Vasopressin 2 receptor antagonist peptides and uses thereof

Abstract

The present disclosure relates to a peptide comprising a sequence having 80% or more amino acid identity with the amino acid sequence of SEQ ID NO: 1: RPSX1CNLPVKPGPCX2GFFSAFYYSQKX3NKCHSFTYGGCAGNANRFSTX4EKCRRTCX5X6, wherein: X1 represents an amino acid residue of F or G; X2 represents any amino acid residue except a basic amino acid residue; X3 represents an amino acid residue of T or D; X4 represents an amino acid residue of I, L, or E; and (i) X5 represents V and X6 represents G, or (ii) X5 represents G and X6 represents V, and the amino acid at position 39 is A. The present disclosure also relates to various diagnostic and therapeutic uses thereof.

Description

[Technical field]

[0001] The present disclosure relates to the field of medicine relating to the treatment of diseases in which the vasopressin 2 receptor is involved. [Background technology]

[0002] The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor encoded by the AVPR2 gene located on chromosome Xq28. V2R belongs to the vasopressin hormone-sensitive receptor family, along with the V1a, V1b and oxytocin receptors. Like many GPCRs, V2R induces pleiotropic actions by both G protein-dependent and G protein-independent mechanisms.

[0003] V2R is expressed especially in the distal part of the nephron. When activated in the collecting duct, the V2R / Gas pathway stimulates intracellular cAMP production, which activates protein kinase A to phosphorylate aquaporin 2, allowing its translocation from intracellular vesicles to the apical membrane via an intracellular calcium-dependent exocytosis mechanism. Furthermore, water can reach the blood thanks to aquaporin 3 and aquaporin 4 on the basolateral membrane after passing aquaporin 2 on the apical membrane from urine to the principal cells. If recirculation is insufficient, V2R is accompanied by long-term activation thanks to the AVP-V2R-beta arrestin activation complex. A third V2 pathway leads to activation of ERK1 / 2 by metalloproteinase-mediated activation of the insulin-like growth factor receptor.

[0004] Both functional variant gains and losses of V2R are associated with human disease. Deficiency in arginine-vasopressin (AVP) secretion is associated with hyponatremia, resulting in plasma sodium levels below 135 mmol / l in humans. This is associated with a number of diseases, including chronic heart failure, liver failure, and chronic kidney disease, where it has been found to be associated with increased risk of death. Ectopic AVP secretion in lung or prostate cancer can also lead to excessive circulating levels of AVP, which can cause hyponatremia. V2R is also a therapeutic target for autosomal dominant polycystic kidney disease (ADPKD). Many therapeutic strategies have been developed for V2R-related diseases, including ADPKD, but only blockade of V2R has demonstrated its efficacy in humans (Nagao et al., 2012, Exp Anim, Vol. 61:477-488).

[0005] Outside the kidney, endothelial cells express V2R, where it is involved in regulating von Willebrand factor expression. In the inner ear, V2R is involved in controlling lymphatic sac volume and is thought to play a role in Meniere's disease (Eckhard et al., 2012, Mol Aspects Med, Vol. 33: 612-637). In bone, V2R may contribute to homeostasis (Tamma et al., 2013, Proc Natl Acad Sci USA, Vol. 110(46): 18644-18649). Neuropeptides such as AVP may be highly expressed in neuroendocrine tumors. V2R is also present ectopically in human lung, breast, pancreatic, colorectal, and gastrointestinal tumors (Pifano et al., 2017, Front Oncol, Vol. 7-Article 11: 1-11).

[0006] In turn, the insufficiency of V2R activity is associated with many diseases, especially in humans. However, despite the crucial physiological and pathological importance of V2R, the track record of the accumulation of therapeutics for this receptor is poor. Only one antagonist molecule, named Tolvaptan, is mainly used in the art, which is a benzazepine-derived molecule belonging to the vaptan family, and acts as an antagonist of V2R with high affinity but moderate selectivity against the other three vasopressin-sensitive receptors. Tolvaptan is currently used to treat hyponatremia and ADPKD, but has many concerns due to hepatotoxicity.

[0007] Excessive secretion of AVP is a key pathogenetic factor in diseases such as hyponatremia, which explains why the use of V2R antagonists is so efficient in pathologies characterized by euvolemic or hypervolemic hyponatremia, such as SIADH (syndrome of inappropriate secretion of antidiuretic hormone), liver cirrhosis, and congestive heart failure (GIF; Ghali et al., Cardiology, 2008, 111, 147-157).

[0008] Congenital nephrogenic diabetes insipidus is associated with inactivating V2R mutations. This causes polyuria with severe dehydration, especially in children. V2R antagonists (vaptans) behave as pharmacological chaperones and are able to enter cells and restore the mutant receptor (Morello et al., J. Clin. Investigation, 2000, 105, 887-895).

[0009] V2R stimulation by interacting with arrestins leads to the activation of signaling pathways involving cAMP and MAP kinases, which support a proliferative response. For example, AVP infusion in rats induces proliferation of renal tubular epithelial cells, which can be inhibited by V2R antagonists (Alonso et al., Endocrinology, 2009, 150, 239-250). Moreover, diuretics such as V2R antagonists were able to inhibit proliferation of renal cancer cells (Bolignano et al., Urol. Oncol., 2010, 28, 642-647) and lung cancer cells (Pequeux et al., Endocr. Relat. Cancer, 2004, 11, 871-885). Such results indicate that V2R antagonists are good therapeutic candidates for various types of cancer.

[0010] Von Willebrand factor (VWF) is involved in primary hemostasis. It has been demonstrated that AVP and the V2R specific agonist dDAVP (Minilin®) also increase the levels of VWF and factor VIII through their interaction with V2R (Kaufmann et al., J. Clin. Invest., 2000, pp. 106, 107-116). Excessive coagulation can lead to thrombosis (blood clots), which can be cured by limiting the secretion of coagulation factors using V2R antagonists.

[0011] Tolvaptan, a V2R antagonist, is also used to treat cirrhosis. A previous meta-analysis showed that treatment with vaptans increases the overall risk of adverse events in cirrhotic patients due to their well-known liver toxicity (Impact of Vaptans on Clinical Outcomes in Cirrhosis Patients: A Meta-Analysis of Randomized Controlled Trials, 2019, Frontiers in Pharmacology).

[0012] Tolvaptan, a V2R antagonist, is also used to treat heart failure. The efficacy and safety of tolvaptan in a real-world clinical setting was confirmed by this large-scale analysis (Tolerability of Tolvaptan in Patients With Heart Failure ― Final Results of the Samsca Post-Marketing Surveillance in Heart Failure (SMILE) Study, 2019, Circulation Journal).

[0013] Although vaptans have clearly demonstrated the therapeutic activity of V2R antagonists against various pathologies, their therapeutic use is limited by some major drawbacks. - Vaptans and their metabolites are hepatotoxic, which requires close patient monitoring and limits their long-term use. Some of them are only administered intravenously, which limits their use to hospitalized patients. - Vaptans have some selectivity for only the V2R, with V2 / Vla selectivity indices ranging from 112 (satavaptan) to 0.15 (conivaptan). - Vaptans are antagonists of MAP kinase activation. Therefore, they are unable to completely block specific V2R-related signaling pathways. Vaptans are poorly soluble in physiological buffers and have limited bioavailability (Bernier et al., JASN, 2006, 17, 591).

[0014] The most selective V2R antagonist known in the art is a peptide originally isolated from mamba snake venom, named mambaquaretin (MQ1). MQ1 is a 57 amino acid residue long peptide that is reticulated by three disulfide bonds and adopts a Kunitz peptide structure (Ciolek et al., 2017, Proc Natl Acad Sic USA, Vol. 114(27):7154-7159). The MQ1 peptide exhibits high affinity for human V2R and no affinity for other 156 GPCRs, including V1aR, V1bR and OTR, at μM concentrations. The MQ1 peptide is a fully competitive antagonist of three pathways related to Gas proteins, interaction with beta-arrestin, and activation of MAP kinases (Ciolek et al., 2017, Proc Natl Acad Sic USA, Vol. 114(27): 7154-7159). Upon administration, the MQ1 peptide induces a pure aquaresis, i.e., a reduction in water but not in any electrolytes. The efficacy of MQ1 in cystic kidney disease has been shown (Ciolek et al., 2017, Proc Natl Acad Sic USA, Vol. 114(27): 7154-7159). [Prior art documents] [Patent documents]

[0015] [Patent Document 1] International Publication No. 2014 / 041526 [Non-patent literature]

[0016] [Non-Patent Document 1] Nagao et al., 2012, Exp Anim, Vol. 61:477-488 [Non-Patent Document 2] Eckhard et al., 2012, Mol Aspects Med, Vol. 33: pp. 612-637 [Non-Patent Document 3] Tammaら, 2013, Proc Natl Acad Sci USA, Vol.110(46):18644~18649 pages

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[0017] There remains a need for alternative or improved V2R antagonists compared to known V2R antagonists, especially for medical use. [Means for solving the problem]

[0018] The present disclosure provides the amino acid sequence of SEQ ID NO:1:

[0019] [ka]

[0020] A peptide comprising a sequence having 80% or more amino acid identity with During the ceremony, - X1 represents an amino acid residue of F or G, - X2 represents any amino acid residue except a basic amino acid residue, - X3 represents an amino acid residue of T or D, - X4 represents an amino acid residue of I, L or E, - (i) X5 means V and X6 means G, or (ii) X5 means G and X6 means V, - relating to a peptide, wherein the amino acid at position 39 is A.

