherein the peptide consists of at least 7 and at most 100 amino acid residues comprising the sequence SEQ ID NO:2, for example, at least 7 and at most 70 amino acid residues comprising the sequence SEQ ID NO:2, for example, at least 7 and at most 50 amino acid residues comprising the sequence SEQ ID NO:2, for example, at least 7 and at most 30 amino acid residues comprising the sequence SEQ ID NO:2, for example, at least 7 and at most 15 amino acid residues comprising the sequence SEQ ID NO:2.

[0097] Therefore, in some embodiments, the peptide agonist consists of a peptide optionally linked to a conjugation portion, wherein the peptide consists of a continuous sequence of SEQ ID NO:2 in the range of 7 to 130 amino acids, for example, in the range of 7 to 100 consecutive amino acids, for example, in the range of 7 to 50 consecutive amino acids, for example, in the range of 7 to 15 consecutive amino acids, wherein at most 2, for example, at most 1 amino acid has been replaced with a standard or non-standard amino acid.

[0098] In a preferred embodiment, the peptide agonist consists of a peptide optionally linked to a conjugation moiety, wherein the peptide consists of the sequence SEQ ID NO:2.

[0099] In another embodiment, the peptide agonist of Tacr2 consists of a peptide optionally linked to a conjugation portion, wherein the peptide comprises at least 10 and at most 130 amino acid residues comprising the sequence SEQ ID NO:1, wherein at most 2, for example, at most 1, amino acid has been replaced with a standard or non-standard amino acid. Specifically, the peptide agonist of Tacr2 may consist of a peptide optionally linked to a conjugation portion, wherein the peptide comprises at least 10 and at most 100 amino acid residues comprising the sequence SEQ ID NO:1, for example, at least 10 and at most 70 amino acid residues comprising the sequence SEQ ID NO:1, for example, at least 10 and at most 50 amino acid residues comprising the sequence SEQ ID NO:1, for example, at least 10 and at most 30 amino acid residues comprising the sequence SEQ ID NO:1, for example, at least 10 and at most 15 amino acid residues comprising the sequence SEQ ID NO:1.

[0100] Therefore, in some embodiments, the peptide agonist consists of a peptide optionally linked to a conjugation portion, wherein the peptide consists of a continuous sequence of SEQ ID NO:1 in the range of 10 to 130 amino acids, for example, in the range of 10 to 100 consecutive amino acids, for example, in the range of 10 to 50 consecutive amino acids, for example, in the range of 10 to 15 consecutive amino acids, wherein at most 2, for example, at most 1 amino acid has been replaced with a standard or non-standard amino acid.

[0101] In a preferred embodiment, the peptide agonist comprises a peptide optionally linked to a conjugation portion, wherein the peptide comprises the sequence SEQ ID NO:1, wherein at most two, for example, at most one, amino acid has been substituted. In a preferred embodiment, the peptide agonist comprises a peptide optionally linked to a conjugation portion, wherein the peptide comprises the sequence SEQ ID NO:1.

[0102] In some embodiments, the peptide agonist comprises or consists of the sequence DSFVGLM (SEQ ID NO:2). Preferably, the peptide agonist comprises or consists of neurokinin A (SEQ ID NO:1). Preferably, the peptide agonist is neurokinin A (SEQ ID NO:1).

[0103] In another embodiment, the peptide agonist of Tacr2 consists of a peptide optionally linked to a conjugation portion, wherein the peptide consists of at least 10 and at most 130 amino acid residues comprising a sequence selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, wherein at most 2, for example, at most 1, amino acid has been replaced with a standard or non-standard amino acid. Specifically, the peptide agonist of Tacr2 may consist of a peptide optionally linked to a conjugation portion, wherein the peptide comprises at least 10 and at most 100 amino acid residues comprising a sequence selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, for example, comprising at least 10 and at most 70 amino acid residues comprising a sequence selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, for example, comprising at least 10 and at most 5 ... The sequences SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27 contain at least 10 and at most 30 amino acid residues, for example, at least 10 and at most 15 amino acid residues selected from the sequences SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

[0104] Therefore, in some embodiments, the peptide agonist comprises a peptide optionally linked to a conjugation portion, wherein the peptide comprises a continuous sequence of 10 to 130 amino acids, for example, 10 to 100 consecutive amino acids, for example, 10 to 50 consecutive amino acids, for example, 10 to 15 consecutive amino acids, selected from the sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, wherein at most 2, for example, at most 1 amino acid has been replaced with a standard or non-standard amino acid.

[0105] In a preferred embodiment, the peptide agonist comprises a peptide optionally linked to a conjugation portion, wherein the peptide comprises a sequence selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

[0106] In some embodiments, the peptide agonist comprises or consists of sequences selected from the following: SKTDSFVGLM (SEQ ID NO:21), HKTDSFVGLX (SEQ ID NO:22), HKTESFVGLM (SEQ ID NO:23), HKTESFVGLX (SEQ ID NO:24), KTDSFVGLM (SEQ ID NO:25), KDSFVGLM (SEQ ID NO:26), and KESFVGLM (SEQ ID NO:27).

[0107] Conjugated moiety

[0108] A agonist of Tacr2 may comprise or consist of a peptide covalently linked to or composed of the conjugated moiety. For example, a peptide agonist may be any peptide agonist described in the “Peptide Agonists” section above, wherein the conjugated moiety may be any conjugated moiety described in this section.

[0109] In one embodiment, the conjugated moiety can be a peptide, sugar, lipid, or any other chemical group that can be covalently linked to the peptide. Preferably, the conjugated moiety improves the agonistic behavior of the agonist towards Tacr2. The conjugated moiety can also improve the physical properties of the Tacr2 agonist, such as its solubility, stability, or half-life.

[0110] In one embodiment, the conjugated portion may be a compound that masks the agonist relative to the host immune system, such as a polyethylene glycol (PEG) polymer chain or a modified PEG, such as NPEG.

[0111] In some embodiments, a PEG moiety comprising 1 to 50 ethylene glycol units is conjugated to a peptide agonist. The PEG moiety may contain at least 2 ethylene glycol units, such as at least 4 ethylene glycol units, or at least 6 ethylene glycol units. The PEG moiety may reduce the host's immune response to the present peptide agonist, or it may increase its hydrodynamic size and thus its circulation time.

[0112] In some embodiments, the conjugation moiety promotes the interaction of the agonist with membranes and other biological structures. For example, one or more lipids, such as one or more fatty acids, can be conjugated to the peptide agonist. The conjugated fatty acid can enhance the hydrophobicity of the peptide agonist, thereby enhancing its interaction with the membrane. Examples of lipids that can be conjugated to peptide agonists are palmitoleyl, isoprenyl, and myristoyl. Conjugation with other lipid groups is also possible.

[0113] In some embodiments, the conjugated moiety is a peptide, such as a peptide that promotes cell penetration, like polyarginine. Preferably, the conjugated moiety is a peptide that promotes the interaction of Tacr2 agonists with proteins and / or peptides present in the biological system. Advantageously, the conjugated moiety may be a hormone fragment.

[0114] In some implementations, the agonist may be glycosylated, such as N-glycosylated or O-glycosylated. Glycosylation of the agonist can facilitate proper folding or facilitate the recognition of other carbohydrate moieties present in the biological system, such as other glycosylated moieties. Some examples of glycans that can be conjugated to peptides or that can be included in Tacr2 agonists are glycans comprising N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fructose, mannose, and other monosaccharides.

[0115] In some implementations, the conjugated part is a sugar (saccharide), such as a monosaccharide, disaccharide, or polysaccharide.

[0116] In some embodiments, the conjugated portion is a sugar, and the sugar is conjugated to an oxygen atom in an amino acid residue of the peptide. For example, the sugar may be conjugated to a hydroxyl group of serine.

[0117] In some embodiments, the conjugated portion is a sugar, and the sugar is conjugated to a nitrogen atom in an amino acid residue of the peptide. For example, the sugar may be conjugated to an amino group of arginine.

[0118] In some embodiments, the conjugated portion is a sugar, such as mannose (Man). For example, the conjugated peptide agonist comprises or consists of the sequence SEQ ID NO:21 and has mannose conjugated to the serine residue at position 12 of SEQ ID NO:21.

