Selective cyclized peptide kappa-opioid receptor antagonists

Abstract

New PCT Patent Application Medizinische Universität Wien, et al. Vossius Ref.: AG2593 PCT S3 ABSTRACT The present invention relates to a novel cyclic peptide / cyclized dynorphin A analog (dynA analog) which acts a specific antagonist of the kappa opioid receptor (KOR). The present invention further relates to a pharmaceutical composition comprising the novel cyclic peptide / cyclized dynA analog of the invention. Moreover, the present invention further relates to a pharmaceutical composition comprising the novel cyclic peptide / cyclized dynA analog of the invention for use in treating, inhibiting or preventing depression (major depressive disorder (MDD)), a (depression-related) neuropsychiatric disease, stress related disorders (e.g. post- traumatic stress disorder and / or phobias), anxiety, and / or a drug addiction (like opiate, methamphetamine, alcohol, nicotine, ecstasy and / or cocaine / cocaine derivative addiction). The present invention also relates to a method of preparing the novel cyclic peptide / cyclized dynA analog, for example by (late-stage) functionalization with cysteine staples.

Description

[0001] Selective cyclized peptide K-opioid receptor antagonists

[0002] The present invention relates to a novel compound, namely a novel cyclic peptide / cyclized dynorphin A analog (cyclized dynA analog). The cyclized dynA analog of the invention acts a specific ligand of the kappa opioid receptor (K-opioid receptor; KOR). In particular, the cyclic peptide / dynA analog of the invention acts a specific antagonist of the KOR. The present invention further relates to a pharmaceutical composition comprising the cyclized dynA analog of the invention. Moreover, the present invention further relates to a pharmaceutical composition comprising the cyclic peptide / cyclized dynA analog of the invention for use in treating, inhibiting or preventing KOR-activity-related diseases / disorders, including depression (major depressive disorder (MDD)), a (depression-related) neuropsychiatric disease, stress related disorders (e.g. post-traumatic stress disorder and / or phobias), anxiety, and / or a drug addiction (like anopiate, methamphetamine, alcohol, nicotine, ecstasy and / or cocaine / cocaine derivative addiction). The present invention also relates to a method of preparing the cyclized dynA analog, for example by (late-stage) functionalization with cysteine staples.

[0003] The KOR and its endogenous opioid peptide dynorphin A (dynA) mediate a diverse array of physiological actions including nociception, reward and stress (Bruchas, Brain Res 1314, 2010, 44-55). The wide expression of the KOR throughout the central (CNS) and peripheral (PNS) nervous systems, and its involvement in the control of important physiological processes (antinociception, motivation, emotion, cognition etc.), have stimulated the development of KOR ligands as novel drugs; for example for the treatment of pain, itch and neuropsychiatric disorders (Carroll, J Med Chem 56, 2013, 2178-95; Dalefield, Front Pharmacol 13, 2022, 837671). The neurobiological functions of KOR allow potential utilization of KOR pharmacology for numerous therapeutic applications, ranging from the use of KOR agonists as effective, non- addictive pain medications (Machelska, Front Pharmacol 9, 2018, 1388) to the use of KOR antagonists for neuropsychiatric and neurological disorders including depression, anxiety or drug abuse (Carroll, loc. cit.).

[0004] Specific KOR agonists provide promising potential to develop safer and more effective analgesics since they lack the undesired effects which are typical for agonists of the p- or 6- opioid receptor (MOR or DOR, respectively), including respiratory depression, constipation, tolerance or addiction (Darcq, Nat Rev Neurosci 19, 2018, 499-514). Nevertheless, opioid- mediated activation of KOR is often associated with sedation, dysphoria and hallucinations, which have precluded clinical development of KOR agonists (White, J Pharmacol Exp Ther 352, 2015, 98-109; Brust, Sci Signal 9, 2016, rall7). Over recent years, biased opioid ligands selectively activating G protein over p-a rrestin signaling pathways have provided an avenue to develop therapeutics with reduced KOR-related adverse effects (Machelska, loc. cit.). However, it remains uncertain whether biased agonism offers therapeutic benefits (Che, Sci Signal 14, 2021, eaav0320). Enadoline (CI-977) and spiradoline (U-62066E) are small-molecule KOR agonists, which failed in clinical trials owing to their centrally mediated side effects and significant CNS permeability (Albert -Vartanian, J Clin Pharm Ther 41, 2016, 371-82). In that regard, restricting KOR to the PNS by designing agonists with limited access to the CNS has emerged as an alternative strategy to bypass adverse KOR effects (Davenport, Nat Rev Drug Discov 19, 2020, 389-413). For example, Difeli kefa li n is a selective and peripherally acting KOR agonist that was recently approved for the treatment of pruritis associated with chronic kidney disease (Fishbane, N Engl J Med 382, 2020, 222-32). W02013 / 063160 proposes a combination of diketopiperazines and peptidic opioid receptor agonists as analgesic compositions effective in the PNS or CNS. However, when peripherally activity is desired, the lack of brain penetration frequently observed for peptides may be an advantage over small molecules (Davenport, loc. cit.).

[0005] In addition to pain relieving KOR agonists, there is considerable interest in developing selective KOR-antagonistic therapeutic agents for the treatment of neuropsychiatric disorders (Carroll, loc. cit.). Notably, KOR antagonists have demonstrated the potential to relieve symptoms of depression, anxiety or drug abuse (Carroll, loc. cit.). However, the vast majority of these ligands are small molecules, and only a few peptidic KOR antagonists have been developed to date (Carroll, loc. cit.; Aldrich, Handb Exp Pharmacol 271, 2022, 197-220; Aldrich, The AAPS Journal 11(2), 2009, 312-22). For example, WO2021 / 207678 proposes small-molecule KOR antagonists like norbinaltorphimine, CYM 53,093 / BTRX-3315040 or CERC-501 in the treatment of secondary sleep disturbance induced by chronic pain. On the other hand, Dynantin, a linear Dynorphin Ai- 17 (dynAi-17) analog, is a selective peptide KOR antagonist with antidepressant and anxiolyticlike therapeutic potential. However, Dynantin's unsatisfactory plasma stability and permeability to enter the CNS were required to be improved by using a glyco-liposome delivery system (Lu, J Med Chem 44, 2001, 3048-53; Lewicky, EurJ Pharm Biopharm 154, 2020, 290-6).

[0006] While multiple small molecules are available as research tools or drugs, peptide ligands of KOR that have advanced into the clinic or have become reliable chemical probes are rare (Davenport, loc. cit.; Aldrich, Handb Exp Pharmacol 271, loc. cit.; DeMarco, J Med Chem 61 (13), 2018, 5751- 7; Bedini, Front Pharmacol 11, 2020, 188). Reasons include the intrinsic lack of stability and low membrane permeability of peptides, resulting in unfavorable pharmacokinetic properties (Muratspahic, RSC Chem Biol 1, 2020, 177-91).