[0021] In some embodiments of the peptide, X1 represents an amino acid residue of G.

[0022] In some embodiments of the peptide, X2 represents an amino acid residue selected from the group consisting of A, D, E, F, G, I, L, N, M, Q, S, T, V and Y.

[0023] In some embodiments of the peptide, X3 represents an amino acid residue of D.

[0024] In some embodiments of the peptide, X4 represents an E or L amino acid residue.

[0025] In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO:1.

[0026] In some embodiments, the peptide has 80% or more amino acid identity with a peptide selected in the group comprising: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the peptide comprises an amino acid sequence selected in the group comprising: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the peptide consists of an amino acid sequence selected in the group comprising: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0027] The peptide may be labeled with a detectable molecule.

[0028] The present disclosure also relates to a nucleic acid encoding the above peptide, which may be included in an expression vector.

[0029] The present disclosure further relates to a pharmaceutical composition comprising a peptide as defined above and a physiologically acceptable carrier.

[0030] The present disclosure also relates to the above peptides for use as a medicament, and for use in treating a disease selected from the group consisting of normovolemic or hypovolemic hyponatremia, renal syndrome of antidiuretic incompatibility, congenital nephrogenic diabetes insipidus, polycystic kidney disease, cancer, thrombosis, Meniere's disease, refractory liver disease, and conditions characterized by heart failure.

[0031] The present disclosure also relates to the in vitro use of the above-described labeled peptides to detect cells expressing the Vasopressin 2 receptor.

[0032] The present disclosure provides an in vitro method for detecting dysregulation of Vasopressin 2 receptor cellular expression, comprising: a) providing a cell to be tested; b) contacting the cells provided in step a) with a labeled peptide according to claim 11; c) measuring the expression level of vasopressin 2 receptor cellular expression; d) comparing the expression level values ​​obtained in step c) with reference expression level values; e) determining the incidence of dysregulation of Vasopressin 2 receptor cell expression; The present invention further relates to a method comprising the steps of:

[0033] The present disclosure also relates to diagnostic reagents comprising the above-described labeled peptides. [Brief description of the drawings]

[0034] [Figure 1]FIG. 1 illustrates the in silico prediction of immunogenicity of a known MQ1 V2R antagonist peptide (also called "U-Da2a") of SEQ ID NO: 7 and a V2R antagonist called MQ-LEAD according to the present disclosure of SEQ ID NO: 6. The sequences of the two peptides were entered into the NETMHCNETMHCPAN3.2 software (www.IEDB.org) by selecting HLA-DR molecules among the most frequent molecules in European and North American populations. The NETMHC software allows prediction of the possible association of a sequence with HLA class II molecules. The selected class II HLA molecules are among the most frequent molecules in European and North American populations. The prediction score is a percentile, which is reported for each sequence of 9 amino acids relative to the first amino acid. The lower the score, the stronger the association of the sequence with the HLA class II molecule is expected. In FIG. 1, each numbered column corresponds to each amino acid residue of a peptide of 57 amino acid residues in length. Top panel: known MQ1 V2R antagonist peptide. Bottom panel: MQ-LEAD peptide of SEQ ID NO: 6. In the rows named "DRBx_nnnn", DRBx is the HLA-DRB locus designation and nnnn is the number of alleles. Immunogenicity scores: (i) score less than 10% (black), (ii) 10-20% (dark grey), and (iii) 20%-30% (light grey). The bold and underlined positions in the bottom panel correspond to the differences in amino acid residues of the MQ-LEAD peptide compared to the MQ1 peptide. [Diagram 2] FIG. 1 illustrates the binding of V2R antagonist peptides according to the present disclosure to V2R in comparison with the V2R binding properties of known MQ1 peptides. Abscissa: log of molar concentration of V2R antagonist peptide or MQ1 peptide tested. Ordinate: 3H-AVP binding, % of control. [Figure 3A]FIG. 1 illustrates the effect of V2R antagonist peptides on diuresis. Rats were injected daily ip at 3 nmol / kg with the V2R antagonist peptides being tested (Sprague-Dawley) and diuresis was followed over time. Abscissa: duration in hours. Ordinate: diuresis in ml / h / kg. Test conditions: (i) MQ1 peptide [black circle "●", dashed line], (ii) MQ-LEAD [black square "□", solid line] and (iii) MQ K39A [black triangle "

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[0035] definition The term "subject" refers to any single subject seeking treatment or participating in a clinical trial, epidemiological study, or used as a control, and includes human and mammalian patients, such as cattle, horses, dogs, and cats. In certain preferred embodiments, the subject is a human.

[0036] The term "treat" or "treating" cancer, as used herein, means administering a combination therapy according to the present invention to a subject with cancer.

[0037] The term "pharmaceutical acceptable" refers generally to materials that are safe, non-toxic, and free of biological or other undesirable substances, useful in the preparation of pharmaceutical compositions, and includes materials acceptable for pharmaceutical use for domestic animals as well as humans.

[0038] In the scope of the present invention, the "percent identity" between two sequences of nucleic acids or peptides / proteins means the percentage of identical nucleotides or amino acid residues between the two sequences being compared, obtained after optimal alignment, this percentage being purely statistical, the differences between the two sequences being randomly distributed along their length. The comparison of sequences is traditionally carried out by comparing the sequences after optimally aligning them, said comparison can be carried out by using segments, or "alignment windows". Optimal alignment of sequences for comparison can be performed by manual comparison, as well as by the local homology algorithm of Smith and Waterman (1981), the local homology algorithm of Neddleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), or computer software employing such algorithms (GAP, BESTFIT, FASTA and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, Inc., 575 Science Dr., Madison, WI, or the comparison software BLAST NR or BLAST P).

[0039] The percent identity between two sequences is determined by comparison of the two optimally aligned sequences, where the sequences being compared may have additions or deletions compared to a reference sequence of optimal alignment between the two sequences. The percent identity is calculated by determining the number of positions where nucleotides or amino acid residues are identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window, and multiplying this result by 100 to obtain the percent identity between the two sequences.

[0040] As used herein, "V2R antagonist activity" refers to the activity of a substance, e.g., a peptide, that (i) binds to V2R, most preferably human V2R, with a binding affinity value of less than 100 nM, and (ii) inhibits cAMP production with an IC50 value of less than 1000 nM.

[0041] Disclosure Details The present disclosure relates to a family of vasopressin 2 receptor antagonists, which may be used primarily for medical and diagnostic purposes.

[0042] The present inventors have identified a family of peptides sharing specific structural features that behave as highly potent vasopressin 2 antagonists both in vitro and in vivo. Such peptides are derived from the known MQ1 peptide (also called "U-Da2a") described in PCT published application WO 2014 / 041526.

[0043] The present inventors have found that several MQ1-derived peptides, which share a common feature of having a lysine (K) residue at amino acid position 39, inhibit the activity of human vasopressin 2 receptor (also referred to herein as "V2R") at nanomolar levels. Notably, the disclosed family of peptides has a significantly higher V2R inhibitory potency than that of known MQ1 peptides. Such peptides have several-fold higher V2R affinity than known MQ1 peptides, for example, 8-fold higher V2R affinity compared to known MQ1 peptides.

[0044] Such peptides disclosed herein behave as potent antagonist compounds of the Vasopressin 2 receptor and are collectively referred to herein as "V2R antagonist peptides."

[0045] As shown in the Examples, the V2R antagonist peptides disclosed herein potently inhibit cAMP production by V2R-expressing cells under vasopressin activity at concentrations much lower than those of the known MQ1 peptide.

[0046] In vivo, the V2R antagonist peptides of the present disclosure do indeed induce aquaresis. Notably, the V2R antagonist peptides disclosed herein are approximately 500 times more potent than tolvaptan, the most therapeutically used V2R antagonist compound for treating hyponatremia, i.e., in this physiological context, they are in fact approximately 500 times more potent than the leading V2R antagonist compounds used to date.

[0047] V2R antagonist peptides The present disclosure provides the amino acid sequence of SEQ ID NO:1:

[0048] [ka]

[0049] A peptide comprising a sequence having 80% or more amino acid identity with During the ceremony, - X1 represents an amino acid residue of F or G, - X2 represents any amino acid residue except a basic amino acid residue, - X3 represents an amino acid residue of T or D, - X4 represents an amino acid residue of I, L or E, - (i) X5 means V and X6 means G, or (ii) X5 means G and X6 means V, - relating to a peptide, wherein the amino acid at position 39 is A.