[0119] In some embodiments, the conjugation moiety is an amino group. For example, in embodiments where the agonist of the present invention is a peptide agonist, the peptide may contain C-terminal amidation, for example, the C-terminal OH group of the peptide may be replaced with an amino group.

[0120] In some embodiments, the conjugation moiety is an acetyl group. For example, in embodiments where the agonist of the present invention is a peptide agonist, the peptide may contain an N-terminal acetylation, for example, an N-terminal amine may be acetylated.

[0121] In some implementations, the conjugated portion binds to the terminal amino acid of the peptide agonist.

[0122] In some embodiments, the conjugated portion binds to the terminal amino acid of the peptide agonist, wherein the peptide agonist comprises or consists of sequences selected from or composed of sequences selected from the following: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:30.

[0123] In some embodiments, the conjugation moiety binds to the amino group of the side chain of a non-terminal amino acid of a peptide agonist as defined herein, such as γ-N binding to lysine or arginine.

[0124] In some embodiments, the conjugated moiety is a lipid, and it binds to the peptide agonist directly or via another amino acid. In one embodiment, the conjugated moiety is a fatty acid; for example, the conjugated moiety could be C... 6-40 -alkylene-COOH, for example, C 8-30 -alkylene-COOH, for example, C 10-25 -alkylene-COOH. In one embodiment, the lipid is selected from decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid.

[0125] In some embodiments, the conjugated moiety is a fatty acid linked to an amino acid; for example, the conjugated moiety could be -Naa-C. 6-40 -alkylene-COOH, for example –Naa-C 8-30 -alkylene-COOH, for example –Naa-C 10-25 -alkylene-COOH, where Naa can be any amino acid, such as any protein amino acid, such as Glu or Asp.

[0126] In some embodiments, the conjugated portion is -Naa-myristic acid, and it is bound to the γ-amino group of a non-terminal lysine residue of the peptide agonist. For example, in one embodiment, the conjugated peptide agonist is the peptide of SEQ ID NO:1, wherein glutamate is linked to the γ-N group of the side chain of the lysine residue at position 2 of SEQ ID NO:1, and wherein myristic acid is linked to the amino group of said glutamate.

[0127] In some embodiments, the conjugated moiety is -Naa-palmitic acid, and it is bound to the γ-amino group of a non-terminal lysine residue of the peptide agonist. For example, in one embodiment, the conjugated peptide agonist is the peptide of SEQ ID NO:1, wherein glutamate is linked to the γ-N group of the side chain of the lysine residue at position 2 of SEQ ID NO:1, and wherein palmitic acid is linked to the amino group of said glutamate.

[0128] In some embodiments, the conjugated moiety is -Naa-stearic acid, and it is bound to the γ-amino group of a non-terminal lysine residue of the peptide agonist. For example, in one embodiment, the conjugated peptide agonist is the peptide of SEQ ID NO:1, wherein glutamate is linked to the γ-N group of the side chain of the lysine residue at position 2 of SEQ ID NO:1, and wherein stearic acid is linked to the amino group of the glutamate.

[0129] In some embodiments, the conjugated moiety is palmitic acid, which is bound to the amino group of the N-terminal amino acid of the peptide agonist. For example, in one embodiment, the conjugated peptide agonist is the peptide of SEQ ID NO:1, wherein palmitic acid is linked to the amino group of the N-terminal amino acid of the peptide.

[0130] Tacr2

[0131] The agonist is characterized by a high affinity for and is an agonist of the tachykinin receptor 2 (Tacr2). Tacr2 is a G protein-coupled receptor (GPCR) that the inventors found to be induced in brown and beige adipose tissue upon cold exposure. The inventors discovered that increasing the expression and presence of Tacr2 on the membranes of brown and beige adipocytes, and increasing its activity through the interaction of the peptide agonist with Tacr2, leads to an increase in basal oxygen consumption (OCR), norepinephrine (NE)-induced OCR, and maximal respiration in brown and beige adipocytes. Brown and beige adipocytes, upon NE activation, consume fat and glucose to generate heat.

[0132] The Tacr2 agonist of the present invention can interact with human Tacr2 composed of SEQ ID NO:10 and mouse Tacr2 composed of SEQ ID NO:11.

[0133] Pharmaceutical Composition

[0134] One aspect of this disclosure relates to a pharmaceutical composition, and more particularly to a pharmaceutical composition comprising an agonist of Tacr2 as defined herein or a pharmaceutically acceptable salt thereof, for treating obesity and / or diabetes in an individual with this need.

[0135] An agonist, as defined in the “Agonists of Tacr2” section above, or a pharmaceutically acceptable salt thereof, may be part of a pharmaceutical composition and thus administered to an individual affected by obesity and / or diabetes, as described in the “Methods of Treating Diabetes and / or Obesity” section below.

[0136] Another aspect of this disclosure relates to the use of an agonist of Tacr2, or a pharmaceutically acceptable salt thereof, as defined in the foregoing sections, in the preparation of a medicament for the treatment of diabetes and / or obesity.

[0137] Although the compounds or salts of the present invention can potentially be administered as unprocessed peptide agonists, they are preferably presented in the form of pharmaceutical formulations. Therefore, the present invention further provides pharmaceutical formulations comprising the compounds of the present invention as defined herein, or pharmaceutically acceptable salts or esters thereof, and pharmaceutically acceptable carriers therefor. Pharmaceutical formulations can be prepared using conventional techniques, such as those described by Remington (2005).

[0138] Pharmaceutical compositions may comprise an agonist of Tacr2 as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or diluent. Pharmaceutically acceptable carriers and / or diluents are well known to those skilled in the art. For compositions formulated as liquid solutions, acceptable carriers and / or diluents include saline and sterile water, and may optionally include antioxidants, buffers, antibacterial agents, and other commonly used additives.

[0139] In particular, pharmaceutical compositions comprising the Tacr2 agonist of the present invention or a pharmaceutically acceptable salt thereof can be formulated for parenteral administration. Therefore, the pharmaceutical compositions of the present invention can be formulated into a variety of parenteral formulations.

[0140] For injection and infusion, formulations can be in the form of suspensions, solutions, or emulsions in oily or aqueous media, such as solutions in aqueous polyethylene glycol. Alternatively, the active ingredient can be in powder form, obtained through aseptic separation of a sterile solid or by lyophilization from a solution, for reconstitution with a suitable media such as sterile, pyrogen-free water prior to use. Formulations can be presented in single-dose or multi-dose sealed containers, such as ampoules, vials, pre-filled syringes, infusion bags, or can be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid excipient for injection, such as water, immediately before use. Injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0141] Examples of oily or non-aqueous carriers, diluents, solvents, or media include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, and may contain formulation agents such as preservatives, wetting agents, emulsifiers or suspending agents, stabilizers, and / or dispersants.

[0142] The pharmaceutical composition can be formulated for any type of parenteral administration. In a preferred embodiment, the pharmaceutical composition can be prepared for subcutaneous injection into an individual in need.

[0143] Treatment methods for insulin resistance, diabetes and / or obesity

[0144] One aspect of this disclosure relates to a method for treating insulin resistance, diabetes, and / or obesity in an individual with this need, said method comprising administering to said individual a therapeutically effective amount of a Tacr2 agonist as defined in the “Tacr2 Agonists” section. The invention also provides Tacr2 agonists for use in such methods, for example, for use in the methods described in this section.

[0145] Another aspect of this disclosure relates to a method for treating insulin resistance, diabetes, and / or obesity in an individual with this need, wherein the method includes enhancing the activity of brown adipose tissue cells in the individual by administering a therapeutically effective amount of a Tacr2 agonist as defined in the “Tacr2 agonist” section.

[0146] Another aspect of this disclosure relates to a method for inducing weight loss in an individual with this need, wherein the method comprises administering to the individual a therapeutically effective amount of a Tacr2 agonist as defined in the “Tacr2 agonist” section.

[0147] Another aspect of this disclosure relates to a method for inducing weight loss in an individual with this need, wherein the method includes enhancing the activity of brown adipose tissue cells in the individual by administering a therapeutically effective amount of a Tacr2 agonist, as defined in the “Tacr2 agonist” section, to the individual.