[0007] Multiple synthetic strategies have been developed in the context of attempts to improve metabolic stability of KOR peptide ligands, including substitution of natural L-amino acids with D- or non-canonical amino acids, peptide backbone modifications and cyclization (Arttamangkul, J Med Chem 38, 1995, 2410-2417; Arttamangkul, J Med Chem 40, 1997, 1211- 18; Fang, J Med Chem 52, 2009, 5619-25; Muratspahic, J Med Chem 64, 2021, 9042-55).

[0008] Further, more generally, some constrained peptides have evolved into a promising class of nextgeneration therapeutics (Valeur, Angew Chem Int Ed Engl 56, 2017, 10294-323). Such constrained peptides have either been approved or are entering late-stage clinical trials for a range of therapeutic applications (Muttenthaler, Nat Rev Drug Discov 20, 2021, 309-25).

[0009] Peptide stapling has been widely utilized to develop constrained peptides to improve their 'drug-like' pharmacokinetic and pharmacodynamic properties (Li, Chem Rev 120, 2020, 10079- 144; Golosov, J Med Chem 64, 2021, 2622-33; Miles, Chem Sci 7, 2016, 3694-702; Stefanucci, ACS Med Chem Lett 10, 2019, 450-6). Linear peptides comprise high degree of conformational flexibility. Hence, they may exist in high entropy states and sample a variety of conformations in solution (Li, loc. cit.). Conformational restriction is considered to reduce flexibility of peptides and thus to minimize the entropy loss upon binding which often results in enhanced stability and membrane permeability as well as increased affinity and selectivity of stapled peptides to their target proteins (Li, loc. cit.; Golosov, loc. cit.; Miles, loc. cit.). The strategy of peptide stapling has been applied to design ligands of other G protein-coupled receptors (GPCRs), in particular galanin and neuropeptide Y receptors (Green, Bioorg Med Chem 21, 2013, 303-10), orexin receptors, (Karhu, Peptides 102, 2018, 54-60) oxytocin and vasopressin receptors (Muttenthaler, J Med Chem 53, 2010, 8585-96) and the melanocortin 1 receptor (White, J Med Chem 65, 2022, 12956-69).

[0010] Peptide stapling using cysteine crosslinking has emerged as one potential approach to induce conformational restraint of peptides (cf. Fairlie, Peptide Science 106(6), 2016, 843-52). For example, it has been reported that cysteine stapling may improve stability of some peptides (Li, loc. cit.; Golosov, loc. cit.). Hitherto, however, cysteine stapling, especially with late-stage functionalization moieties, has only sparsely been employed for opioid receptor ligands. DynAi-i? (YGGFLRRIRPKLKWDNQ) is the endogenous peptide KOR ligand that plays a pivotal role in a plethora of physiological functions. (Caudle, Pain 87, 2000, 235-9). DynAi-17 comprises an N-terminal "message" motif (YGGF), common to all endogenous opioid peptides, and a C- terminal "address" sequence (LRRIRPKLKWDNQ) required for binding and activation of KOR (Chavkin, Proc Natl Acad Sci U S A 78, 1981, 6543-7). Structure-activity relationship studies of dynAi-17 have revealed residues and motifs essential for its pharmacological activity at the KOR (Naqvi, Peptides 19, 1998, 1277-1292). For example, the extracellular loop 2 of KOR that is rich in negatively charged residues (O'Connor, Proc Natl Acad Sci USA 112, 2015, 11852-7) has been suggested to play an essential role in selectivity and maintenance of high affinity of dynA at the KOR (Wang, J Biol Chem 269, 1994, 25966-9; Paterlini, J Med Chem 40, 1997, 3254-62; Bjorneras, FEBS J 281, 2014, 814-24). However, dynAi-17 does not have 'drug-like' properties: (i) it is linear and flexible, and can adopt multiple conformational states, which may explain why it displays significant affinity towards MOR and DOR (O'Connor, loc. cit.; Gomes, Proc Natl Acad Sci USA 117, 2020, 11820-8); (ii) dynA fragments are able to signal via all three opioid receptors (Gomes, loc. cit.; White, Mol Pharmacol 85, 2014, 83-90), and (iii) it has a short half-live and susceptibility to metabolic degradation - common to linear peptides in general (Vlieghe, Drug Discov Today 15, 2010, 40-56).

[0011] To leverage dynAi-17 (or its shorter variant dynAi-13 (YGGFLRRIRPKLK); both sometimes also simply termed "dynA") as a template for KOR ligand design, a range of chemical strategies, including cyclization, N- or C-terminal modification, incorporation of non-canonical and D- amino acids, and / or peptide backbone modifications, are required (Vlieghe, loc. cit.; Davenport, loc. cit.; Muttenthaler, Nat Rev Drug Discov 20, loc. cit.; Aldrich, The AAPS Journal 11(2), loc. cit.). Further, the development of selective peptide KOR ligands using dynA as a design template is challenging due to the high degree of sequence homology between KOR, MOR and DOR binding pockets (Aldrich, Handb Exp Pharmacol 271, loc. cit.).

[0012] Previous studies reported that C-terminal amidation of some dynA-based ligands may contribute to their affinity at the KOR (Chavkin, loc. cit.; Naqvi, loc. cit.). Lactam bridges, disulfide linkages, and carbon-carbon crosslinks have previously been explored to constrain dynA analogs with only mixed success (Schiller, Tetrahedron Vol. 44, 1988, 73-74; Kawasaki, J Med Chem 33, 1990, 1874-9; Arttamangkul, J Med Chem 38, loc. cit.; Arttamangkul, J Med Chem 40, loc. cit.; Fang, loc. cit.). Further, these strategies do not provide synthetic flexibility to rapidly diversify analogs with varying crosslinker functionality. Cysteine staples have emerged as a latestage functionalization strategy that may, in principle, be used to modulate GPCR pharmacology (White, J Med Chem 65, loc. cit.). However, cysteine stapling hitherto is underutilized in the field of opioid receptors targeting and has not been investigated in the design of KOR ligands, not to mention in the design of KOR antagonists.

[0013] A recent study demonstrated that grafting dynA epitopes onto a plant-derived, cyclic peptide scaffold (SFTI-1) is a successful strategy to design potent peptide KOR agonists with strong peripheral KOR activity. In this study, efficacy of one resulting peptide KOR agonist (helianorphin-19) was verified in a mouse model of chronic visceral pain, and no signs of central effects were observed, considering motor coordination or sedation (Muratspahic, J Med Chem 64, loc. cit.).