[0050] As used herein, "X n " and "Xn" may be used indiscriminately to refer to the same variable amino acid residue.

[0051] As used herein, amino acid residues include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V), as well as D. The amino acid may be selected from the group consisting of beta-alanine, gamma-aminobutyric acid, delta-aminolevulinic acid, 4-aminobenzoic acid, aminoisobutyric acid, dehydroalanine, cystine, cystathionine, lanthionine, dienkholic acid, diaminopimelic acid, norleucine, alloisoleucine, isoserine, N-ethylglycine, N-propylglycine, N-isopropylglycine, N-methylalanine, N-ethylalanine, and amino acid analogs including isoserine.

[0052] However, the amino acid residue is most preferably selected from the group consisting of the 20 conventional amino acid residues.

[0053] All amino acids in the peptides of the present invention may be in either the D or L form, although the naturally occurring L form is preferred.

[0054] Without wishing to be bound by any particular theory, the inventors believe that there is a direct correlation between the high V2R antagonist properties of the peptides disclosed herein and the presence of an alanine residue (A) at amino acid position 39 of SEQ ID NO:1.

[0055] As shown in the examples herein, the V2R antagonist peptides disclosed herein are also expected to be endowed with reduced immunogenicity, which may make them eligible for use in the treatment of various pathologies associated with V2R dysfunction, particularly those that require their repeated administration to a subject, such as, illustratively, antidiuretic incompatible nephrogenic syndrome or congenital nephrogenic diabetes insipidus.

[0056] Furthermore, without wishing to be bound by any particular theory, the inventors believe that the V2R antagonist peptides according to the present disclosure are highly selective for V2R, as shown for the known MQ1 peptide. Indeed, the high selectivity of the V2R antagonist peptides for V2R means that they are expected to have low undesirable effects when administered for therapeutic purposes, in contrast to, for example, tolvaptan, a much used V2R antagonist, which has a number of reported undesirable effects due to its moderate selectivity for V2R.

[0057] The inventors believe that the affinity of the V2R antagonist peptides disclosed herein is not substantially affected by the identity of the X2 amino acid residue, except when the X2 amino acid residue consists of a basic amino acid residue. In contrast, the presence of a basic amino acid residue as the X2 amino acid residue negatively affects the affinity of the peptide for V2R, resulting in a change in the binding ability to V2R.

[0058] As used herein, including in the case of the X2 amino acid residue, a basic amino acid residue consists of an amino acid residue, either a conventional or unconventional amino acid residue, which is positively charged at pH 7.0. Basic conventional amino acid residues are selected from the group consisting of K (lysine), R (arginine) and H (histidine).

[0059] Thus, in a preferred embodiment, X2 means any amino acid residue except K, R and H.

[0060] In a preferred embodiment, X2 represents an amino acid residue selected from the group consisting of A, D, E, F, G, I, L, N, M, Q, S, T, V and Y.

[0061] In another preferred embodiment, X2 represents an amino acid residue selected from the group consisting of N, A, E, F and I.

[0062] In a further preferred embodiment, X2 represents an N or A amino acid residue.

[0063] Similarly, the identity of the two naturally occurring amino acids X5 and X6 located at the C-terminus of the V2R antagonist peptides disclosed herein does not substantially affect the binding ability of the peptides to V2R.

[0064] In a most preferred embodiment, the present disclosure relates to a peptide having 80% or more amino acid identity with the peptide of SEQ ID NO: 1, and which does not contain deletions or additions of amino acid residues compared to the peptide of SEQ ID NO: 1. Thus, most preferably, the V2R antagonist peptide according to the present disclosure has a length of 57 amino acids, the amino acid sequence of which does not contain deletions or additions of amino acids compared to SEQ ID NO: 1.

[0065] A peptide having an amino acid sequence with 80% amino acid identity with SEQ ID NO:1 most preferably consists of a peptide that differs exclusively from the peptide of SEQ ID NO:1 by the presence of one or more substitutions of amino acid residues compared to SEQ ID NO:1, whereby the peptide has a length of 57 amino acid residues and contains one or more amino acid substitutions compared to SEQ ID NO:1.

[0066] A peptide whose sequence has 80% amino acid identity with SEQ ID NO:1 can contain up to 11 amino acid substitutions compared to SEQ ID NO:1. The disclosure encompasses peptides that have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 substitutions of an amino acid residue compared to SEQ ID NO:1.

[0067] Thus, the present disclosure encompasses V2R antagonist peptides comprising an amino acid sequence having 80% or more amino acid identity with SEQ ID NO:1 and comprising one or more modifications at one or more amino acid residues, peptide bonds, N- and / or C-terminus of the protein, so long as the V2R antagonist activity is maintained. Such modifications, introduced into the peptide by conventional methods known to those skilled in the art, include, but are not limited to, the substitution of natural amino acids with non-proteinogenic amino acids (D-amino acids or amino acid analogs), the modification of peptide bonds, particularly by retro or retro-inverso bonds or bonds other than peptide bonds, cyclization, and the addition of chemical groups to the side chain or terminus(s) of the protein for coupling of the agent of interest to the V2R peptide antagonists described herein, among others. Such modifications may be used to label the V2R antagonist peptide or to further improve its affinity for V2R, bioavailability, and / or stability.

[0068] In some embodiments, in the V2R antagonist peptides disclosed herein, one or more amino acid residues targeted by endoproteases are replaced by their corresponding non-natural D-forms. For example, one or more arginine and / or lysine residues targeted by trypsin can be replaced by their corresponding non-natural D-forms.

[0069] In some embodiments, in the V2R antagonist peptides disclosed herein, one or more disulfide bonds are replaced by non-natural bonds. Preferably, the non-natural bonds are resistant to reduction, such as thiazolidine linkers. Such linkers improve the resistance of the proteins of the invention to reducing agents present in biological fluids.

[0070] Most preferably, V2R antagonist peptides according to the present disclosure, including peptides containing one or more amino acid substitutions compared to SEQ ID NO:1, contain cysteine ​​residues located at amino acid positions corresponding to positions 5 and 55 of SEQ ID NO:1.

[0071] Most preferably, V2R antagonist peptides according to the present disclosure, including peptides containing one or more amino acid substitutions compared to SEQ ID NO:1, contain cysteine ​​residues located at amino acid positions corresponding to positions 14 and 38 of SEQ ID NO:1.

[0072] Most preferably, V2R antagonist peptides according to the present disclosure, including peptides containing one or more amino acid substitutions compared to SEQ ID NO:1, contain cysteine ​​residues located at amino acid positions corresponding to positions 30 and 51 of SEQ ID NO:1.

[0073] In some embodiments of the peptides disclosed herein, X1 represents an amino acid residue of G (glycine).

[0074] In some embodiments of the peptides disclosed herein, X2 refers to an amino acid residue of A (alanine).

[0075] In some embodiments of the peptides disclosed herein, X3 refers to the amino acid D (aspartic acid).

[0076] In some embodiments of the peptides disclosed herein, X4 represents an amino acid residue of E (glutamic acid) or L (leucine).

[0077] The present disclosure also relates to a peptide comprising the amino acid sequence of SEQ ID NO: 1, in which X1 to X6 each have, independently of the others, the meaning described for SEQ ID NO: 1. It also relates to a peptide consisting of the amino acid sequence of SEQ ID NO: 1, in which X1 to X6 each have, independently of the others, the meaning described for SEQ ID NO: 1.

[0078] The present disclosure also relates to peptides having 80% or more amino acid identity with the amino acid sequence of SEQ ID NO:1, where X1 means F, X2 means N, X3 means T, X4 means I, and X5 and X6 have the same meaning as for SEQ ID NO:1. Most preferably, X5 means V and X6 means G. The present disclosure further relates to peptides having 80% amino acid identity with the peptide of SEQ ID NO:2, which includes a peptide consisting of SEQ ID NO:2.

[0079] The present disclosure also relates to a peptide having 80% or more amino acid identity with the amino acid sequence of SEQ ID NO: 1, where X1 means F, X2 means N, X3 means T, X4 means L, and X5 and X6 have the same meaning as for SEQ ID NO: 1. Most preferably, X5 means V and X6 means G. The present disclosure further relates to a peptide having 80% or more amino acid identity with the peptide of SEQ ID NO: 3. This also relates to a peptide consisting of the amino acid sequence of SEQ ID NO: 3.

[0080] The present disclosure also relates to a peptide having 80% or more amino acid identity with the amino acid sequence of SEQ ID NO: 1, where X1 means F, X2 means A, X3 means T, X4 means L, and X5 and X6 have the same meaning as for SEQ ID NO: 1. Most preferably, X5 means V and X6 means G. The present disclosure further relates to a peptide having 80% or more amino acid identity with SEQ ID NO: 4. This also relates to a peptide consisting of the amino acid sequence of SEQ ID NO: 4.