[0148] In some embodiments, the agonist is administered in the form of a pharmaceutical composition as defined in the "Pharmaceutical Compositions" section. Typically, the agonist is administered parenterally. For example, the agonist can be administered by injection. The agonist can be administered by subcutaneous injection.

[0149] In some implementations, the applied agonist binds to Tacr2 in brown and beige adipocytes.

[0150] The applied agonist can have multiple effects on Tacr2. Therefore, it preferably stimulates Tacr2 expression and localization on the cell membrane in brown and beige adipocytes. It also preferably increases glucose uptake, particularly in brown adipocytes. It also preferably increases lipid uptake, particularly in brown adipocytes. It also preferably increases energy expenditure, particularly in brown adipocytes. It also preferably increases oxygen consumption, particularly in brown adipocytes. It also preferably increases thermogenesis, particularly in brown adipocytes. These effects are beneficial for the treatment of obesity and / or diabetes.

[0151] In some embodiments, methods for treating diabetes and / or obesity in individuals with this need include increasing the basal oxygen consumption rate in primary brown adipocytes. Oxygen consumption in adipocytes can be measured using methods known in the art, such as using a Seahorse XF-96 Flux Analyzer, also described in Example 2. Methods for treating diabetes and / or obesity in individuals with this need may also include increasing the norepinephrine-induced oxygen consumption rate in primary brown adipocytes. Methods for treating diabetes and / or obesity in individuals with this need may also include increasing the maximal oxygen consumption rate in primary brown adipocytes.

[0152] In some implementations, methods for treating insulin resistance, diabetes, and / or obesity in individuals with this need include increasing glucose uptake / absorption in brown and beige adipocytes. Therefore, these methods can lead to a decrease in blood glucose levels. Blood glucose levels can be assessed using methods known in the art, such as the Glucose Tolerance Test (GTT) (also described in Example 4), or the Oral Glucose Challenge Test (OGCT), or other tests deemed appropriate by the technician.

[0153] In some embodiments, methods for treating insulin resistance, diabetes, and / or obesity in individuals with this need include increasing energy expenditure of brown and beige adipocytes. Methods for measuring cellular energy expenditure are readily available in the art; for example, energy expenditure can be measured by indirect calorimetry (i.e., O2 consumption / CO2 production) in a metabolic cage on a live animal, as described, for example, in Example 5. Energy expenditure can also be measured by a thermometer implanted in animal tissue that records the temperature of the animal's core and brown adipose tissue in response to an increase in neurokinin A.

[0154] In some implementations, methods for treating individuals with insulin resistance, diabetes, and / or obesity include increasing energy expenditure in brown and beige adipocytes by administering an agonist of Tacr2 as disclosed herein, without desensitizing the brown and beige adipocytes to the Tacr2 agonist.

[0155] In some implementations, methods for treating insulin resistance, diabetes, and / or obesity in individuals with this need include stimulating the release of endogenous neurokinin A from brown and beige adipocytes.

[0156] In some implementations, methods for treating individuals with insulin resistance, diabetes, and / or obesity include increasing Tacr2 expression in brown and beige adipocytes and thus increasing their localization on the cell membrane.

[0157] In some implementations, methods for treating diabetes and / or obesity in individuals with this need include inducing weight loss in the individual.

[0158] In some implementations, the induced weight loss corresponds to a reduction in white adipose tissue.

[0159] In some implementations, the induced weight loss does not correspond to a reduction in brown and beige adipose tissue.

[0160] The dosage of a Tacr2 agonist can vary depending on the specific agonist and the individual. In some embodiments, a Tacr2 agonist, particularly a Tacr2 agonist, can be administered to a human individual, for example, at a dose below 2 μg / kg body weight. In some embodiments, the agonist can be administered to a human individual, for example, at a dose between 0.05 and 2 μg / kg body weight, for example, between 0.1 and 2 μg / kg body weight, for example, between 0.5 and 2 μg / kg body weight, for example, between 1 and 2 μg / kg body weight, for example, between 0.1 and 1.5 μg / kg body weight, for example, between 0.1 and 1 μg / kg body weight, for example, between 0.1 and 0.5 μg / kg body weight.

[0161] In some implementations, the individual in need is affected by obesity and / or diabetes. The individual can be any individual, such as a mammal, particularly a human.

[0162] In one embodiment, the present invention relates to a method for treating diabetes, wherein the diabetes is selected, for example, from type 1 diabetes, type 2 diabetes, and gestational diabetes. The diabetes may, in particular, be diabetes related to insulin resistance.

[0163] In some embodiments, methods for treating diabetes and / or obesity in individuals with this need include increasing the individual's insulin sensitivity. Insulin sensitivity can be determined using methods known in the art, such as the insulin tolerance test (ITT) described in Examples 4 and 5.

[0164] In some embodiments, the present invention provides methods for increasing insulin sensitivity and / or treating insulin resistance. Insulin resistance may be a precursor to type 2 diabetes, or it may be associated with type 2 diabetes. Therefore, methods for treating diabetes and / or obesity may include increasing the insulin sensitivity of the individual. Methods for measuring insulin sensitivity are known in the art and may be, for example, an insulin tolerance test (ITT), in which blood glucose levels are measured after insulin administration. Available ITTs are described in Examples 4 and 5, and those skilled in the art will be able to apply the methods described therein to other mammals, such as humans. In one embodiment, insulin resistance is determined by measuring the systemic glucose clearance rate (GDR) using a hyperinsulinic-euglycemic clamp. GDR reflects the amount of exogenous glucose required to fully compensate for hyperinsulinemia. Whether a particular individual has insulin resistance can be determined, for example, as described in Tam et al., DiabetesCare. 2012 Jul; 35(7):1605-10. doi:10.2337 / dc11-2339.

[0165] Normally, blood glucose levels decrease due to insulin administration and reach C60. min After a period of time, blood glucose levels usually rise again, returning to the levels recorded before insulin administration. Individuals with insulin resistance respond to insulin administration in different ways, and specifically, their responses are characterized by the following:

[0166] -C min C is higher than in individuals without insulin resistance min ; and / or

[0167] - Compared to individuals without insulin resistance, reaching C min The time interval between returning to the "pre-insulin glucose level" is short, which is the blood glucose level recorded before insulin administration.

[0168] According to the method of this disclosure, a Tacr2 agonist is administered to treat insulin resistance, diabetes, and / or obesity in individuals in need, for example, to individuals with or suspected of having insulin resistance. Individuals with or suspected of having insulin resistance and treated according to the method of this disclosure (i.e., by administration of a Tacr2 agonist) respond differently to insulin administration than individuals with or suspected of having insulin resistance who have not been administered a Tacr2 agonist as disclosed herein. Specifically, individuals who have received administration of a Tacr2 agonist as described herein have a response characterized by the following:

[0169] -C min Lower; and / or

[0170] - Reaching C min The time interval between returning to pre-insulin glucose levels is relatively long.

[0171] The response can be compared to an individual who has or is suspected of having insulin resistance but has not been given a Tacr2 agonist as disclosed herein, or to the same individual before treatment.

[0172] Therefore, in some implementations, methods for treating diabetes and / or obesity in individuals with this need include lowering blood glucose C. min .

[0173] In some implementations, methods for treating diabetes and / or obesity in individuals with this need include increasing blood glucose C10 levels in response to insulin. min The time interval between returning to the pre-insulin glucose level (i.e., the blood glucose level recorded before insulin administration), wherein the insulin is endogenous or exogenous.

[0174] In some embodiments, this disclosure relates to a method of treating obesity and / or obesity-related diseases in an individual with this need, wherein the method includes administering to the individual a therapeutically effective amount of a Tacr2 agonist as defined in the “Tacr2 Agonists” section.

[0175] In other embodiments, this disclosure relates to a method of treating obesity and obesity-related diseases in an individual with such need, wherein the method includes enhancing the activity of brown adipose tissue cells in the individual by administering to the individual a therapeutically effective amount of a Tacr2 agonist as defined in the “Tacr2 agonist” section.

[0176] In a further embodiment, this disclosure relates to a method for inducing weight loss, wherein the method includes administering to the individual a therapeutically effective amount of a Tacr2 agonist as defined in the “Tacr2 agonist” section.