[0014] Further, the dynA analog zyklophin with a small intramolecular cyclic structure was shown to exhibit antagonistic activity on KOR following systemic administration in vivo (Aldrich, Proc Natl Acad Sci U S A 106, 2009, 18396-401; Patkar, J Med Chem 52(21), 2009, 6814-21; W02009 / 049233). Zyklophin is thus one example of a stabilized peptide KOR antagonist with an advantage over small molecules: its effects have a shorter duration as compared to long- lasting small molecule KOR antagonists (Aldrich, Proc Natl Acad Sci U S A loc. cit.). However, significant drawbacks with respect to zyklophin were reported. For example, zyklophin was reported to induce scratching / itch in mice upon administration, likely due to off-target effects (DiMattio, Neuroscience Letters 563, 2014, 155-9). Specificity of zyklophin for KOR over MOR and DOR was also merely moderate (e.g. DiMattio loc. cit.).

[0015] There is still the need in the art to advance the status of peptide KOR ligands, in particular peptide KOR antagonists, with 'drug-like' features, and to expand the repertoire of these ligands as pharmacological tools to interrogate KOR signaling and as therapeutic candidates. Likewise, there is still the unmet need to design more promising and useful peptide KOR ligands, in particular peptide KOR antagonists, with desired pharmacological properties (e.g. high selectivity and / or higher stability) to interrogate KOR pharmacology and to develop useful and improved therapeutic leads for KOR-mediated disorders.

[0016] The problem underlying the present invention is thus the provision of medically useful means and methods for a pharmaceutical intervention in KOR-related diseases, defects or disorders, in particular diseases, defects or disorders which can be treated by KOR antagonists. More specifically, the problem underlying the present invention is the provision of medically useful KOR antagonists. The technical problem is solved by the provision of the embodiments characterized in the claims.

[0017] The present invention relates to a compound of Formula (I), below: Formula (I), wherein

[0018] (i) SQ1consists of 5 to 8 amino acid residues and comprises

[0019] (I) at its N-terminus, the amino acid sequence YGGF (SEQ ID NO.l; most preferred), or the amino acid sequence YGGF having 1, 2, 3 or 4 conservative amino acid substitutions (for example, the amino acid sequence YGGF having Y1 substituted by / Va-AcTyrl, DTyrl, Phel or Phe(p-Br)l and / or G2 substituted by DAIa2); and

[0020] (II) at its C-terminus, 1, 2, 3 (preferred) or 4 (consecutive) amino acid residue(s) of the amino acid sequence LRR (most preferred) or LRRI (SEQ ID NO.2), or 1, 2, 3 (preferred) or 4 (consecutive) amino acid residue(s) of the amino acid sequence LRR or LRRI having 1, 2, 3 or 4 conservative amino acid substitution(s);

[0021] (ii) SQ2consists of 4 to 6 amino acid residues and comprises 1, 2, 3, 4 or 5 (preferred) (consecutive) amino acid residue(s) of the amino acid sequence RPKLK (SEQ ID NO.3; most preferred) or IRPKLK (SEQ ID NO.4), or comprises 1, 2, 3, 4 or 5 (preferred) (consecutive) amino acid residue(s) of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s);

[0022] (iii) each of L1, L2and L3is independently selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2-CH2-, -CH(CH3)-CH2-, -CH2-CH(CH3)- and -CH(CH3)-CH(CH3)-, preferably -CH2-; and

[0023] (iv) R is selected from H, methyl and ethyl, preferably selected from H and methyl, more preferably R is H.

[0024] The above compound is also termed cyclic peptide of the invention or cyclized dynorphin A analog (dynA analog) of the invention.

[0025] In the context of the invention, constrained KOR-targeting dynAi-13 analogs were developed by using diverse peptide stapling approaches. In particular stapling with diverse cysteine stapled moieties including methylene, acetone, m-xylene and tetrazine stapled motifs was applied (cf. Figure 1). Further, in the context of designing the constrained KOR-targeting dynAi-13 analogs, a stapling strategy was utilized that could be implemented at a late stage, on unprotected peptide scaffolds. This stapling strategy is amenable to diversification (late-stage functionalization; cf. Figure 1). Further, C-terminal amidation was applied and the resulting effects were tested.

[0026] Evidence is provided in the context of the invention, and by the appended examples, that the cyclized dynA analogs as claimed and as provided / disclosed herein (particular constrained cyclized dynAi-13 analogs; also termed "cysteine stapled dynA analogs (CSD analogs)") exhibit the desired and even surprisingly improved pharmacological properties. The present invention thus provides for useful, and an even improved, means and methods for medical / pharmaceutical intervention in KOR-related diseases, defects or disorders, in particular diseases, defects or disorders which can be treated by KOR antagonists. More specifically, present invention provides for even improved KOR antagonists.

[0027] The pharmacological properties of the cyclized dynA analogs of the invention include high affinity to KOR, high antagonizing activity / potency, high stability, (extraordinary) high specificity to KOR (approx, three orders of magnitude; as compared to other opioid receptors, e.g. MOR and / or DOR) and / or less adverse effects (e.g. no itch / scratching). In one embodiment, the pharmacological properties of the cyclized dynA analogs of the invention (also) include central activity / capability of modulating KOR function within the CNS. In the context of this embodiment, the cyclized dynA analogs of the invention may even be capable of crossing the blood-brain barrier (BBB) / entering the CNS. The cyclized dynA analogs of the invention are improved with respect to the affinity to KOR, the KOR-antagonizing activity / potency, the stability, the specificity to KOR and / or the adverse effects (e.g. no itch / scratching). In one embodiment, the cyclized dynA analogs of the invention may (also) be improved with respect to the central activity / capability of modulating KOR function within the CNS; or even with respect to, and / or the central activity / crossing the blood-brain barrier (BBB) / entering the CNS. In particular, these improvements apply when being compared to the known small-molecule KOR antagonists or known peptide KOR antagonists (like Zyklophin).

[0028] In particular, it is documented herein below and in the appended examples, that the constrained dynAi-13 analog termed "CSD-CH2(i,8)-NH2" (see, for example, Figure 1 and Formula( IF) herein below) targets the KOR as a stable (competitive) antagonist with nanomolar affinity and an up to and even more than 1000-fold enhanced selectivity for KOR over MOR and / or DOR (as compared to dynAi-13 as such). For example, the selectivity of CSD-CH2(i,8)-NH2 for KOR is shown to be about 1 / 1000 / 1000 (KOR / MOR / DOR). In contrast, the selectivity of zyklophin for KOR is only about l / 194 / >330 (KOR / MOR / DOR; cf. DiMattio loc. cit.). Without being bound by theory, the C-terminal amide of CSD-CH2(i,8)-NH2 may interact with the acidic residues in the extracellular loop of KOR, thereby leading to, for example, an increase in KOR affinity and specificity (e.g. as compared to other KOR ligands; like U50,488 or the other KOR ligands disclosed herein (see, for example, Figure 1)). Further, also without being bound by theory, CSD-CH2(i,8)-NH2 may, upon binding to KOR, stabilize the inactive state of KOR and promote a more inactive KOR-CSD-CH2(i,8)-NH2 complex. Also shown herein is the improved stability of CSD-CH2(i,8)-NH2 towards proteolytic degradation in human serum. In addition, the ability of CSD-CH2(i,8)-NH2to retain inhibitory KOR activity in a DRG neuronal primary cell model is shown.