[0081] The present disclosure also relates to a peptide having 80% or more amino acid identity with the amino acid sequence of SEQ ID NO: 1, where X1 means G, X2 means N, X3 means D, X4 means I, and X5 and X6 have the same meaning as for SEQ ID NO: 1. Most preferably, X5 means V and X6 means G. The present disclosure further relates to a peptide having 80% or more amino acid identity with the peptide of SEQ ID NO: 5. This also relates to a peptide consisting of the amino acid sequence of SEQ ID NO: 5.

[0082] The present disclosure also relates to a peptide having 80% or more amino acid identity with the amino acid sequence of SEQ ID NO: 1, where X1 means G, X2 means N, X3 means D, X4 means E, and X5 and X6 have the same meaning as for SEQ ID NO: 1. Most preferably, X5 means V and X6 means G. The present disclosure further relates to a peptide having 80% or more amino acid identity with the peptide of SEQ ID NO: 6. This also relates to a peptide consisting of the amino acid sequence of SEQ ID NO: 6.

[0083] The present disclosure also provides a compound of formula (I): [Nt-EXT]n-L1x-[V2R-AP]-L2y-[Ct-EXT]m (I) A compound of the formula: - [Nt-EXT] means a chemical moiety covalently attached to the N-terminus of a V2R antagonist peptide disclosed herein; - [V2R-AP] means a V2R antagonist peptide according to the present disclosure; - [Ct-EXT] means a chemical moiety covalently attached to the C-terminus of a V2R antagonist peptide disclosed herein; - n and m each independently represent an integer equal to 0 or 1; - each of L1 and L2, when present, is a linker moiety that can avoid inhibition of the interactions between (i) [Nt-EXT] and [V2R-AP] and (ii) [Ct-EXT] and [V2R-AP], respectively, and more importantly, each of L1 and L2, when present, is useful to prevent either [Nt-EXT] or [Ct-EXT] from affecting the binding of the V2R antagonist peptide [V2R-AP] to V2R; - relating to compounds in which x and y each independently represent an integer equal to 0 or 1.

[0084] In some preferred embodiments, [Nt-EXT] and / or [Ct-EXT], when present, independently refer to a non-proteinaceous chemical moiety, such as a non-proteinaceous detectable molecule radioactive or fluorophore-containing chemical moiety, or a stabilizing moiety, such as ZZ, DsBa, and DsBb, used to label the V2R antagonist peptide. In some such embodiments, [Nt-EXT] and / or [Ct-EXT] are agents that improve the bioavailability of the V2R antagonist peptide described herein, and in particular reduce its urinary elimination, such as, for example, a polyethylene glycol molecule. In some preferred embodiments, [Nt-EXT] and / or [Ct-EXT], when present, independently refer to a polyalkylene glycol, in particular a polyethylene glycol. Such preferred embodiments include polyethylene glycol of the formula H-(O-CH2-CH2)nO-, where n is an integer ranging from 5 to 20, advantageously from 10 to 15, for example, n is an integer ranging from 12.

[0085] In some other preferred embodiments, [Nt-EXT] and / or [Ct-EXT], if present, refer independently to protein moieties, including protein / peptide moieties that allow purification, detection, fixation, and / or cellular targeting of the protein of the invention, and / or improve the affinity, bioavailability, production in an expression system, and / or stability of the V2R antagonist peptide. Such protein moieties may be selected from (i) labeling moieties, such as fluorescent proteins (GFP and its derivatives, BFP and YFP), (ii) reporter moieties, such as enzyme tags (luciferase, alkaline phosphatase, glutathione-transferase (GST), β-galactosidase), (ii) binding moieties for fixation on a support, such as epitope tags (polyHis6, FLAG, HA, myc), DNA binding domains, hormone binding domains, poly-lysine tags, and (iii) targeting moieties for localizing the compound of formula (I) to a specific cell type or cell compartment. In addition, the compound of formula (I) advantageously comprises a linker (L1, L2) connecting each of [Nt-EXT] and / or [Ct-EXT] to [V2R-AP], which, if present, is of sufficient length to avoid inhibition of the interaction between the sequence of [V2R-AP] and each of [Nt-EXT] and / or [Ct-EXT]. The L1 and / or L2 linkers may also comprise a protease recognition site, for example for removing affinity tags and stabilizing moieties from the purified chimeric protein according to the invention.

[0086] In some embodiments, the L1 and / or L2 linker moieties may include a protease recognition site, for example, for removing affinity tags and stabilizing moieties from compounds of Formula (I) according to the present disclosure.

[0087] In addition, some embodiments of the V2R antagonist peptides described herein, or compounds of formula (I), may be advantageously modified by means well known to the skilled artisan in order to alter their physiological properties, in particular to improve their half-life in the organism (glycosylation: HAUBNE R. et al., J. Nucl. Med., 2001, 42, 326-36; conjugation with PEG: KIM TH. et al., Biomaterials, 2002, 23, 2311-7), solubility (hybridization with albumin: KOEHLER MF. et al., Bioorg. Med. Chem. Lett., 2002, 12, 2883-6), resistance to proteases (unnatural amino acids (e.g. D-configuration) and / or intestinal absorption (Lien et al., TIB, 2003, 21, 556-).

[0088] Synthesis of V2R antagonist peptides The V2R antagonist peptides described herein can be produced by known cloning techniques or by chemical synthesis.

[0089] For example, DNA encoding the V2R antagonist peptide is prepared by using cloning technology and inserted into a self-replicating vector to prepare recombinant DNA. The recombinant DNA is introduced into a suitable host, such as Escherichia coli, Bacillus subtilis, Actinomyces, yeast, filamentous fungi, plant cells, insect cells, and animal cells to obtain a transformant. The V2R antagonist peptide disclosed herein can be obtained from the culture product of the transformant. Alternatively, DNA encoding the V2R antagonist peptide is prepared and subjected to a cell-free protein synthesis system using cell extracts derived from wheat germ and E. coli to synthesize the peptide according to the present disclosure.

[0090] The present disclosure encompasses the use of a nucleic acid encoding a V2R antagonist peptide described herein in an expressible form, or a recombinant vector comprising said nucleic acid. A nucleic acid encoding a V2R antagonist peptide in an expressible form refers to a nucleic acid molecule that upon expression in a cell or cell-free system results in a functional peptide.

[0091] Indeed, in embodiments in which the compound of formula (I) comprises a protein, said compound of formula (I) may also be produced as a recombinant protein, following the same methods described above.

[0092] Moreover, for the V2R antagonist peptides disclosed herein, conventional chemical synthesis methods, such as the "solid phase method" or the "liquid phase method", are used to successively bond amino acids and elongate them by dehydration / condensation. Methods for synthesizing V2R antagonist peptides by chemical synthesis are described in the Examples of the present specification.

[0093] Pharmaceutical Compositions The present disclosure further relates to the use of the V2R antagonist peptides described herein or, in some embodiments, compounds of formula (I) comprising said V2R antagonist peptides for medical purposes, in particular for treating pathologies involving the V2R pathway.

[0094] The present disclosure also encompasses the use of nucleic acids encoding in an expressible form some embodiments of the V2R antagonist peptides or proteins of the compounds of formula (I) described herein, or recombinant vectors containing said nucleic acids. Nucleic acids encoding such proteins in an expressible form refer to nucleic acid molecules that upon expression in a cell or cell-free system result in a functional protein, i.e., a protein consisting of a V2R antagonist.

[0095] Thus, in accordance with the present disclosure, the protein, the nucleic acid, and / or the recombinant vector may be comprised in a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0096] Pharmaceutical compositions according to the present disclosure may be formulated for administration by a number of routes, including, but not limited to, enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, subcutaneous, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical, mucosal, nasal, buccal, sublingual, and / or by inhalation, and / or as a mouth spray, nasal spray, and / or aerosol.

[0097] In some embodiments, pharmaceutical compositions according to the present disclosure may be in any suitable form encompassed by the prior art, such as injectable solutions or suspensions, tablets, coated tablets, capsules, syrups, suppositories, creams, ointments, lotions, and the like.

[0098] Pharmaceutical compositions containing V2R antagonist peptides or selected compounds of formula (I) may be in the form of a powder of lyophilizate, where the active ingredient is mixed with sugar, for example, mannitol. For this use, such pharmaceutical compositions are generally reconstituted with an appropriate volume of water or sodium chloride solution. The resulting liquid pharmaceutical composition can then be administered by a suitable route of administration.

[0099] In the pharmaceutical composition, the V2R antagonist peptide or selected compound of formula (I) is mixed with one or more pharma- ceutically acceptable carriers and, optionally, a sustained release matrix, such as a biodegradable polymer, to form a therapeutic composition.

[0100] In certain embodiments, the pharmaceutical composition may further comprise one or more salts, one or more buffering agents, and / or one or more preservatives.