[0177] In other embodiments, this disclosure relates to a method for inducing weight loss in an individual with this need, wherein the method includes enhancing the activity of brown adipose tissue cells in the individual by administering a therapeutically effective amount of a Tacr2 agonist, as defined in the “Tacr2 agonist” section, to the individual.

[0178] In some implementations, obesity and / or obesity-related conditions are treated by reducing the weight of the individual in need by at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as 10% or more.

[0179] Therefore, in some embodiments of this disclosure, an agonist of Tacr2 or a pharmaceutically acceptable salt thereof is administered to an individual who has or is suspected of having obesity and / or obesity-related diseases, resulting in a weight loss of at least 5%, for example at least 6%, for example at least 7%, for example at least 8%, for example at least 10% or more.

[0180] In one implementation scheme, the individual requiring treatment is one who is overweight, obese, and / or has obesity-related conditions such as diabetes or heart disease.

[0181] In one embodiment, the individual requiring treatment has a BMI of 25 or higher, such as 30 or higher, 35 or higher, or 40 or higher. In another embodiment, the individual requiring treatment has a waist-to-hip ratio of at least 0.80, such as 0.80-0.84, such as at least 0.85 (female), or at least 0.90, such as 0.9-0.99, such as above 1.00 (male). In a further embodiment, the individual requiring treatment has a fasting blood glucose level of at least 6.1 mmol / L, such as at least 7.0 mmol / L. In an even further embodiment, the individual requiring treatment has a glycated hemoglobin level of at least 42 mmol / mol, such as 42 to 46 mmol / mol, or at least 48 mmol / mol.

[0182] Individuals requiring treatment using the disclosed methods may exhibit one or more of the following symptoms:

[0183] • Elevated blood pressure: ≥140 / 90 mmHg;

[0184] • Dyslipidemia: Triglycerides (TG): ≥1.695mmol / L, and high-density lipoprotein cholesterol (HDL-C) ≤0.9mmol / L (men), ≤1.0mmol / L (women);

[0185] • Central obesity: waist-to-hip ratio >0.90 (men); >0.85 (women), or body mass index >30 kg / m2;

[0186] Microalbuminuria: urinary albumin excretion rate ≥20 μg / min or albumin:creatinine ratio ≥30 mg / g;

[0187] • Hyperglycemia: Blood glucose levels above 11.1 mmol / L (200 mg / dL) or chronic sugar levels of 5.6-7 mmol / L (100-126 mg / dL).

[0188] Therefore, obesity-related diseases can be diseases associated with the above symptoms.

[0189] sequence

[0190]

[0191]

[0192]

[0193]

[0194] Example

[0195] Example 1: Tacr2 expression in adipose tissue in response to cold exposure and a high-fat diet

[0196] animal

[0197] All animal experiments were conducted in accordance with Danish animal regulations, with free access to food and water and a 12-hour light-dark cycle. Male C57Bl / 6 mice were purchased from Taconic (Denmark) at 4 weeks of age and acclimatized for 1 week prior to experiments. For cold exposure studies, mice were randomly assigned to groups based on their body weight and body composition as determined by MRI scans (EchoMRI, TX, USA) and then housed individually in thermoneutral (approximately 28°C) conditions for at least three weeks prior to cold exposure. For high-fat diet studies, male C57Bl / 6 mice were housed at room temperature and fed a 60% high-fat diet (Research diets, D12492, Denmark) for 7 weeks.

[0198] Adipocyte Fractionation

[0199] Interscapular BAT from 4-week-old male C57Bl / 6 mice raised under thermoneutral or cold conditions was digested with type II collagenase (Sigma-Aldrich, Denmark) in a horizontally shaking water bath at 37°C for 30 minutes, with vortexing every 10 minutes. The stromal vascular fraction (SVF) and adipocyte fraction were separated by centrifugation.

[0200] RNA extraction

[0201] Tissue was lysed using Tri-Reagent (Invitrogen, Denmark) and homogenized using Tissue Lyser (Thermo, Denmark). RNA was extracted using the Qiagen RNeasy Mini Kit (Germany) according to the manufacturer's protocol.

[0202] qPCR

[0203] According to the manufacturer's instructions, complementary DNA was prepared using the AB High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Denmark), and qPCR was performed on a Roche LC480 (Roche, Switzerland) using Sybr Green (Primer Design, UK). Primers were purchased from Tag Copenhagen (Denmark), see Table 2:

[0204] Table 2: Primers used for qPCR

[0205] 36B4 Fwd Tcatccagcaggtgtttgaca 36B4 Rev Ggcaccgaggcaacagtt Tacr2 Fwd Gcctccccacaatgtttcta Tacr2 Rev Tgaggacaaacaccaccaga

[0206] result

[0207] In vivo experiments showed that Tacr2 was significantly induced in interscapular brown adipose tissue (iBAT) and inguinal white adipose tissue (iWAT) after cold exposure (4°C) compared to thermoneutral exposure (30°C). Notably, cold-induced Tacr2 expression in iBAT and iWAT remained significantly induced at the end of the 1-week cold exposure period, while epididymal white adipose tissue (eWAT), which did not exhibit browning or significant energy expenditure, did not induce Tacr2 expression under cold provocation. Figure 1a -c). Cold-mediated Tacr2 induction in BAT occurs in both mature adipocytes and preadipocyte stem cells ( Figure 2a -b). Furthermore, a 60% high-fat diet stimulation increased Tacr2 expression levels in brown adipocytes, suggesting therapeutic sensitization to Tacr2 agonists such as NKA in obese individuals. Figure 2c ).

[0208] Results: Tacr2 expression in brown adipocytes, preadipocyte stem cells, and iWAT was induced in response to cold exposure. Tacr2 expression levels in brown adipocytes also increased in response to a high-fat diet.

[0209] Example 2: Inositol 3-phosphate (IP3) levels and O2 consumption in primary brown adipocytes treated with NKA

[0210] Isolation, differentiation and culture of primary adipocytes

[0211] Primary brown preadipocytes were isolated from the interscapular brown fat pad by digestion with type 1 collagenase (Worthington, NJ, USA) at 37°C in a horizontal shaking water bath for 50 minutes, with vortexing every 10 minutes. The collagenase was diluted 1:10 in KRB with free fatty acid-free BSA (Sigma-Aldrich, Denmark). The enzymatic reaction was stopped by adding growth medium, the cell suspension was precipitated, resuspended in growth medium, and then filtered through a 40 μm nylon mesh (VWR, Denmark) to avoid tissue clumps. Preadipocytes were expanded in DMEM (Gibco, Denmark) supplemented with 10% fetal bovine serum (Invitrogen, Denmark) and 1% penicillin and streptomycin (Lonza, Denmark) until confluence was achieved. On day one after reaching confluence, primary brown preadipocytes were differentiated for two days using a mixture of insulin (0.5 mg / mL), rosiglitazone (1 mM), dexamethasone (100 μM), IBMX (50 mM), and T3 (1 μM). Thereafter, cells were maintained in growth medium containing insulin and T3 throughout the experiment. The maintenance medium was changed every two days. Cells were grown at 37°C and 10% CO2.

[0212] Instantaneous transfection and processing

[0213] On day 3 of differentiation, cells were transiently transfected or reseeded via electroporation for experiments. For transient transfection, pcDNA3.1 containing the mouse Tacr2 coding sequence or empty pcDNA3.1 (Genscript, NJ, USA) was electroporated into adipocytes using an electroporation machine program A34 and kit from Lonza (Denmark). Adipocytes were then treated with neurokinin A (10⁻⁷ M) for a specified time.

[0214] IP3 Measurement

[0215] On day one after electroporation, primary brown adipocytes were loaded with 5 μCi of myo-[3H]inositol (Perkin Elmer, Denmark) in complete maintenance medium and incubated overnight. Cells were then treated with NKA at 37°C for 90 min in HBSS supplemented with 10 mM LiCl. After treatment, cells were extracted with 10 mM formic acid, and the cell membrane was captured using poly-L-lysine-coated SPA beads (Perkin Elmer, Denmark) and analyzed on a Top Counter (Perkin Elmer, Denmark).