[0029] Moreover, in the context of the invention, CSD-CH2(i,8)-NH2 was shown in vivo to be centrally active; i.e.to modulate KOR function within the CNS. This even provides for evidence that CSD- CH2(i,8)-NH2 is capable of crossing the BBB and of entering the CNS, respectively. In particular, CSD-CH2(i,8)-NH2is shown herein to antagonize centrally-mediated effects of the KOR by the small molecule KOR agonist U50,488 in mice. More particular, the in vivo KOR antagonism of CSD-CH2(i,8)-NH2in mice after s.c. administration is demonstrated herein based on its ability to reverse antinociception and sedation / motor impairment caused by U50,488.

[0030] Furthermore, in the context of the invention, CSD-CH2(i,8)-NH2 was shown not to induce scratching / itch in vivo (based on behavioural observations in mice (data not shown); e.g. in contrast to zyklophin (cf. DiMattio loc. cit.))

[0031] In conclusion, by using the endogenous dynAi-13 as a molecular template, peptide cysteine stapling (in particular with a methylene or similar group), and the introduction of a C-terminal N-group (in particular -NH2), the safe, potent, stable, highly specific and( / or) centrally-acting KOR antagonists of the invention were developed; and are provided herewith.

[0032] The compound of the invention, i.e. the KOR antagonist provided herewith, comprises a peptide backbone (linear peptide precursor) which is derived from dynA, in particular from dynAi-13 (YGGFLRRIRPKLK; SEQ ID NO.7). The compond of the invention is thus also termed herein "dynA analog". The peptide backbone of the compound of the invention is highly similar on a structural basis to the amino acid sequence of dynAi-13. The peptide backbone of the compound of the invention may comprise (in consecutive order) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues that are identical to (or constitute at least conservative substitutions of) the corresponding amino acid residues of dynAi-13 at the respective position. It is preferred that the non-identical amino acid residues are conservative substitutions. For example, in the peptide backbone of the dynA analog of the invention, the amino acid residue corresponding to Y at position 1 of dynAi-13. (Yl) may be substituted by / Va-AcTyrl, DTyrl, Phel or Phe(p-Br)l and / or the amino acid residue corresponding to G at position 2 (and / or 3) of dynAi-13 (G2 (and / or G3)) may be substituted by DAIa2. The peptide backbone of the compound of the invention may be at least 53.9%, 61.5%, 69.2%, 76.9%, 84.6% or 92.3% identical to dynAi-13. Generally, a higher degree of identity, and of structural similarity, for the peptide backbone of the herein disclosed dynA analog as compared to dynAi-13 is preferred (e.g. the higher amounts of identical amino acid residues and / or the higher values of identity are preferred).

[0033] The compound of the invention, i.e. the KOR antagonist provided herewith, further comprises an intra-molecular cyclic structure. The compond is thus also termed herein "cyclic peptide" or "cyclized dynA analog". The cyclic structure of the compound of the invention is preferably formed between the N-terminal amino acid residue (e.g. the Yl residue) and an (inner) amino acid residue at position 7, 8 or 9, most preferably at position 8 (i.e. corresponding to the I residue at position 8 of dynAi-13), of the peptide backbone. The compound of the invention is not envisaged to constitute a head-to tail cyclized dynA analog (i.e. with the cyclic structure being formed between the N-terminal and C-terminal amino acid residue). In the cyclized dynA analog of the invention, in particular in the peptide backbone thereof, the (inner) amino acid residue which corresponds to the amino acid residue at the respective position of dynAi-13 (e.g. the I residue at position 8 of dynAi-13) is a residue which is suitable for forming the intramolecular cyclic structure. Most preferably, this residue is a Cysteine residue (most preferred) or a Homocysteine residue (e.g in lieu of the I residue at position 8 of dynAi-13).

[0034] In the cyclized dynA analog of the invention (see also Formula (I) and Formula (II)), SQ1consists of 5 to 8 amino acid residues, i.e. 5, 6, 7 or 8 amino acid residues. Preferably, SQ1consists of 6 to 8 or 5 to 7 amino acid residues. Most preferably, SQ1consists of 7 amino acid residues.

[0035] Further, in the cyclized dynA analog of the invention (see also Formula (I) and Formula (II)), SQ1comprises

[0036] (I) at its N-terminus, a biologically active "message" motif of dynAi-17 or dynAi-13 (cf. Chavkin loc. cit.; e.g. SEQ ID NO.l); and

[0037] (II) at its C-terminus, 1, 2, 3 or 4 amino acid residue(s), preferably 2 to 4 amino acid residues, most preferably 3 amino acid residues, of the N-terminal part of a biologically active "address" sequence of dynAi-17 or dynAi-13 (cf. Chavkin loc. cit.; e.g. SEQ ID NO.8); e.g. the first three (most preferred) or first four N-terminal amino acid residues of a biologically active "address" sequence of dynAi-17 or dynAi-13. It is preferred that the amino acid residues are consecutive amino acid residues of the N-terminal part of a biologically active "address" sequence of dynAi-17 or dynAi-13.

[0038] It is preferrd that SQ1consist of the following components:

[0039] (I) at its N-terminus, the amino acid sequence YGGF (SEQ ID NO.l; most preferred), or the amino acid sequence YGGF having 1, 2, 3 or 4 conservative amino acid substitutions (for example, the amino acid sequence YGGF having Y1 substituted by / Va-AcTyr, DTyr, Phe or Phe(p-Br) and / or G2 substituted by DAIa2); and

[0040] (II) at its C-terminus, 1, 2, 3 or 4 amino acid residue(s), preferably 2 to 4 amino acid residues, most preferably 3 amino acid residues, of the amino acid sequence LRR (preferred) or LRRI (SEQ ID NO.2), or 1, 2, 3 or 4 amino acid residue(s), preferably 2 to 4 amino acid residues, most preferably 3 amino acid residues, of the amino acid sequence LRR or LRRI having 1, 2, 3 or 4 conservative amino acid substitution(s). 1, 2 or 3 (preferred) amino acid residue(s) of the amino acid sequence LRR is more preferred. The amino acid sequence LRR is most preferred. It is preferred that the amino acid residues are consecutive amino acid residues of the respective reference sequence.

[0041] SQ1may consist of the amino acid sequence YGGFLRR with 1, 2, 3, 4, 5, 6 or 7 conservative amino acid substitution(s) (preferred); or of the amino acid sequence YGGFLRRI with 1, 2, 3, 4, 5, 6, 7 or 8 conservative amino acid substitution(s). Preferred examples of SQ1are the amino acid sequences YGGFLRR (SEQ ID NO.5) (most preferred) or YGGFLRRI (SEQ ID NO.6).