[0101] Pharmaceutically acceptable carriers are those well known to those skilled in the art and are conventionally used.

[0102] Within the scope of this disclosure, a "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating agent, that is involved in the carrying or delivery of a prophylactically or therapeutically active agent.

[0103] In certain embodiments, suitable pharma- ceutically acceptable carriers may be selected in the group including sugars, e.g., lactose, glucose and sucrose, glycols, e.g., propylene glycol, polyols, e.g., glycerin, sorbitol, mannitol and polyethylene glycol, esters, e.g., ethyl oleate and ethyl laurate, buffers, e.g., magnesium hydroxide and aluminum hydroxide, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffers, and the like.

[0104] The pharmaceutical compositions according to the present disclosure contain a therapeutically effective amount of the V2R antagonist peptide or selected embodiment of the compound of formula (I) described herein, or a nucleic acid or recombinant vector encoding the same, e.g., an amount sufficient to provide a medical benefit to the individual to whom it is administered. The pharma- ceutically effective dose depends on the type of composition used, the route of administration, whether administration is in a single or multiple doses, the mammalian species (human or non-human mammal) to be treated, the physical characteristics of the individual parameters including age, health, size, weight, concomitant drug treatment, and other factors recognized by those skilled in the medical art. Thus, within the scope of the present disclosure, the effective amount of the active ingredient to be administered, in particular the amount of the V2R antagonist peptide or selected compound of formula (I), may be determined by a physician or recognized skilled artisan and may be suitably adapted within the time course of treatment.

[0105] The V2R antagonist peptide or selected compound of formula (I) may be administered in an amount ranging from 0.1 to 300 nanomoles of selected V2R antagonist peptide per kg of body weight per administration step, depending on the individual parameters described above. The inventors believe that administration of the V2R antagonist peptides disclosed herein in amounts less than 0.1 nanomoles per kg of body weight is not therapeutically effective. The inventors also believe that administration of the V2R antagonist peptides disclosed herein in amounts greater than 300 nanomoles per kg of body weight may result in undesirable effects, possibly some toxic effects, in the subject.

[0106] Indeed, the exact molecular weight (Mw) of each of the disclosed V2R antagonist peptides can always be determined.

[0107] Assuming that a V2R antagonist peptide according to the present disclosure has a Mw of 6500, administration of 0.1 nmoles / kg thereof to a subject having a body weight of 80 kg would consist of administering 52 ng of said peptide to said subject. Assuming still that a V2R antagonist peptide according to the present disclosure has a Mw of 6500, administration of 300 nmoles / kg thereof to a subject having a body weight of 80 kg would consist of administering 0.156 mg of said peptide to said subject.

[0108] For clarity, in some embodiments where the selected active ingredient comprises a V2R antagonist peptide according to the present disclosure, the amount of the active ingredient administered to a subject in need thereof is calculated based on the Mw value of the V2R antagonist peptide contained therein, and not on the Mw value of the active ingredient itself. Illustratively, in some embodiments where the active ingredient is a compound of formula (I) disclosed herein, the amount of the active ingredient administered to a subject in need thereof is calculated based on the Mw value of the [V2R-AP] moiety contained therein, and not on the Mw value of the active ingredient itself.

[0109] As used herein, an amount of a selected V2R antagonist peptide of 0.1 nmole / kg or more includes an amount of said selected V2R antagonist peptide of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nmole / kg or more.

[0110] As used herein, an amount of the selected V2R antagonist peptide of 300 nmole / kg or less includes amounts of the selected V2R antagonist peptide of 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 150 nmole / kg or less.

[0111] Medical Use The present disclosure relates to the use of the V2R antagonist peptides described herein or selected compounds of formula (I) described herein for the preparation of a medicament. It also relates to the V2R antagonist peptides described herein or selected compounds of formula (I) described herein for use as a medicament.

[0112] The present disclosure also relates to a method for treating a subject suffering from a condition involving the V2R pathway, comprising administering to said subject a V2R antagonist peptide as described herein, or a selected compound of formula (I) as described herein, or a nucleic acid or recombinant vector encoding same.

[0113] The present disclosure also relates to the use of a V2R antagonist peptide as described herein, or a selected compound of formula (I) as described herein, or a nucleic acid or recombinant vector encoding same, for the preparation of a medicament for treating a subject suffering from a condition involving the V2R pathway.

[0114] The present disclosure further relates to a V2R antagonist peptide as described herein, or a selected compound of formula (I) as described herein, or a nucleic acid or recombinant vector encoding same, for use in treating a subject suffering from a condition involving the V2R pathway.

[0115] In some embodiments, the active ingredient, i.e., the V2R antagonist peptide or selected compound of formula (I) described herein, may be administered as a single bolus, as several divided doses administered over time, or the dose may be proportionally reduced or increased as the exigencies of the therapeutic situation dictate. It may be particularly advantageous to formulate the therapeutic agent into a unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to a physically discrete unit suitable as a dosage unit for the mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present disclosure are dictated by and may depend directly on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the field of compounding technology of such active ingredients to address the sensitivities in the individual.

[0116] Thus, one of skill in the art will understand, based on the disclosure provided herein, that doses and administration schedules will be adjusted according to methods well known in the therapeutic arts, i.e., maximum tolerated doses can be readily established, and effective amounts that provide a detectable therapeutic benefit to a subject can also be determined, as can the time requirements for administration of each agent that provides a detectable therapeutic benefit to a subject.

[0117] The determination of appropriate dosages and schedules for administration of chemotherapeutic agents is well known in the relevant art, and it will be understood by those of skill in the art, given the teachings disclosed herein, that this determination is encompassed by the present disclosure.

[0118] Pathologies involving the V2R pathway include, but are not limited to, (i) conditions characterized by normovolemic or hypovolemic hyponatremia, such as congestive heart failure (CHF), cirrhosis, syndrome of inappropriate antidiuretic hormone secretion (SIADH) and cerebral edema, (ii) renal syndrome of inappropriate antidiuretic hormone secretion (NSIAD), (iii) congenital nephrogenic diabetes insipidus (cNDI), (iv) polycystic kidney disease, (v) cancer, including kidney and lung cancer, (vi) thrombosis, (vii) Meniere's disease, (viii) refractory liver disease, and (ix) heart failure.

[0119] Diagnostics According to a further aspect, the present disclosure relates to the use of V2R antagonist peptides or selected compounds of formula (I) as diagnostic or imaging reagents applicable in optical imaging, magnetic resonance imaging (MRI) and positron emission tomography (PET) for detecting V2R cellular expression under physiological or pathological conditions or in response to endogenous or exogenous stimuli for diagnostic or research purposes, which may also be used as a drug screening tool to screen V2R ligands, including V2R agonists and antagonists.

[0120] In a preferred embodiment, the V2R antagonist peptide is labeled by coupling to a detectable molecule.

[0121] The present disclosure then further relates to the labeled V2R antagonist peptides described herein.

[0122] Conventional detectable molecules (labels) capable of producing a detectable signal, alone or in concert with other compositions or compounds, may be used. Suitable detection methods include, for example, detection of agents tagged directly or indirectly with fluorescent labels by immunofluorescence microscopy, including confocal microscopy, or flow cytometry (FACscan), detection of radiolabeled agents by autoradiography, electron microscopy, immunostaining, subcellular fractionation, and the like. In some embodiments, radioactive elements (e.g., radioactive amino acids) are incorporated directly into the peptide chain, while in other embodiments, fluorescent labels may be attached to the peptide via biotin / avidin interactions, such as attachment to fluorescein-conjugated peptides.

[0123] In embodiments of the present disclosure, the detection procedure involves visually inspecting the labeled agent for a color change, or inspecting the labeled V2R peptide antagonist for a physicochemical change, which may occur through oxidation or other chemical reactions, which may be detected by eye, using a spectrophotometer, or the like.

[0124] Thus, detectable molecules useful for labeling the V2R antagonist peptides described herein are preferably labeling agents that generate a detectable and / or quantifiable signal, in particular radioactive, magnetic, or luminescent (radioluminescent, chemiluminescent, bioluminescent, fluorescent, or phosphorescent) agents. Labeled proteins can be directly or indirectly labeled via covalent or non-covalent attachment using standard conjugation techniques well known to those skilled in the art. Examples of labeling agents include radioisotopes, such as technetium 99 ( 99 Tc), fluorine 18( 18 F), tritium ( 3 H) and iodine-125( 125 I) Luminescent agents such as AlexaFluor, FITC and Cyanine 3, paramagnetic contrast agents such as gadolinium compounds, and superparamagnetic contrast agents such as iron oxide nanoparticles.

[0125] For clarity, the V2R antagonist peptides disclosed herein coupled to a detectable molecule may consist of an embodiment of a compound of formula (I), in which at least one of [Nt-EXT] or [Ct-EXT] is present (i.e. has at least one of the integers m or n that equals 1) and comprises or consists of said detectable molecule.