[0216] Oxygen consumption

[0217] Oxygen consumption was measured using a Seahorse XF-96 flux analyzer (Seahorse Bioscience, Denmark). Primary brown adipocytes were seeded in complete maintenance medium in Seahorse 96-well cell culture plates (SeahorseBiosciences, Denmark) on day one after electroporation and cultured for an additional four days prior to the experiment. Cells were allowed to equilibrate in Flux medium consisting of phenol red-free, unbuffered DMEM (Sigma-Aldrich, Denmark) supplemented with 0.5% free fatty acid-free BSA (Sigma-Aldrich, Denmark), 25 mM glucose, and 1 mM pyruvate (Company, Country), and adjusted to pH 7.4. All readings were taken over two minutes, with mixing over three minutes. Readings were taken 24 hours before IP3 measurement and 72 hours before the start of oxygen consumption measurement. Figure 3 At time 0), neurokinin A (NKA) containing C-terminal amidation of SEQ ID NO:1 was added to cells. The compound was diluted in Flux medium and... Figure 3 The cells were injected into the cell culture at the time shown in b to achieve the following final concentrations: norepinephrine (5 μM), oligomycin (1 μM), FCCP (1 μM), antimycin A (1 μM), and rotenone (1 μM). The experiment was performed on primary adipocytes exposed to NKA for 72 hours. Figure 3 b).

[0218] result:

[0219] In vitro experiments showed that 24-hour NKA treatment increased IP3 levels in primary brown adipocytes overexpressing Tacr2. Figure 3 a). Experiments also showed that 72 hours of NKA treatment increased oxygen consumption (OCR) in primary brown adipocytes not transfected with Tacr2. Figure 3 b).

[0220] Increased oxygen consumption indicates that brown adipocytes are responding to NKA by increasing energy consumption.

[0221] Example 3

[0222] In the Tacr2 loss-of-function study, glucose uptake in primary brown and beige adipocytes was investigated using conditional and systemic knockout mice after administration of neurokinin A (NKA) containing C-terminal amidation of SEQ ID NO:1 or a mediator. Furthermore, the agonistic effect of NKA on the respiratory capacity of human brown and beige adipocytes was tested. Energy expenditure was assessed using PET scans.

[0223] Example 4: Effects of TACR2 agonism on thermogenesis and reversal of obesity and diabetes-related complications

[0224] High-fat diet (HFD)

[0225] Five-week-old male C57Bl / 6 mice were fed HFD, in which 60% of the energy came from fat, until the mice became obese (50g) and glucose intolerant, as a model of type 2 diabetes (T2D).

[0226] Leptin-deficient (Ob / Ob) mice

[0227] Ob / Ob mice were fed a normal diet until they became obese and glucose intolerant, serving as a genetic model of T2D.

[0228] Multiple Low-Dose Streptozotocin (MLDS)

[0229] Seven-week-old male C57Bl / 6 mice fed a normal diet were injected daily with streptozotocin (35 mg / kg) for 5 consecutive days as a model of type 1 diabetes (T1D).

[0230] Insulin Tolerance Test (ITT)

[0231] Insulin was injected intraperitoneally (ip) into obese mice, and glucose levels were assessed within two hours.

[0232] Glucose tolerance test (GTT)

[0233] Inject glucose into the peritoneum of obese mice or administer glucose orally, and assess glucose levels within two hours.

[0234] Weight and body composition

[0235] Weight and body composition were monitored throughout the experiment (via MRI scans).

[0236] Energy consumption

[0237] Throughout the treatment with NKA / mediator, mice were housed in a TSE indirect calorimetric system to measure their food and water intake, physical activity, O2 consumption, and CO2 production.

[0238] Example 5

[0239] animal

[0240] All animal experiments were conducted according to Danish animal regulations, with free access to food and water and a 12-hour light-dark cycle. Male C57Bl / 6 mice were purchased from Janvier (Denmark) at 4 weeks of age and acclimatized for 1 week prior to experiments. For the high-fat diet study, male C57Bl / 6 mice were housed at room temperature and fed a 60% high-fat diet (Research diets, D12492, Denmark) for 18 weeks. Following this diet, the mice became highly obese, typically weighing between 42 and 45 grams. These mice were also referred to as diet-induced obesity (DIO) mice. Mice were randomly assigned to groups based on their weight and body composition as determined by MRI scans (EchoMRI, TX, USA) and then housed individually for at least one week prior to treatment. Oxygen consumption and respiratory efficiency rate (RER) were measured using a PhenoMaster (TSE-Systems, Germany). Food consumption was manually assessed every other day.

[0241] Neurokinin A treatment (therapy)

[0242] The neurokinin A (NKA) of SEQ ID NO:1 was custom-synthesized by Almac Group (UK) and dissolved in sterile saline solution. For treatment, the NKA was dissolved in... Diluted in (B. Braun, DK). Mice were treated twice daily for 9 days.

[0243] Insulin tolerance test

[0244] Mice were fasted for 3 hours prior to the intraperitoneal insulin tolerance test (IPITT). Insulin (Actrapid, Novo Nordisk, DK) was administered at a dose of 1 IU per kg of lean body weight as determined pre-treatment. Blood glucose was measured using a Roche glucometer. Lower blood glucose levels indicated higher insulin sensitivity.

[0245] result

[0246] Neurokinin A treatment increased calorie burning in diet-induced obese (DIO) mice. Figure 4 a) and improved its metabolic parameters. Interestingly, calorie burning was not accompanied by desensitization to NKA; in fact, Figure 4 The results shown in figure a represent the results of 9 consecutive days of treatment. A significant decrease in body weight was also observed. Figure 4 b) This is attributed to the reduction of white adipose tissue rather than brown adipose tissue, such as Figure 4As shown in d. Furthermore, ITT showed that treated mice had significantly lower blood glucose levels after insulin treatment and therefore higher insulin sensitivity than control mice. Interestingly, blood glucose levels were not only lower, but also... min After that, it will return to the base level at a slower pace.

[0247] Example 6

[0248] Peptide Design and Synthesis

[0249] NKA was designed to bind to albumin via N-terminal fatty acid acylation or to bind albumin to γ-Glu on Lys2. The peptide was labeled with myristic acid (C14), palmitic acid (C16), or stearic acid (C18). NKA was also glycosylated using mannose coupled to Ser residues.

[0250] The peptides were synthesized by Almac Group (Scotland) using resin synthesis and purified by RP-HPLC. The peptides were analyzed by HPLC and mass spectrometry.

[0251] Example 7

[0252] IP3 Measurement

[0253] HEK293 cells were transiently transfected using a Lipofectamine 2000 (Thermo Fisher, Denmark) with a plasmid (pcDNA3.1, Genscript, NJ, USA) encoding human or mouse Tacr1-3. Six hours after transfection, cells were loaded with 5 μCi of myo-[3H]inositol (Perkin Elmer, Denmark) under complete maintenance conditions and cultured overnight. Cells were then treated with an NKA analogue at 37°C for 90 min in HBSS supplemented with 10 mM LiCl. Following treatment, cells were extracted with 10 mM formic acid, and the cell membrane was captured using poly-L-lysine-coated SPA beads (Perkin Elmer, Denmark) and analyzed on a Top Counter (Perkin Elmer, Denmark).

[0254] The NKA analog was dissolved in DMSO and diluted in PBS containing 0.2% or 2% w / v bovine serum albumin (BSA) (Sigma, Denmark) without free fatty acids (FFA).

[0255] The data was analyzed using GraphPad Prism 7 (GraphPad, CA, USA), with a 4-parameter logistic curve fitting. Data analysis included calculating the highest and lowest values, hill slope, and logEC50.

[0256] Weight loss

[0257] Diet-induced obese (DIO) mice were prepared as described in Example 5 above. DIO mice were treated once daily by subcutaneous injection (sc) of 1 mg / kg [Lys2-γ-Glu-C16]NKA or a mediator (a sterile filtered saline solution containing 3% w / v FFA-free BSA and 1.25% DMSO) for three consecutive days, followed by a washout period and four additional treatments. Mice were monitored post-treatment to observe the persistence of the treatment effect. Food intake and calorie burn were monitored using a PhenoMaster (TSE-Systems, Germany).