[0042] In the cyclized dynA analog of the invention (see also Formula (I) and Formula (II)), SQ2consists of 4 to 6 amino acid residues, i.e. 4, 5 or 6 amino acid residues. Preferably, SQ2consists of 5 to 6 or 4 to 5 amino acid residues. Most preferably, SQ2consists of 5 amino acid residues.

[0043] Further, in the cyclized dynA analog of the invention (see also Formula (I) and Formula (II)), SQ2comprises 1, 2, 3, 4 or 5 amino acid residue(s), preferably 4 or 5 amino acid residues, most preferably 5 amino acid residues, of the C-terminal part of a biologically active "address" sequence of dynAi-13 (cf. Chavkin loc. cit. ; e.g. SEQ ID NO.8). It is most preferred that the amino acid residues are consecutive amino acid residues of the C-terminal part of a biologically active "address" sequence of dynAi-13. In the cyclized dynA analog of the invention (see also Formula (I) and Formula (II)), SQ2may consist of 4 to 6 amino acid residues and comprises 1, 2, 3, 4 or 5 amino acid residue(s), preferably 4 or 5 amino acid residues, most preferably 5 amino acid residues, of the amino acid sequence RPKLK (SEQ ID NO.3) (preferred) or IRPKLK (SEQ ID NO.4); or may consist of 1, 2, 3, 4 or 5 amino acid residue(s), preferably 4 or 5 amino acid residues, most preferably 5 amino acid residues, of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s). It is preferred that the amino acid residues are consecutive amino acid residues of the respective reference sequence.

[0044] SQ2may consist of the amino acid sequence RPKLK with 1, 2, 3, 4 or 5 conservative amino acid substitution(s) (preferred) or of the amino acid sequence IRPKLK with 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s). Preferred examples of SQ2are the amino acid sequences RPKLK (SEQ ID NO.3) (most preferred) or IRPKLK (SEQ ID NO.4).

[0045] It is envisaged that peptide backbone of the cyclized dynA analog of the present invention comprises, as its N-terminal part, a biologically active "message" motif of dynAi-17 or dynAi-13 (cf. Chavkin loc. cit.; e.g. SEQ ID NO.l) and, as its C-terminal part, a biologically active "address" sequence of dynAi-13 (cf. Chavkin loc. cit.; e.g. SEQ ID NO.8). A biologically active "message" motif of dynAi-17 or dynAi-13 and a biologically active "address" sequence of dynAi-13 is known in the art (cf. Chavkin loc. cit. (e.g. Fig. 1)) and is disclosed herein (see, for example, SEQ ID NOs. 1, 8 and 10). The skilled person is readily able to test whether a given amino acid stretch (within (a peptide backbone of) a cyclized dynA analog of the invention) is a biologically active "message" motif or a biologically active "address" sequence. Respective guidance and assays are provided herein and in the appended examples and also in the art (e.g. Chavkin loc. cit.). For example, a biologically active "message" motif results in naloxone-reversible opioid activity (e.g. Chavkin loc. cit.).

[0046] Examples of the peptide backbone of a cyclized dynA analog of the invention are YGGFLRRCRPKLK (SEQ ID NO.ll), YGGFLRRICPKLK (SEQ ID NO.12) and YGGFLRCIRPKLK (SEQ ID NO.13). The most preferred peptide backbone of a cyclized dynA analog of the invention consists of the amino acid sequence YGGFLRRCRPKLK (SEQ ID NO.ll).

[0047] The present invention also encompasses (a) conservative amino acid substitution(s) within these particular peptide backbones ( e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 conservative amino acid substitution(s)), provided that an (inner) cycle-forming residue is maintained (e.g. the depicted C residue). What is said with respect to conservative amino acid substitution(s) herein elsewhere also applies here, mutantis mutandis. The technical meaning of conservative amino acid substitution(s) is well known in the art. For example, an amino acid substitution is conservative (in the context of the invention), when the replacement amino acid(s) belong(s) to the same category of amino acids than the amino acid(s) to be replaced. For example, an acidic amino acid may be replaced by another acidic amino acid, a basic amino acid may be replaced by another basic amino acid, an aliphatic amino acid may be replaced by another aliphatic amino acid, and / or a polar amino acid may be replaced by another polar amino acid. The meaning of the terms "acidic amino acid(s)", "basic amino acid(s)", "aliphatic amino acid(s)" and "polar amino acid(s)" are known in the art (see, for example, Stryer, Biochemie, Spectrum Akad. Verlag, 1991, Item I. 2.). These terms are correspondingly used throughout the invention.

[0048] In particular, the term "acidic amino acid(s)" as used herein is intended to mean an amino acid selected from the group comprising Asp, Asn, Glu, and Gin, the term "basic amino acid(s)" as used herein is intended to mean an amino acid selected from the group comprising Arg, Lys and His, the term "aliphatic amino acid(s)" as used herein is intended to mean any amino acid selected from the group comprising Gly, Ala, Ser, Thr, Vai, Leu, He, Asp, Asn, Glu, Gin, Arg, Lys, Cys and Met, and the term "polar amino acid(s)"as used herein is intended to mean any amino acid selected from the group comprising Cys, Met, Ser, Tyr, Gin, Asn and Trp.

[0049] Further, a conservative amino acid substitution in accordance with the invention may mean that F is replaced by I, L or Y; G is replaced by A, C, D, E or R; I is replaced by F, L, M, N or V; K is replaced by E, M, N, Q, R or T; L is replaced by F, H, I, M, P, Q, R, V or W; P is replaced by H, L, Q, R or S; R is replaced by C, G, H, K, L, M, P, Q, T or W; Y is replaced by C, D, F, H, N or S. Particular (conservative) amino acid substitutions which may occur in accordance with the invention are the substitution of Y by / Va-AcTyr, DTyr, Phe or Phe(p-Br) and / or the substitution of G by DAIa.

[0050] In principle, any amino acid configuration may be present in the context of the peptide backbone (linear peptide precursor) or in the dynA analog of the invention. In general, most naturally occurring amino acids are in the L-configuration. Thus, also in the context of the invention, there is a general preference for amino acids in the L-configuration. In the context of the invention, however, one or more of the amino acid residues of the provided cyclized dynA analog may also be in the D-configuration. The respective peptide backbone (linear peptide precursor) in the cyclized dynA analog may thus have a certain pattern of D- and L-amino acid residues. Also in this context, however, the resulting dynA analog must be a dynA analog in accordance with the invention and function accordingly (for example as described herein elsewhere, e.g. as in one, more or all of items (a) to (g), below). The skilled person is readily in the position to test which amino acid residue(s) of a dynA analog should be in the L-configuration and which may be in the D-configuration, i.e. to figure out a suitable pattern of D-and L-amino acid residues (i.e. a pattern which results in a functional dynA analog in accordance with the invention). In this context, the skilled person may, for example, rely on the tests / assays disclosed herein and in the appended examples (e.g. one or more of the (pharmacological) tests / assays for testing whether a dynA analog is a dynA analog of the invention as described with respect to items (a) to (g), below, and in appended Example 1, infra).