[0126] In some preferred embodiments, in the labeled agent, the V2R antagonist peptide is covalently linked to a radioactive or fluorescent agent.

[0127] Covalent attachment of a labeling agent, e.g., a fluorescent or radioactive agent, to a V2R antagonist peptide can be achieved by (i) incorporating the labeling agent at the N- or C-terminus of the protein during its chemical synthesis, or (ii) incorporating a reactive group (e.g., a free cysteine, biotinyl, azide moiety) into a recombinant or synthetic protein and then using this group to covalently attach the labeling agent.

[0128] Preferably, the labeling agent is covalently attached to the N- or C-terminus of the protein, since neither the N- nor the C-terminus of the V2R antagonist peptide is involved in its binding to V2R.

[0129] The present disclosure relates to the use of a labeled V2R antagonist peptide as described herein for detecting cells expressing V2R in vitro or in vivo, and to the use of a labeled V2R antagonist peptide as described herein for measuring V2R expression levels by cells expressing V2R, in vitro or in vivo.

[0130] The present disclosure also provides an in vitro method for detecting dysregulation of Vasopressin 2 receptor cellular expression, comprising: a) providing a cell to be tested; b) contacting the cells provided in step a) with a labeled V2R antagonist peptide according to the present disclosure; c) measuring the expression level of the vasopressin 2 receptor by the cells provided at the end of step b); d) comparing the expression level values ​​obtained in step c) with reference expression level values; e) determining the incidence of dysregulation of Vasopressin 2 receptor cell expression; The present invention relates to a method comprising the steps of:

[0131] In some embodiments, the reference expression level value used in step d) consists of the average V2R expression level value expected in cells of a subject not suffering from dysregulated V2R cellular expression, i.e. a subject not suffering from a disorder or disease in which the V2R pathway is involved.

[0132] In some other embodiments, the reference expression level value used in step d) consists of a known, otherwise determined or determinable V2R expression value in cells of a subject suffering from dysregulated V2R cellular expression, i.e., a subject suffering from a disorder or disease in which the V2R pathway is involved.

[0133] As is well known in the art, ectopic expression of AVP and its receptor (North et al., 1999, Peptides, Vol. 20:837-842; Sinha et al., 2019, Oncogene, Vol. 30(6):1231-1245) has been shown to mediate the potential antiproliferative effects of V2R agonists in breast, pancreatic, colorectal, and lung cancers (Garona et al., 2015, Int J Oncol, Vol. 46:2335-2345; Ripoll et al., 2013, Breast Cancer Res Treat, Vol. 142:9-18; Iannucci et al., 2011, Future Med Chem, Vol. 3:1987-1993; Garona et al., 2018, Cancer Res Treat, Vol. 51(2): 438-450; Pifano et al., 2017, Front Oncol, 7: 11; Garona et al., 2020, in Vitamins and Hormones, Vol. 113: 259-289) as well as the antiproliferative effect of V2R antagonists in human renal carcinoma (Sinha et al., 2019, Oncogene, Vol. 30(6): 1231-1245). In this context, the V2R antagonist peptides according to the present disclosure are easily interesting probes for imaging cancer cells that highly express V2R.

[0134] Another subject of the present disclosure relates to the use of the V2R antagonist peptides described herein in in vitro diagnostic methods.

[0135] Also, another subject of the present disclosure is the in vitro or in vivo use of the V2R antagonist peptides described herein or selected compounds of formula (I) described herein for diagnosing pathologies involving increased or decreased V2R expression levels.

[0136] A further subject of the present disclosure is a V2R antagonist peptide or a selected compound of formula (I) as described herein for in vitro or in vivo use for diagnosing a pathology involving increased or decreased V2R expression levels.

[0137] In some diagnostic applications, labeled V2R antagonist peptides are used to visualize V2R expression in tissues from patients in vitro or in vivo, and the expression levels are assessed in comparison to the same type of tissue from healthy individuals. High expression of V2R indicates a pathology such as cancer, while low expression of V2R indicates a pathology such as congenital nephrogenic diabetes insipidus (cNDI). Once diagnostic methods have been established, it is possible to determine effective treatments for diagnosed patients, including, for example, the use of V2R antagonists to treat cancer or cNDI.

[0138] The subject of the present invention is also the use of V2R antagonist peptides as research tools for studying V2R.

[0139] Another subject of the present disclosure is a method for producing a cellular membrane comprising: a) contacting the cells to be analyzed with a labeled V2R antagonist peptide; b) detecting the labeled cells; A method for detecting V2R in vitro or in vivo, comprising:

[0140] The cellular label is in particular a fluorescent or magnetic label detectable by any technique known to the person skilled in the art (fluorescence microscopy, flow cytometry, magnetic resonance imaging).

[0141] Detection of V2R receptors in a mammal in vivo (cellular imaging), particularly in real time, includes a preliminary step of administering the labeled V2R antagonist peptide to the mammal (parenteral injection, oral administration).

[0142] Another subject of the present disclosure is the use of the V2R antagonist peptides disclosed herein for screening V2R ligands.

[0143] The subject matter of the present disclosure also includes a) incubating the V2R with a test molecule and a labeled V2R antagonist peptide of the present disclosure; b) measuring the signal obtained in the presence and absence of the test molecule, respectively, where a lower signal in the presence of the molecule compared to a control without the test molecule indicates that the test molecule is a V2R ligand; A method for screening a V2R ligand, comprising:

[0144] The identified ligands are then tested for agonistic or antagonistic effects on V2R in cells expressing V2R using pharmacological assays well known in the art, such as those disclosed in the Examples of this application.

[0145] The present disclosure also provides a kit comprising: (a) a first container containing one or more of the V2R antagonist peptides described herein, nucleic acids or recombinant vectors encoding same, modified host cells, pharmaceutical compositions, diagnostic or imaging reagents, in solution or lyophilized form; (b) a second container, optionally containing a diluent or a reconstitution solution for the lyophilized formulation; (c) a third container, optionally containing an isolated V2R receptor, or a host cell capable of expressing V2R, in solution or lyophilized form, and optionally instructions for use of the solution(s) and / or reconstitution and / or use of the lyophilized formulation(s).

[0146] The present disclosure is further illustrated herein by the examples, but is in no way limited thereto. EXAMPLES

[0147] Example 1 Activity of V2R antagonist peptides A. Materials and Methods Chemical synthesis of peptides Peptide synthesis was performed on a 12.5 μmol scale Gyros Protein Technologies, Inc. Prelude synthesizer and was deprotected, purified, and folded as described (Ciolek et al., 2017, Proc Natl Acad Sci USA, Vol. 114:7154-7159).

[0148] Peptide batches with a purity of more than 95% were used in all experiments. After automated synthesis of MQ1 and deprotection of the N-terminal amine function, 6-azidohexane was coupled onto the resin. 2 eq of 6-azidohexane was coupled twice for 60 min with the coupling agent HCTU (1.9 eq) in the presence of 2 eq of diisopropylethylamine. 6-azidohexane-MQ1 was cleaved from the resin and the MQ1 V2R peptide antagonist was purified and oxidized. 10 eq of DFO-DBCO (p-isothiocyanatobenzyldeferoxamine-diallylbicyclooctyne dissolved in 200 μl of DMF), Cy5.5-DBCO (dissolved in 200 μl of HEPES buffer), or AFDye-488-DBCO (dissolved in 200 μl of HEPES buffer) was mixed with 0.3 μmol of 6-azidohexane-MQ1 dissolved in HEPES buffer (200 μl, pH 7.4), left at room temperature overnight, and purified by HPLC.

[0149] B. Results Various MQ1-derived peptides were tested for their binding affinity to the human vasopressin 2 receptor (V2R).

[0150] The results are summarized in Table 1 below.

[0151] [Table 1]

[0152] In the event of a discrepancy between a sequence disclosed in Table 1 and a sequence set forth in the sequence listing, the correct sequence is the sequence listed in Table 1.

[0153] The results shown in Table 1 show that the IC of 0.5 or less was obtained compared to known MQ1 V2R antagonist peptides. 50 The most efficient peptides tested, which all exhibit ratio values, are peptides in which the lysine residue at amino acid position 39 in a known MQ1 V2R antagonist peptide is replaced by an alanine residue (an amino acid change conventionally designated "K39A"). The most efficient V2R antagonist peptides are the following peptides: - the "K39A" peptide of the amino acid sequence SEQ ID NO: 2, - the "K39A+I48L" peptide of the amino acid sequence SEQ ID NO: 3, - the "N15A+K39A+I48L" peptide of amino acid sequence SEQ ID NO: 4, - the "F4G+K39A+T27D" peptide of amino acid sequence SEQ ID NO:5, and - the "F4G+K39A+T27D I48E" peptide of amino acid sequence SEQ ID NO: 6, also referred to herein as "MQ-LEAD".