[0258] Results: The results of IP3 assays showed that NKA and NKA analogs (SEQ ID NO:1 and 21, labeled with myristic acid (C14), palmitic acid (C16), stearic acid (C18), or mannose) were specific for the Tacr2 receptor in both mouse and human receptors; see Tables 3, 4, and 5.

[0259] Table 3: Tacr1 activation data relative to Tacr1 for NKA, SP, NKA analogs (SEQ ID NO: 1 and 21, labeled with myristic acid (C14), palmitic acid (C16), stearic acid (C18) or mannose) and stable NKA analogs (SEQ ID NO: 2, 5 and 22 to 27).

[0260]

[0261]

[0262] Table 4: Tacr2 activation data for NKA, NKA analogs (SEQ ID NO: 1 and 21, labeled with myristic acid (C14), palmitic acid (C16), stearic acid (C18) or mannose) and stable NKA analogs (SEQ ID NO: 2, 5 and 22 to 27) relative to Tacr2.

[0263]

[0264]

[0265] Table 5: Tacr3 activation data relative to Tacr3 for NKA, NKB, NKA analogs (SEQ ID NO: 1 and 21, labeled with myristic acid (C14), palmitic acid (C16), stearic acid (C18) or mannose) and stable NKA analogs (SEQ ID NO: 2, 5 and 22 to 27).

[0266]

[0267]

[0268] The results of IP3 assays showed that stable NKA analogs (SEQ ID NO: 2, 5 and 22 to 27) were specific for the Tacr2 receptor in both mouse and human receptor cases; see Table 4.

[0269] NKA and NKA analogs (SEQ ID NO: 1 and 21, labeled with myristic acid (C14), palmitic acid (C16) or stearic acid (C18)) have low BSA binding affinity, indicating that they are specific for Tacr2, as shown in Table 5.

[0270] Table 5: Tcar2 activation of BSA. BSA binding of NKA and NKA analogs (SEQ ID NO: 1 and 21, labeled with myristic acid (C14), palmitic acid (C16), or stearic acid (C18)).

[0271]

[0272] As a result of treatment with [Lys2-γ-Glu-C16]NKA, a significant decrease in body weight was observed. Figure 5 a) This is attributed to a reduction in white adipose tissue rather than brown adipose tissue. Food intake was significantly reduced when mice were treated with [Lys2-γ-Glu-C16]NKA. Treatment with [Lys2-γ-Glu-C16]NKA also increased calorie burning, as seen in diet-induced obese (DIO) mice with weight loss. Figure 5 c) and improvements in metabolic parameters are shown.

[0273] References

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[0278] Cypess AM,Weiner LS,Roberts-Toler C,Franquet Elijah E,KesslerSH,Kahn PA,English J,Chatman K,Trauger SA,Doria A,Kolodny GM,2015.Activation of human brown adipose tissue by aβ3-adrenergic receptoragonist.Cell Metabolism,21(1):33-8.

[0279] Deal MJ,Hagan RM,Ireland SJ,Jordan CC,McElroy AB,Porter B,Ross BC,Stephens-Smith M,Ward P.(1992)Conformationally constrained tachykininanalogues:potent and highly selective neurokinin NK-2 receptoragonists.J.Med.Chem.,35(22):4195-204.

[0280] D'Orléans-Juste P,Dion S,Drapeau G,Regoli D.(1986)Different receptorsare involved in the endothelium-mediated relaxation and the smooth musclecontraction of the rabbit pulmonary artery in response to substance P andrelated neurokinins.Eur.J.Pharmacol.,125(1):37-44.

[0281] Douglas S.D.,Leeman S.E.,Barrett J.,Dombrowsky E.,Heyward C.Y.,Remeshwar P.Tachykinin receptors:NK2 receptor.Last modified on 02 / 04 / 2015.Accessed on 17 / 03 / 2016.IUPHAR / BPS Guide to pharmacology,http: / / www.guidetopharmacology.org / GRAC / ObjectDisplayForward?objectId=361.

[0282] Emonds-Alt X,Golliot F,Pointeau P,Le Fur G,Breliere JC.(1993)Characterization of the binding sites of[3H]SR 48968,a potent nonpeptideradioligand antagonist of the neurokinin-2 receptor.Biochem Biophys ResCommun,191:1172-1177.

[0283] Hansen JB,Tonnesen MF,Madsen AN,Hagedorn PH,Friberg J,Grunnet LG,Heller RS,Nielsen J,Baeyens L,Anker-Kitai L,Qvortrup K,Bouwens L,Efrat S,Aalund M,Andrews NC,Billestrup N,Karlsen AE,Holst B,Pociot F,Mandrup-Poulsen T.(2012).Divalent metal transporter 1 regulates iron-mediated ROS andpancreaticβcell fate in response to cytokines.Cell Metab.16(4):449-61.

[0284] Matuszek MA,Zeng XP,Strigas J,Burcher E.(1998)An investigation oftachykinin NK2 receptor subtypes in the rat.Eur.J.Pharmacol.,352(1):103-9.

[0285] Remington:The Science and Practice of Pharmacy 2005,Lippincott,Williams&Wilkins.

[0286] Sarau HM,Griswold DE,Potts W,Foley JJ,Schmidt DB,Webb EF,Martin LD,Brawner ME,Elshourbagy NA,Medhurst AD,Giardina GA,Hay DW.(1997)Nonpeptidetachykinin receptor antagonists:I.Pharmacological and pharmacokineticcharacterization of SB 223412,a novel,potent and selective neurokinin-3receptor antagonist.J Pharmacol Exp Ther,281:1303-1311.

[0287] van Giersbergen PL,Shatzer SA,Burcher E,Buck SH.(1992)Comparison ofthe effects of neuropeptide K and neuropeptide gamma with neurokinin A at NK2receptors in the hamster urinary bladder.Naunyn SchmiedebergsArch.Pharmacol.,345(1):51-6.

[0288] Warner FJ,Comis A,Miller RC,Burcher E.(1999)Characterization of the[125I]-neurokinin A binding site in the circular muscle of humancolon.Br.J.Pharmacol.,127(5):1105-10.