[0051] In general, the meaning of the term "amino acid" or "amino acid residue" is known in the art and is used herein accordingly. When an "amino acid" is a component of a peptide / protein, the term "amino acid" is used herein in the same sense than "amino acid residue". An "amino acid" or "amino acid residue" as referred to herein may be a naturally-occurring amino acid (preferred), more preferably a naturally-occurring L-amino acid. However, as mentioned, an "amino acid" or "amino acid residue" in context of this invention may also be a D-amino acid, or a non-naturally-occurring (i.e. a synthetic) amino acid, like, for example, norleucine, R- alanine, or selenocysteine.

[0052] It will also be acknowledged by the one skilled in the art that one or several of the amino acids as comprised in the peptide backbone (linear peptide precursor) or in the dynA analog of the invention may be modified. In accordance therewith, any amino acid as used / defined herein may, in principle, also represent its modified form. For example, a Cys residue as used herein may be a modified Cys residue like a homocysteine residue. Modifications may, among others, be a methylation or acylation, or the like. In principle, however, non-modified amino acid residues are preferred. In any case, it is envisaged that a cyclized dynA analog described herein which comprises such (a) modification(s) or modified amino acid(s) is still functionally active in accordance with the invention (cf., for example, items (a) to (g), infra). Respective assays for determining whether a given dynA analog fulfils this requirement, are known to the one skilled in the art and are, among others, also described herein, e.g. in the example part and / or in the context of items (a) to (g), infra.

[0053] In the cyclized dynA analog of the invention (see also Formula (I) and Formula (II)), each of L1, L2and L3is independently selected from -CH2-, -CH(CHs)-, -C(CHs)2-, -CH2-CH2-, -CH(CH3)-CH2-, - CH2-CH(CH3)- and -CH(CH3)-CH(CH3)-. Preferably, at least one of L1, L2and L3is is -CH2-. More preferably, at least two of L1, L2and L3are -CH2-. For example, L1and L2may be -CH2-, L2and L3may be -CH2- or L1and L3may be -CH2-. Most preferably, all three of L1, L2and L3are -CH2-. In the cyclized dynA analog of the invention (see also Formula (I) and Formula (II)), R is selected from H, methyl and ethyl. Preferably R is selected from H and methyl. Most preferably, R is H.

[0054] In a preferred embodiment, the cyclized dynA analog according to the invention is of Formula (II), below: Formula (II), wherein SQ1and SQ2are defined as described herein elsewhere. More preferably, SQ1is YGGFLRR (SEQ ID NO.5) or SQ2is RPKLK (SEQ ID NO.3). Most preferably, SQ1is YGGFLRR (SEQ ID NO.5) and SQ2is RPKLK (SEQ ID NO.3).

[0055] The most preferred example of a cyclized dynA analog according to the invention (termed CSD- CH2(i,8)-N H2) is a compound of Formula (I), wherein

[0056] (i) SQ1is YGGFLRR (SEQ ID NO.5);

[0057] (ii) SQ2is RPKLK (SEQ ID NO.3);

[0058] (iii) each of L1, L2and L3is -CH2-; and

[0059] (iv) R is H.

[0060] The valence bound formula of the most preferred example of a cyclized dynA analog according to the invention (termed CSD-CH2(i,8)-NH2) is given below (Formula (II')):

[0061] Formula (IT)

[0062] The invention also provides (the use of) derivatives of the disclosed cyclized dynA analogs, such as salts, e.g. with physiologic organic and inorganic acids like HCI, H2SO4, H3PO4, malic acid, fumaric acid, citronic acid, tatratic acid, acetic acid.

[0063] Generally, the cyclized dynA analog according to the invention is envisaged to exhibit (or is capable of exhibiting) at least one, more (e.g. at least two, three or four), or (preferably) all of the desired and advantageous (biological) functions as described herein, in particular the (biological) functions of CSD-CH2(i,8)-NH2. Most relevantly, the cyclized dynA analog according to the invention is a specific KOR antagonist, preferably with the ability of modulating KOR function within the CNS; or even of crossing the BBB. In particular, the cyclized dynA analog according to the invention is envisaged to bind to KOR (or to be capable of binding to KOR), to be a specific KOR antagonist (in particular as compared to MOR and / or DOR), to modulate KOR function within the CNS (and even cross the BBB and enter the CNS, respectively), and( / or) to be stable (e.g. (human) serum).

[0064] More particular, the cyclized dynA analog according to the invention exhibits (or is capable of exhibiting) at least one, more (e.g. at least two, three or four), or (preferably) all of the following biological functions:

[0065] (a) the cyclized dynA analog modulates KOR function within the CNS (and even crosses the BBB and enters the CNS, respectively);

[0066] (b) the cyclized dynA analog is an antagonist of the KOR; e.g. with a KOR antagonist potency corresponding to an EC50 of <70 nM, <60 nM, <50 nM, <40 nM or < / about 30 nM (in an adenylyl cyclase assay quantifying the reduction of cyclic AMP (cAMP); e.g. as described in https: / / doi.org / 10.1021 / im0501Q5i; Patkar, Med. Chem. 48(14), 2005, 4500-3; higher potency is preferred), an EC50 of <750 nM, <700 nM, <650 nM, <600 nM or < / about 560 nM (in a FMP blue assay; e.g. as described in https: / / doi.Org / 10.1016 / j.biopha.2021.112173; Zhao, Biomedicine & Pharmacotherapy, 112173, 2021, 1-11; higher potency is preferred), and / or an EC50 of <550 nM, <500 nM, <450 nM, <400 nM or < / about 340 nM (in a [35S]GTPgS assay; e.g. as described in https: / / doi.org / 10.1021 / jm501827k; Joshi, J. Med. Chem. 58(22), 2015, 8783-95; higher potency is preferred);

[0067] (c) the cyclized dynA analog specifically binds to KOR (but not to MOR and DOR; e.g. with a >400-fold, >500-fold, >600-fold, >700-fold, >800-fold, >900-fold or > / about 1000-fold selectivity for KOR over MOR and / or DOR; higher selectivity is preferred);

[0068] (d) the cyclized dynA analog binds to KOR with an affinity corresponding to a Ki of <10-8M, preferably <8.0xl0-9M, <7.0xl0-9M or <6.8xl0-9M; and / or

[0069] (e) the cyclized dynA analog is stable in (human) serum for >0.5 h (e.g. >50% (preferably >75%) of the cyclized dynA analog remains in the (human) serum after (about) 0.5 h; >25% (preferably >50%) of the cyclized dynA analog remains in the (human) serum after (about) 1.0 h; >20% (preferably >30%) of the cyclized dynA analog remains in the (human) serum after (about) 1.5 h; and / or >15% (preferably >25%) of the cyclized dynA analog remains in the (human) serum after (about) 2.0 h).