[0154] Example 2 Immunogenicity and selectivity of V2R antagonist peptides A. Materials and Methods A.1. Immunogenicity Immunogenicity of protein drugs remains the main risk of their development failure and is therefore a very important concern in the pharmaceutical industry. Immunogenicity is the ability of a molecule to induce an immune response. The antibodies resulting from this response (anti-drug antibodies, or ADA) can inhibit the therapeutic activity of the protein or even induce allergic symptoms. Because immune responses are species-dependent, animals such as mice and rats are the least predictive immunogenicity models and do not allow the immunogenicity of molecules to be evaluated when injected into humans. Preclinical evaluation of immunogenicity is based on the mechanisms that control the immune response in humans.

[0155] The ADA response to protein drugs primarily involves three different cell types: i) B cells, which produce antibodies; ii) CD4 T cells, which provide the necessary support for B cells to secrete antibodies; and iii) dendritic cells, which present proteins as peptides to T cells, thereby triggering their activation. When proteins are injected into the body, they are taken up by dendritic cells and broken down into peptides. Some of the peptides generated have the appropriate anchor residues to bind to HLA class II molecules and are presented to T cells. The peptides recognized by T cells are called T epitopes. Because HLA class II molecules are polymorphic, T epitopes vary from molecule to molecule and therefore vary between individuals based on these HLA class II molecules.

[0156] Considering the role of T lymphocytes in ADA responses, the immunogenicity of protein preparations is highly dependent on their ability to activate T lymphocytes and therefore contain T epitopes in their sequences. The assessment of T epitope content can be performed using prediction software, the most efficient of which is NETMHC. The presence of potential T epitopes in U-Da2a and MQ-LEAD was examined by NETMHC software.

[0157] A.2. Selectivity The general protocol for the protease assay is as follows. 1. Delivers 2X enzyme 2. Deliver buffer to wells that do not contain enzyme 3. Delivering MQ-WT (U-Da2a) into Water Using Acoustic Technology (Echo550; Nanoliter Range) 4 Incubate for 5-15 minutes. 5. Deliver 2X substrate to initiate the reaction 6 Rotate and shake and start measurements at room temperature in EnVision; 25 times at 5 minute intervals (2 hours) 7 Analyze the data by taking the slope of the linear portion of the measurement *(signal / time) 8. Calculate the slope using Excel and perform curve fitting using Prism software

[0158] Specific specifications are disclosed in Table 2 at the end of this disclosure.

[0159] Buffer: A 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij 35 B' 25 mM MES pH 6, 50 mM NaCl, 0.005% Brij 35, 5 mM DTT B 75 mM Tris pH 7.0, 0.005% Brij 35, 3 mM DTT B+EDTA 75mM Tris pH 7.0, 1mM EDTA, 0.005% Brij35, 3mM DTT C 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM CaCl2, 0.025% CHAPS, 1.5 mM DTT D 25 mM sodium acetate pH 5.5, 0.1 M NaCl, 5 mM DTT E 25 mM sodium acetate pH 3.5, 5 mM DTT F 25 mM Tris pH 9, 150 mM NaCl L 400 mM Na Acetate pH 5.5, 4 mM EDTA, 8 mM DTT X (Furin) 100 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 0.5% TX-100, 1 mM DTT S 0.1 M Sodium Acetate pH 3.5, 0.1 M NaCl TCN 25 mM Tris pH 7.5, 10 mM CaCl2, 150 mM NaCl TCNB 25 mM Tris pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35 Z 25 mM Tris pH 9, 2.5 μM ZnCl2, 0.005% Brij KLK7 Buffer 50 mM Tris, 150 mM NaCl, pH 8.5 ACE2 Buffer 75mM Tris, pH 8.5, 1M NaCl Neprilysin Buffer 50mM Tris, pH 9.0 CTSD buffer 100 mM sodium acetate pH 3.5, 200 mM NaCl, 0.02% Brij 35 CTSE buffer 100 mM sodium acetate pH 3.5, 500 mM NaCl, 0.005% TritonX-100 Elastase Buffer 50 mM Tris, pH 7.5, 1 M NaCl, 0.05% Brij35 1x Caspase Buffer 1 50 mM HEPES pH 7.4, 100 mM NaCl, 0.01% CHAPS, 0.1 mM EDTA, 10 mM DTT 1x Caspase Buffer 2 50 mM HEPES pH 7.4, 1 M Na Citrate, 100 mM NaCl, 0.01% CHAPS, 0.1 mM EDTA, 10 mM DTT MMP Buffer: 50 mM HEPES (pH 7.5), 10 mM CaCl2, 0.01% Brij-35, store at 4° C. Add 0.1 mg / ml BSA to the buffer before use.

[0160] B. Results B.1. Immunogenicity The predicted immunogenicity of the known MQ1 peptide of SEQ ID NO:7 and the MQ LEAD peptide of SEQ ID NO:6 has been calculated.

[0161] The comparison results are shown in Figure 1.

[0162] As shown in FIG. 1, the immunogenic regions present in the known MQ1 peptide are no longer found in the MQ LEAD V2R antagonist peptide.

[0163] In MQ1(U-Da2a), 15 potential epitopes were identified, including six epitopes with high scores (<10% percentile), whereas in the MQ-LEAD sequence, only three potential T epitopes were identified, including three with low scores (20-30% percentile).

[0164] The MQ-LEAD peptide then poses a much lower immunogenicity risk than the known MQ1 peptide.

[0165] B.2. Selectivity The selectivity of the known MQ1 peptide for the vasopressin 2 receptor was examined by assaying binding to a number of molecules.

[0166] The results are shown in Table 3 at the end of this disclosure.

[0167] The results in Table 3 illustrate the selectivity of the MQ1 peptide for the vasopressin 2 receptor.

[0168] Example 3 Pharmacology of V2R antagonist peptides A. Materials and Methods—V2R Binding Assay Cell membranes expressing vasopressin receptors were purchased from PERKINELMER (Courtaboeuf, France). 3 Binding experiments were performed with H-AVP (PERKINELMER, Courtaboeuf, France). The reaction mixture contained 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2 and 1 g / L BSA in a final volume of 100 μL. Plates were incubated for 3 h at room temperature. The binding reaction was stopped by filtration through GF / C filters presoaked in 0.5% polyethyleneimine on a cell harvester (PERKINELMER, Courtaboeuf, France) and the plates were dried. Ultimagold O (25 μl; PERKINELMER) was added to each well and samples were counted (counting yield 50%) using a TopCount counter (PERKINELMER, Courtaboeuf, France). Nonspecific binding was measured in the presence of 1 μM AVP. One-site inhibition mass effect curves were fitted to the inhibition binding data using Kaleidagraph (SYNERGY SOFTWARE, Reading, PA, USA). 50Value K i and comparative experiments were performed using the Cheng-Prusoff equation (Cheng et al., Biochem. Pharmacol., 1973, 22, 3099-3108).

[0169] B. Results Binding of V2R antagonist peptides according to the present disclosure to V2R The results are shown in Figure 2.

[0170] The results in Figure 2 show that the MQ-LEAD peptide according to the present disclosure has a 5-fold increase in affinity for the Vasopressin 2 receptor when compared to the known MQ1 (U-Da2a) peptide. Even better results are obtained with the V2R antagonist peptide MQ K39A.

[0171] These results indicate that the V2R antagonist peptides MQ-LEAD and MQ K39A behave as potent V2R antagonist peptides.

[0172] Example 4 In vivo activity of V2R antagonists according to the present disclosure A. Materials and Methods A.1. In vivo diuresis assay Sprague-Dawley rats aged 6–12 weeks were acclimated for 2 days in metabolic cages (Techniplast France, Lyon, France) with food and water ad libitum and then ip-injected (fixed volume 1 ml) with 3 nmol / kg of different MQs dissolved in 0.9% NaCl (French consent number 2015082111349702v1). Urine was collected at different time points and centrifuged at 20,800 g for 30 min. Urine osmolality was determined with an osmometer (Knauer, Berlin, Germany).