[0289] Warner FJ, Mack P, Comis A, Miller RC, Burcher E. (2001) Structure-activityrelationships of neurokinin A(4-10) at the human tachykinin NK(2)receptor:therole of natural residues and their chirality. Biochem Pharmacol, 61:55-60. sequence list <110> University of Copenhagen <120> Tacr2 agonists <130> P4155PC02 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Human (Homo sapiens) <400> 1 His Lys Thr Asp Ser Phe Val Gly Leu Met 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(7) <223> NKA <400> 2 Asp Ser Phe Val Gly Leu Met 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(8) <223> NKA, X = NH-(R)-gamma-lactam-Leu <400> 3 Lys Asp Ser Phe Val Gly Xaa Met 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(7) <223> NKA, X = beta - Ala <400> 4 Asp Ser Phe Val Xaa Leu Met 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(7) <223> NKA, X=Nle <400> 5 Asp Ser Phe Val Gly Leu Xaa 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(7) <223> NKA, X = beta - Ala <400> 6 Asp Ser Phe Trp Xaa Leu Met 1 5 <210> 7 <211> 11 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(11) <223> NKA <400> 7 Tyr His Lys Thr Asp Ser Phe Val Gly Leu Met 1 5 10 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(8) <223> NKA, X (position 4) = NH - (R) - Phe and X (position 7) = gamma - Lactam-Leu <400> 8 Lys Asp Ser Xaa Val Gly Xaa Met 1 5 <210> 9 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(7) <223> NKA, X(position 6)=methyl-Leu; X(position 7)=Nle <400> 9 Asp Lys Phe Val Gly Xaa Xaa 1 5 <210> 10 <211> 398 <212> PRT <213> Human (Homo sapiens) <400> 10 Met Gly Thr Cys Asp Ile Val Thr Glu Ala Asn Ile Ser Ser Gly Pro 1 5 10 15 Glu Ser Asn Thr Thr Gly Ile Thr Ala Phe Ser Met Pro Ser Trp Gln 20 25 30 Leu Ala Leu Trp Ala Thr Ala Tyr Leu Ala Leu Val Leu Val Ala Val 35 40 45 Thr Gly Asn Ala Ile Val Ile Trp Ile Ile Leu Ala His Arg Arg Met 50 55 60 Arg Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ala Leu Ala Asp Leu 65 70 75 80 Cys Met Ala Ala Phe Asn Ala Ala Phe Asn Phe Val Tyr Ala Ser His 85 90 95 Asn Ile Trp Tyr Phe Gly Arg Ala Phe Cys Tyr Phe Gln Asn Leu Phe 100 105 110 Pro Ile Thr Ala Met Phe Val Ser Ile Tyr Ser Met Thr Ala Ile Ala 115 120 125 Ala Asp Arg Tyr Met Ala Ile Val His Pro Phe Gln Pro Arg Leu Ser 130 135 140 Ala Pro Ser Thr Lys Ala Val Ile Ala Gly Ile Trp Leu Val Ala Leu 145 150 155 160 Ala Leu Ala Ser Pro Gln Cys Phe Tyr Ser Thr Val Thr Met Asp Gln 165 170 175 Gly Ala Thr Lys Cys Val Val Ala Trp Pro Glu Asp Ser Gly Gly Lys 180 185 190 Thr Leu Leu Leu Tyr His Leu Val Val Ile Ala Leu Ile Tyr Phe Leu 195 200 205 Pro Leu Ala Val Met Phe Val Ala Tyr Ser Val Ile Gly Leu Thr Leu 210 215 220 Trp Arg Arg Ala Val Pro Gly His Gln Ala His Gly Ala Asn Leu Arg 225 230 235 240 His Leu Gln Ala Met Lys Lys Phe Val Lys Thr Met Val Leu Val Val 245 250 255 Leu Thr Phe Ala Ile Cys Trp Leu Pro Tyr His Leu Tyr Phe Ile Leu 260 265 270 Gly Ser Phe Gln Glu Asp Ile Tyr Cys His Lys Phe Ile Gln Gln Val 275 280 285 Tyr Leu Ala Leu Phe Trp Leu Ala Met Ser Ser Thr Met Tyr Asn Pro 290 295 300 Ile Ile Tyr Cys Cys Leu Asn His Arg Phe Arg Ser Gly Phe Arg Leu 305 310 315 320 Ala Phe Arg Cys Cys Pro Trp Val Thr Pro Thr Lys Glu Asp Lys Leu 325 330 335 Glu Leu Thr Pro Thr Thr Ser Leu Ser Thr Arg Val Asn Arg Cys His 340 345 350 Thr Lys Glu Thr Leu Phe Met Ala Gly Asp Thr Ala Pro Ser Glu Ala 355 360 365 Thr Ser Gly Glu Ala Gly Arg Pro Gln Asp Gly Ser Gly Leu Trp Phe 370 375 380 Gly Tyr Gly Leu Leu Ala Pro Thr Lys Thr His Val Glu Ile 385 390 395 <210> 11 <211> 384 <212> PRT <213> Mus musculus <400> 11 Met Gly Ala His Ala Ser Val Thr Asp Thr Asn Ile Leu Ser Gly Leu 1 5 10 15 Glu Ser Asn Ala Thr Gly Val Thr Ala Phe Ser Met Pro Gly Trp Gln 20 25 30 Leu Ala Leu Trp Ala Thr Ala Tyr Leu Ala Leu Val Leu Val Ala Val 35 40 45 Thr Gly Asn Ala Thr Val Ile Trp Ile Ile Leu Ala His Glu Arg Met 50 55 60 Arg Thr Val Thr Asn Tyr Phe Ile Ile Asn Leu Ala Leu Ala Asp Leu 65 70 75 80 Cys Met Ala Ala Phe Asn Ala Thr Phe Asn Phe Ile Tyr Ala Ser His 85 90 95 Asn Ile Trp Tyr Phe Gly Ser Thr Phe Cys Tyr Phe Gln Asn Leu Phe 100 105 110 Pro Val Thr Ala Met Phe Val Ser Ile Tyr Ser Met Thr Ala Ile Ala 115 120 125 Ala Asp Arg Tyr Met Ala Ile Val His Pro Phe Gln Pro Arg Leu Ser 130 135 140 Ala Pro Ser Thr Lys Ala Val Ile Ala Val Ile Trp Leu Val Ala Leu 145 150 155 160 Ala Leu Ala Ser Pro Gln Cys Phe Tyr Ser Thr Ile Thr Val Asp Gln 165 170 175 Gly Ala Thr Lys Cys Val Val Ala Trp Pro Asn Asp Asn Gly Gly Lys 180 185 190 Met Leu Leu Tyr His Leu Val Val Phe Val Leu Ile Tyr Phe Leu 195 200 205 Pro Leu Val Val Met Phe Ala Tyr Ser Val Ile Gly Leu Thr Leu 210 215 220 Trp Lys Arg Ala Val Pro Arg His Gln Ala His Gly Ala Asn Leu Arg 225 230 235 240 His Leu Gln Ala Lys Lys Lys Phe Val Lys Ala Met Val Leu Val Val 245 250 255 Val Thr Phe Ala Ile Cys Trp Leu Pro Tyr His Leu Tyr Phe Ile Leu 260 265 270 Gly Thr Phe Gln Glu Asp With Tyr Tyr Arg Lys Phe With Gln Gln Val 275 280 285 Tyr Leu Ala Leu Phe Trp Leu Ala Met Ser Ser Thr Met Tyr Asn Pro 290,295,300 Ile Ile Tyr Cys Cys Leu And His Arg Phe Arg Ser Gly Phe Arg Leu 305 310 315 320 Ala Phe Arg Cys Cys Pro Trp Gly Thr Pro Thr Glu Glu Asp Arg Leu 325 330 335 Glu Leu Thr His Thr Pro Ser Ile Ser Arg Arg Val Asn Arg Cys His 340 345 350 Thr Lys Glu Thr Leu Phe Met Thr Gly Asp Met Thr His Ser Glu Ala 355 360 365 Thr Asn Gly Gln Val Gly Gly Pro Gln Asp Gly Glu Pro Ala Gly Pro 370 375 380 <210> 12 <211> 129 <212> PRT <213> Homo sapiens <400> 12 Met Lys Ile Leu Val Ala Leu Ala Val Phe Phe Leu Val Ser Thr Gln 1 5 10 15 Leu Phe Ala Glu Glu Ile Gly Ala Asn Asp Asp Leu Asn Tyr Trp Ser 20 25 30 Asp Trp Tyr Asp Ser Asp Gln Ile Lys Glu Glu Leu Pro Glu Pro Phe 35 40 45 Glu His Leu Leu Gln Arg Ile Ala Arg Arg Pro Lys Pro Gln Gln Phe 50 55 60 Phe Gly Leu Met Gly Lys Arg Asp Ala Asp Ser Ser Ile Glu Lys Gln 65 70 75 80 Val Ala Leu Leu Lys Ala Leu Tyr Gly His Gly Gln Ile Ser His Lys 85 90 95 Arg His Lys Thr Asp Ser Phe Val Gly Leu Met Gly Lys Arg Ala Leu 100 105 110 Asn Ser Val Ala Tyr Glu Arg Ser Ala Met Gln Asn Tyr Glu Arg Arg 115 120 125 Arg <210> 13 <211> 114 <212> PRT <213> Homo sapiens <400> 13 Met Lys Ile Leu Val Ala Leu Ala Val Phe Phe Leu Val Ser Thr Gln 1 5 10 15 Leu Phe Ala Glu Glu Ile Gly Ala Asn Asp Asp Leu Asn Tyr Trp Ser 20 25 30 Asp Trp Tyr Asp Ser Asp Gln Ile Lys Glu Glu Leu Pro Glu Pro Phe 35 40 45 Glu His Leu Leu Gln Arg Ile Ala Arg Arg Pro Lys Pro Gln Gln Phe 50 55 60 Phe Gly Leu Met Gly Lys Arg Asp Ala Gly His Gly Gln Ile Ser His 65 70 75 80 Lys Arg His Lys Thr Asp Ser Phe Val Gly Leu Met Gly Lys Arg Ala 85 90 95 Leu Asn Ser Val Ala Tyr Glu Arg Ser Ala Met Gln Asn Tyr Glu Arg 100 105 110 Arg Arg <210> 14 <211> 115 <212> PRT <213> Mus musculus <400> 14 Met Lys Ile Leu Val Ala Val Ala Val Phe Phe Leu Val Ser Thr Gln 1 5 10 15 Leu Phe Ala Glu Glu Ile Asp Ala Asn Asp Asp Leu Asn Tyr Trp Ser 20 25 30 Asp Trp Ser Asp Ser Asp Gln Ile Lys Glu Ala Met Pro Glu Pro Phe 35 40 45 Glu His Leu Leu Gln Arg Ile Ala Arg Arg Pro Lys Pro Gln Gln Phe 50 55 60 Phe Gly Leu Met Gly Lys Arg Asp Ala Gly His Gly Gln Ile Ser His 65 70 75 80 Lys Arg His Lys Thr Asp Ser Phe Val Gly Leu Met Gly Lys Arg Ala 85 90 95 Leu Asn Ser Val Ala Tyr Glu Arg Ser Ala Met Gln Asn Tyr Glu Arg 100 105 110 Arg Arg Lys 115 <210> 15 <211> 130 <212> PRT <213> Mus musculus <400> 15 Met Lys Ile Leu Val Ala Val Ala Val Phe Phe Leu Val Ser Thr Gln 1 5 10 15 Leu Phe Ala Glu Glu Ile Asp Ala Asn Asp Asp Leu Asn Tyr Trp Ser 20 25 30 Asp Trp Ser Asp Ser Asp Gln Ile Lys Glu Ala Met Pro Glu Pro Phe 35 40 45 Glu His Leu Leu Gln Arg Ile Ala Arg Arg Pro Lys Pro Gln Gln Phe 50 55 60 Phe Gly Leu Met Gly Lys Arg Asp Ala Asp Ser Ser Val Glu Lys Gln 65 70 75 80 Val Ala Leu Leu Lys Ala Leu Tyr Gly His Gly Gln Ile Ser His Lys 85 90 95 Arg His Lys Thr Asp Ser Phe Val Gly Leu Met Gly Lys Arg Ala Leu 100 105 110 Asn Ser Val Ala Tyr Glu Arg Ser Ala Met Gln Asn Tyr Glu Arg Arg 115 120 125 Arg Lys 130 <210> 16 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(7) <223> X(position 1) = S, K; X(position 2) = V, W; X(position 3) = G, beta - Ala; X(position 4) = Me-Leu, L, gamma-lactam-Leu; X(position 5)=Nle, M <400> 16 Asp Xaa Phe Xaa Xaa Xaa Xaa 1 5 <210> 17 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> misc_feature <222> (1)..(21) <223> 36B4 Fwd <400> 17 tcatccagca ggtgtttgac a 21 <210> 18 <211> 18 <212> DNA <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> misc_feature <222> (1)..(18) <223> 36B4 Rev <400> 18 ggcaccgagg caacagtt 18 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> misc_feature <222> (1)..(20) <223> Tacr2 Fwd <400> 19 gcctccccac aatgtttcta 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> misc_feature <222> (1)..(20) <223> Tacr2 Rev <400> 20 tgaggacaaa caccaccaga 20 <210> twenty one <211> 10 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(10) <223> NKA <400> twenty one Ser Lys Thr Asp Ser Phe Val Gly Leu Met 1 5 10 <210> twenty two <211> 10 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(10) <223> X = Leucine <400> twenty two His Lys Thr Asp Ser Phe Val Gly Leu Xaa 1 5 10 <210> twenty three <211> 10 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(10) <223> NKA <400> twenty three His Lys Thr Glu Ser Phe Val Gly Leu Met 1 5 10 <210> twenty four <211> 10 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(10) <223> X = Leucine <400> twenty four His Lys Thr Glu Ser Phe Val Gly Leu Xaa 1 5 10 <210> 25 <211> 9 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(9) <223> NKA <400> 25 Lys Thr Asp Ser Phe Val Gly Leu Met 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(8) <223> NKA <400> 26 Lys Asp Ser Phe Val Gly Leu Met 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(8) <223> NKA <400> 27 Lys Glu Ser Phe Val Gly Leu Met 1 5 <210> 28 <211> 11 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> peptides <222> (1)..(11) <223> SP <400> 28 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10 <210> 29 <211> 10 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(10) <223> NKB <400> 29 Asp Met His Asp Phe Phe Val Gly Leu Met 1 5 10 <210> 30 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NKA-related sequences <220> <221> peptides <222> (1)..(7) <223> X (position 1) = E or D; X (position 2) = S or K; X (position 3) = V or W; X (position 4) = G or beta-alanine; X (position 5) = methyl-leucine Or L or gamma-lactam-leucine; X (position 6) = Nle or M <400> 30 Xaa Xaa Phe Xaa Xaa Xaa Xaa 1 5