[0070] The cyclized dynA analogs of the invention may be particularly stable; and / or may have a high / improved retention and / or half life time (for example in a patient's body (e.g. in blood or serum; see, for example, (e), above). As such, cyclized dynA analogs are preferred to lack sites susceptible for hydrolysis or cleaving proteases, like, for example, serum proteases. The meanings of the terms "hydrolysis" and "(serum) proteases" and the structure of the respective sites are well known in the art.

[0071] Further, (f), the cyclized dynA analog according to the invention may not induce scratching / itch (for example in mice upon administration; e.g. at the administration site). This can, for example, be tested on the basis of behavioral observations and / or as in DiMattio loc. cit.

[0072] Further, (g), the cyclized dynA analog according to the invention may exhibit at least one, more (e.g. at least two, three or four), or (preferably) all of its (biological) functions rapidly upon adminisration (for example upon s.c. administration). This can, for example, be tested (e.g. in mice) as described in Examples 1 and 6 (cf. also Figure 5A). For example, a (competitive) KOR- antagonizing effect (e.g. reduction in antinociception; e.g. as induced by U50,488) of the cyclized dynA analog according to the invention (and / or at least one or more of its other (biological) functions) may occur and / or be detectable (e.g. in mice) after <lh, <45 min, <30 min, <15 min or even <10 min upon administration (e.g. s.c.); and / or before a comparable effect / function(s) of, for example, zyklophin would occur and / or would be detectable.

[0073] The skilled person is readily able to test whether a given (cyclic) peptide or (cyclized) dynA analog, respectively, exhibits any of the herein described (biological) functions; like the herein described (biological) functions of CSD-CH2(i,8)-NH2. DynA (e.g. dynAi-13, dynAi-17, dynAi-i3-NH2 or dynAi-i7-NH2) and / or CSD-CH2(i,8)-NH2 (or any other CSD analog disclosed herein; cf. Figure 1) may be used as controls in this respect (for example, dynA as negative control and / or CSD- CH2(i,8)-NH2 as a positive control). Guidance and assays for testing the (biological) functions are known in the art (see, for example, the respective scientific papers and / or patent literature cited herein) and are provided hierein (see, for example, the above described (biological) functions, like the ones as described in items (a) to (g), supra) and in the appended examples (see, for example, Example 1).

[0074] For example, whether a (cyclized) dynA analog modulates KOR function within the CNS and / or crosses the BBB and enters the CNS, respectively, can be tested as described in Erli (J Med Chem 60, 2017, 7579-90), and in the appended Examples 1 and 6 (cf. also Figure 5B). For example, motor coordination in, for example, the rota rod test after, for example, s.c. administration (e.g. in mice) may be applied in this respect. In addition, whether a (cyclized) dynA analog is capable of modulating KOR function within the CNS and / or passing the BBB and entering the CNS, respectively, can, for example, be determined / tested by (i) in vivo MS imaging using a labelled (cyclized) dynA analog of interest; (ii) label-free quantification of the (cyclized) dynA analog in CSF (and or brain homogenate, for instance via mass spectrometry; and / or (iii) systemic in vivo administration (s.c., i.p., i.v. or p.o.) followed by assessing central effects (such as analgesia via central KOR receptors). Respective means and methods are known in the art (Muratspahic, loc. cit.; Thell, Proc Natl Acad Sci USA 113(15), 2016, 3960-5).

[0075] For example, whether a (cyclized) dynA analog is an antagonist of the KOR / antagonizes the KOR can be tested as described in Muratspahic (J Med Chem 64, loc. cit.) or Duerrauer, (Sci Rep 9, 2019, 19295), and in appended Examples 1, 3, 5 and 6 (cf. also Table 1 and Figures 2, 4 and 5B). For example, a cAMP assay (e.g. according to the Cisbio protocol) and / or Schild regression analysis may be applied in this respect.

[0076] For example, whether a (cyclized) dynA analog specifically binds to KOR (but not to MOR and DOR) can be tested as described in Muratspahic (J Med Chem 64, loc. cit.), and in appended Examples 1 and 4 (cf. also Figure 3A and 3B). For example, whether a (cyclized) dynA analog binds to KOR can be tested as described in Chavkin (loc. cit.) and Naqvi (loc. cit.), and in appended Examples 1 and 3 (cf. also Table 1 and Figure 2).

[0077] For example, whether a (cyclized) dynA analog is stable in (human) serum can be tested as described in Mu ratspa hie (J Med Chem 64, loc. cit.) and in appended Examples 1 and 4 (cf. also Figure 3C).

[0078] For example, a (cyclized) dynA analog according to the invention may exhibit an anxiolytic effect This can also be tested by relying on respective tests / assays described herein and in the art. For example, the elevated plus maze (EPM) test may be used in this respect (e.g. as an animal behavioral model of anxiety / anxiety disorders or anxiety conditions). This is, for example, described in Danduga (Methods Mol Biol 2761, 2024, 93-6).

[0079] The KOR, in particular, the human KOR (hKOR), is well known in the art and is, for example, described in Lalanne (Front Psychiatry 5, 2014, 170) and Du (Nat. Common. 7, 2016, 11120). In principle, when reference to KOR is made herein, any KOR is meant, in particular a KOR of any (animal) species. A KOR referenced herein may, for example, be a KOR of human, mouse, rat, rabbit, monkey or goat etc. origin (see, for example, UniProt entries: OPRK_MOUSE P33534, OPRK_RAT P34975). Preferably, the KOR is hKOR (see, for example, UniProt entry: OPRK_HUMAN P41145). A detailed characterization of the hKOR, including the particular amino acid sequence information, is derivable from the database entry https: / / www.uniprot.org / uniprot / P41145. In general, it is preferred that the KOR to be targeted by the cyclized dynA analog of the invention is the KOR of a patient to be treated. For example, if the patient to be treated is a human, the KOR to be targeted is preferably hKOR.

[0080] Assays for testing the relevant biological function(s) of the KOR are disclosed herein and known in the art. Respective assays are, for example, described in the appended examples (e.g. Example 1).

[0081] Further, multiple antagonists of the KOR are known in the art (see, e.g. https: / / www.guidetopharmacology.org / GRAC / ObjectDisplayForward?objectld=318&familyld= 50&familyType=GPCR). Antagonists of the KOR are known in the art may, for example, be used as reference compounds / controls when testing / assaying whether a dynA analog functions in accordance with the invention, i.e. exhibits the relevant / advantageous property(ies) / (biological)function(s) of the cyclic peptides / cyclized dynA analogs of the invention (e.g. acting as a (superior) KOR antagonist; cf. also one, more, or all of items (a) to (g), supra). For example, a KOR antagonist known in the art may be used a control research tool / pharmacological probe testing whether a given dynA analog (as, for example, structurally defined herein) reverses an effect produced by a KOR agonist (e.g. U50,488) just like the known KOR antagonist; or, if CSD-CH2(i,8)-NH2 is used as a further control research tool / pharmacological probe, like CSD-CH2(i,8)-NH2 itself.