[0173] A.2. In vivo assays for experimental models of hyponatremia Hyponatremia rat model. Specific pathogen-free adult male Sprague-Dawley rats (body weight 445-525 g) were obtained from Janvier laboratories (Le Genest St.Isle, 53941, Saint Berthevin Cedex, France) and were acclimated to the animal house conditions for 1 week. We established an experimental protocol according to a previous paper (Miyazaki T, Yamamura Y, Onogawa T, Nakamura S, Kinoshita S, Nakayama S, et al. Therapeutic effects of tolvaptan, a potent, selective nonpeptide vasopressin V2 receptor antagonist, in rats with acute and chronic severe hyponatremia. Endocrinology. 2005;146:3037-43) to induce a hyponatremia rat model approved by the French Ministry of Education and Research (n°18604-2019010915104191). A stock solution of 2.2 mg / mL desmopressin (dDAVP) was prepared by dissolving 5 mg of drug in saline. dDAVP was administered using subcutaneous ALZET osmotic minipumps (2002 model, DURECT Corporation, Cupertino, CA95014, USA) previously filled with a solution of dDAVP (pump flow rate 0.47 ± 0.02 μL / h and mean filling volume 233.6 ± 4.7 μL). The dose of dDAVP (10 ng / h) was determined in a preliminary experiment. Buprenorphine (Centravet, 03120 Lapalisse, France) was administered sc at a dose of 0.02 mg / kg once daily from day 0 to day 3 to suppress postoperative pain. Water gavage (30 mL / kg) was performed twice daily (9 am and 4 pm) at 9 am on the first 3 and 4 days after ALZET pump implantation. Body weight was measured every day and the volume of water administered was adjusted. Rats were allowed to freely consume standard rat chow and water in their cages. MQ1 dissolved in saline was administered at 10 am on the 2nd, 3rd, and 4th days at 10 or 100 μg / kg (sc route, 0.8 mL / kg).At 9 am on days 0, 2, 3 and 4, 400 μL of blood was collected from the tail vein of rats under isoflurane anesthesia with lithium heparinate (Sanofi-Aventis, Gentilly, France). Samples were centrifuged (4°C, 2000g, 5 min). Sodium determination was performed by the ENVT central laboratory, Toulouse, France, on a VITROS 250 / 350 / 950 / 5,1 FS, 4600 and an integrated system VITROS 5600 (Ortho-Clinical Diagnostics, Buckinghamshire, UK). P<0.05, multiple factor ANOVA followed by Tukey's test.

[0174] B. Results B.1. Effect of two V2R antagonist peptides according to the present disclosure on diuresis The results are shown in Figure 3 (Figures 3A and 3B). The results in Figure 3 show that both peptides of the present disclosure, MQ-LEAD and MQ K39A, improve diuresis.

[0175] B.2. Effect of V2R Antagonist Peptides According to the Present Disclosure on Hyponatremia The aim of the present study was to examine the effect of a peptide derived from mambacaretin (MQ-LEAD), a potent and selective antagonist of the V2 receptor, on DDAVP-induced hyponatremia in normal adult male rats of the CD strain (Sprague Dawley).

[0176] The MQ-LEAD peptide was tested in a rat model of hyponatremia. The in vivo assay schedule is shown in FIG.

[0177] The results are shown in Figure 5.

[0178] The results shown in FIG. 5 showed that the effect of tolvaptan (10 mg / kg, oral route) was significantly different from the vehicle-treated group at D4. The mean value of sodium in the tolvaptan-treated group (137.6±3.3 mM). At D4, the plasma sodium concentrations of the three doses of MQ-LEAD (20, 60 and 200 μg / kg, sc) were significantly higher than the corresponding values ​​of the vehicle-treated group. A direct comparison with the dose of tolvaptan (10 mg / kg, po) suggests that MQ-LEAD may be 500 times more potent than tolvaptan. The sodium values ​​after 20 and 60 μg / kg MQ-LEAD at D3 and D4 were elevated but not statistically different from the basal values ​​(D0).

[0179] Example 5 Inhibition of cAMP Production by the V2R Antagonist Peptides of the Disclosure A. Materials and Methods A.1. The antagonistic effects of MQ-WT, MQ-18(K39A) and MQ-232(MQ-LEAD) were examined in the U2OS AVPR2A Nomad cell line stably expressing human arginine vasopressin. The U2OS AVPR2 Nomad cell line contains U2OS cells that stably express human arginine vasopressin receptor 2A (AVPR2A) without tags. This cell line was designed to assay compounds or analyze their ability to modulate arginine vasopressin receptor 2A (AVPR2A). When agonists bind to AVPR2a, Gs protein is activated, which then triggers a cellular response mediated by cAMP. This cellular response can be measured by quantifying the increase in fluorescence intensity and its cellular distribution.

[0180] A.2. Receptor 2A (V2R) Test items were assayed using a fluorescence-based assay at eight concentrations (1 μM to 1 nM in triplicate) using 1 nM human Arg8-vasopressin as the agonist. - Day 1. Nomad AVPR2 U2OS cell line was thawed (2x106 cells / T25). - Day 2. Cells were maintained in DMEM-F12 supplemented with 10% FBS at 37°C in a humidified 5% CO2 atmosphere. - Day 3. Cells were seeded in 96-well plates at a density of 20.000 cells / well (+ / - 2000 cells). Cells were maintained in DMEM-F12 medium supplemented with 10% FBS for 24 hours at 37°C in a humidified 5% CO2 atmosphere. - Day 4. Cells were incubated for 24 hours with different concentrations of test compounds (1000, 333.33, 111.11, 37.04, 12.37, 4.12, 1.37 and 0.46 nM) dissolved in Opti-MEM and 1 nM Arg8-vasopressin. Opti-MEM with vehicle (water) was added to non-stimulated control cells, 1 nM Arg8-vasopressin to positive control cells and 1 nM Arg8-vasopressin together with 100 nM conivaptan to antagonism control. Experiments were performed at least in triplicate. - Day 5. Nomad biosensor activation was quantified after formaldehyde fixation of cells (3.7 wt.%, 20 min). DAPI (2 μg / ml) was used to stain nuclei and fluorescence was measured using a Thermofisher Cell Insight High-Content Bioimager. Filters used to detect DAPI were 380 / 10 and 460 / 10 nm for excitation and emission, respectively, and filters to detect Nomad biosensor were 548 / 20 and 645 / 75 nm, respectively. Images were obtained using a 20× objective and 9 pictures were taken for each well. Cell quantification was performed by defining a region of interest for the nucleus (stained with DAPI) and averaging each of the triplicates after quantification. Granule quantification was also performed using Thermofisher Cellomics Scan Viewer 6.1.1. Two regions of interest, nucleus and cytoplasm, were defined by the Spot Detection application from Cell Software. This software application quantified the number of granules per nucleus and calculated the average number of granules per cytoplasm for each well. Triplicates were then averaged. Data management was performed using both Excel 2003 and Sigmaplot 9.0.

[0181] B. Results As shown in Figure 5, the V2R antagonist peptides of the present disclosure, i.e., K39A peptide and MQ-LEAD peptide, are more potent inhibitors of cAMP production when compared to the parent MQ1 peptide (MQ-WT peptide). The V2R antagonist peptides dose-dependently inhibit cAMP production under Arg8-vasopressin activation conditions.

[0182] The cAMP production inhibitory properties of the V2R antagonist peptides of the present disclosure are further illustrated in Table 3 below.

[0183] [Table 2]

[0184]

Table 3A

[0185]

Table 3B

[0186]

Table 3C

[0187]

Table 3D

[0188]

Table 3E

[0189]

Table 3F

[0190]

Table 3G

[0191]

Table 3H

[0192]

Table 4A

[0193]

Table 4B

[0194]

Table 4C

[0195]

Table 4D

Claims

1. The amino acid sequence of SEQ ID NO:1: 【Chemistry 1】 A peptide comprising a sequence having 100% amino acid identity with During the ceremony, - X1 represents an amino acid residue of F or G, - X2 represents an amino acid residue of N or A, - X3 represents an amino acid residue of T or D, - X4 represents an amino acid residue of I, L or E, - X5 means V, X6 means G, - A peptide, wherein the amino acid at position 39 is A.

2. 2. The peptide of claim 1, wherein X1 represents an amino acid residue of G.

3. 2. The peptide of claim 1, wherein X3 represents an amino acid residue of D.

4. The peptide of claim 1, wherein X4 represents an amino acid residue of E or L.

5. 2. The peptide of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:

6.

6. 6. A peptide according to any one of claims 1 to 5, labelled with a detectable molecule.

7. A nucleic acid encoding a peptide according to any one of claims 1 to 5.

8. The nucleic acid of claim 7, contained in an expression vector.

9. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 5 and a physiologically acceptable carrier.

10. A pharmaceutical comprising a peptide according to any one of claims 1 to 5.

11. A pharmaceutical comprising a peptide according to any one of claims 1 to 5 for treating a disease selected from the group consisting of conditions characterized by normovolemic or hypovolemic hyponatremia, antidiuretic incompatible renal syndrome, polycystic kidney disease, cancer, thrombosis, Meniere's disease, refractory liver disease, and heart failure.

12. 10. In vitro use of the labeled peptide of claim 6 for detecting cells expressing the Vasopressin 2 receptor.

13. 1. An in vitro method for detecting dysregulation of Vasopressin 2 receptor cellular expression, comprising: b) contacting the cells to be examined with a labeled peptide according to claim 6; c) measuring the expression level of vasopressin 2 receptor cellular expression; d) comparing the expression level values ​​obtained in step c) with reference expression level values; e) determining the incidence of dysregulation of Vasopressin 2 receptor cell expression; The method includes:

14. 10. Use of a peptide according to any one of claims 1 to 6 in an in vitro detection method.

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