Claims

1. Use of a Tacr2 agonist or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating insulin resistance, diabetes and / or obesity in an individual with such need, wherein the agonist comprises or is composed of a peptide, wherein the sequence of the peptide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:

27.

2. Use of a Tacr2 agonist or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inducing weight loss in an individual with this need, wherein the agonist comprises or is composed of a peptide, wherein the sequence of the peptide is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:

27.

3. The use as described in any one of claims 1 to 2, wherein the agonist is a peptide optionally linked to a conjugation moiety.

4. The use as claimed in claim 1, wherein the peptide contains C-terminal amidation or N-terminal acetylation.

5. The use as described in claim 3, wherein the conjugated portion is selected from peptides, lipids, polysaccharides, sugars, and PEG.

6. The use as claimed in claim 3, wherein the conjugated portion is sugar.

7. The use as claimed in claim 3, wherein the conjugated portion is mannose.

8. The use as claimed in claim 3, wherein the conjugated portion is mannose, and wherein the mannose is conjugated to the N-terminal amino acid of the peptide.

9. The use as described in claim 3, wherein the conjugated portion is a fatty acid selected from decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid.

10. The use as claimed in claim 3, wherein the conjugated portion is bound to a terminal or non-terminal amino acid of the peptide according to any one of claims 1 to 2.

11. The use as claimed in claim 3, wherein the conjugated portion is selected from -Naa-myristic acid, -Naa-palmitic acid, -Naa-stearic acid, wherein -Naa- is any protein amino acid, and wherein the conjugated portion is bound to the lysine residue at position 2 of the peptide of SEQ ID NO:

1.

12. The use as claimed in claim 3, wherein the conjugated portion is selected from myristic acid, palmitic acid, stearic acid, and wherein the conjugated portion is bound to the N-terminal amino acid of the peptide of SEQ ID NO:

1.

13. The use as claimed in claim 3, wherein the conjugated portion is mannose, and wherein the mannose is conjugated to the N-terminal serine of the peptide of SEQ ID NO:

21.

14. The use as described in any one of claims 1 to 2, wherein the agonist has a higher affinity for Tacr2 than for Tacr1.

15. The use as claimed in claim 1, wherein the diabetes is any one of type 1 diabetes, type 2 diabetes, and gestational diabetes.

16. The use as described in any one of claims 1 to 2, wherein the agonist lowers the minimum blood glucose concentration C of the individual in need. min .

17. The use as claimed in any one of claims 1 to 2, wherein the agonist increases the individual's response to insulin to achieve a minimum blood glucose concentration C. min The time interval between returning to pre-insulin glucose levels.

18. The use as described in any one of claims 1 to 2, wherein the agonist is administered to the individual via parenteral administration.

19. The use as claimed in any one of claims 1 to 2, wherein the treatment or induction further comprises binding the peptide agonist to the Tacr2.

20. The use as described in any one of claims 1 to 2, wherein the treatment or induction further comprises increasing the basal oxygen consumption rate and / or norepinephrine-induced oxygen consumption rate and / or maximal oxygen consumption rate in primary brown adipocytes.

21. The use as described in any one of claims 1 to 2, wherein the treatment or induction further comprises increasing glucose uptake in brown and beige adipocytes.

22. The use as described in any one of claims 1 to 2, wherein the treatment or induction further comprises increasing energy consumption in brown and beige adipocytes.

23. The use as described in any one of claims 1 to 2, wherein the treatment or induction further comprises increasing the individual's insulin sensitivity.

24. The use as described in any one of claims 1 to 2, wherein the treatment or induction further comprises stimulating the release of endogenous neurokinin A from brown and beige adipocytes.

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