[0082] As mentioned, the cyclized dynA analog of the invention is envisaged to act as a (h)KOR antagonist / antagonist of (h)KOR. The meaning of "(h)KOR antagonist" and "antagonist of the (h)KOR" is known in the art and the respective terms are used herein accordingly (see above and, e.g., https: / / www.guidetopharmacology.org / GRAC / ObjectDisplayForward?objectld=318 &familyld=50&familyType=GPCR).

[0083] In the context of the invention, being an "antagonist" of the KOR and exhibiting "antagonistic" function on the KOR, respectively, means that the KOR activity, i.e. the relevant biological function(s) of the KOR is(are) reduced, inhibited or even entirely blocked. For example, a KOR ligand is antagonizing, if it antagonizes KOR with the same, or a similar (e.g. ± 1-40%), potency and / or efficacy as a known KOR antagonist (e.g. nor-BN I ).). In particular, being an "antagonist" of the KOR and exhibiting "antagonistic" function on the KOR, respectively, means in the context of the invention that the activity of (an) agonist(s) at the KOR is (competitively) reduced, inhibited or even entirely blocked (non limiting examples of KOR agonists are U50,488, dynA (e.g. dynAi-17 or dynAi-13), nalfurafine etc.; see also https: / / www.guidetopharmacology.org / GRAC / ObjectDisplayForward?objectld=318&familyld= 50&familyType=GPCR). More particular, being an "antagonist" of the KOR and exhibiting "antagonistic" function on the KOR, respectively, means in the context of the invention that the response of KOR to an opioid stimulus (e.g. by a KOR agonist), e.g. the intracellular cAMP reduction, is reduced, inhibited or even entirely blocked. This, for example, leads to a nonactivation, de-activation or reduced activation of the RAF / MEK1 / 2 / ERK1 / 2 or the JAK2 / STAT3 signalling cascades (https: / / pubmed.ncbi.nlm.nih.gov / 27881770-enhancing-remyelination- through-a-novel-opioid-receptor-pathway / ?from_single_result=borniger+and+hesp; Borniger, J. Neurosci 36(47), 2016, 3). Means and methods for assaying the KOR-antagonizing activity of a given compound, e.g. (cyclized) dynA analog, are known in the art and are disclosed herein (see, for example, Examples 1, 5 and 6)

[0084] If not indicated differently herein elsewhere, "inhibiting", "decreasing", "blocking", "suppressing" or "reducing" and the like, in the context of the present invention is envisaged to mean that the initial status (for example status of KOR affinity, antagonizing potency and / or efficacy of a dynA analog, activity of a dynA analog, stability of a dynA analog etc.; and the respective KOR function) is lowered (in vitro and / or in vivo) by, for example, at least 10%, by at least 20%, by at least 30%, by at least 50%, by at least 80%, by at least 90%, at least 95%, by at least 99% or even by 100% (in principle, the higher values of percentage are preferred). The skilled person is readily in the position to test the respective degree of "inhibiting", "decreasing", "blocking", "suppressing" or "reducing". Moreover, the skilled person is readily in the position to determine for a given drug (e.g. dynA analog as defined (e.g. by a structure) or as disclosed herein) the IC50 for the respective "inhibiting", "decreasing", "blocking", "suppressing" or "reducing" effect / activity.

[0085] Likewise, if not indicated differently herein elsewhere, "increasing", "inducing" or "improving", and the like, in the context of the present invention particularly means that the initial status (for example status of KOR affinity, antagonizing potency and / or efficacy of a dynA analog, activity of a dynA analog, stability of a dynA analog etc.; and the respective KOR function) is increased (in vitro and / or in vivo) by, for example, at least 10%, by at least 20%, by at least 30%, by at least 50%, by at least 80%, by at least 90%, at least 95%, by at least 99% or by at least 100%; or by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold (in principle, the higher values of percentage are preferred). In particular, "increasing" means that there is already an initial degree of activity / status (baseline) which is further "increased". In particular, "inducing" means that there is (substantially) no initial degree of activity / status which is then "induced". The skilled person is readily in the position to test the respective degree of "increasing", "inducing" or "improving". Moreover, the skilled person is readily in the position to determine for a given drug (e.g. dynA analog as defined (e.g. by a structure) or as disclosed herein) the IC50 for the respective "increasing", "inducing" or "improving" effect / activity.

[0086] The present invention further relates to a pharmaceutical composition comprising the cyclized dynA analog according to the invention. The pharmaceutical composition may further comprise a pharmaceutical carrier.

[0087] The cyclized dynA analog of the invention, or the pharmaceutical composition comprising it, may be for use in the treatment of a KOR-activity-related disease, in particular of a disease resulting from or coming along with an increased KOR activity and / or which is treatable by KOR antagonism / a decreased KOR activity. Medical / pharmaceutical treatments of diseases / disorders are a predominant aspect of the invention.

[0088] The present invention further relates to a pharmaceutical composition comprising the cyclized dynA analog according to the invention, and optionally a pharmaceutical carrier, for use in treating, ameliorating or preventing (i) depression (major depressive disorder (MDD)) and / or a (depression-related) neuropsychiatric disease (e.g. stress-mediated symptoms in depression and mood disorders);

[0089] (ii) adverse sequelae arising from stress during depression, impairment in reward-related function in patients with mood- and anxiety spectrum disorders as well as schizophrenia (e.g. anhedonia);

[0090] (iii) stress related disorders (e.g. post-traumatic stress disorder (PTSD) and / or phobias (e.g. social phobias, agoraphobias, specific phobias, social anxiety disorders));

[0091] (iv) anxiety / anxiety disoders (e.g. panic disoders, obsessive-compulsive disorder (OCD), PTSD; social phobia / social anxiety disorders, generalized anxiety disorder (GAD)); and / or

[0092] (v) drug addiction (e.g. opiate, methamphetamine, alcohol, nicotine, ecstasy and / or cocaine / cocaine derivative addiction).

[0093] Examples of phobias which may be treated, ameliorated or prevented in accordance with the invention include arachnophobia (the fear of spiders), ophidiophobia (the fear of snakes), glossophobia (the fear of public speaking), acrophobia (the fear of heights), and social phobia (social anxiety disorder; e.g. the fear of social interactions); see also https: / / www.verywellmind.com / social-phobia-or-anxiety-disorder-3024447.

[0094] Drug "addiction" in accordance with the invention may include drug "seeking behavior" (like opiate, methamphetamine, alcohol, nicotine, ecstasy and / or cocaine / cocaine derivative seeking behavior).

[0095] As mentioned, medical / pharmaceutical treatments of diseases / disorders are a predominant aspect of the invention. In other words, a pathological status is to be treated predominantly in accordance with the invention (for example, a pathological status like the diseases / disorders mentioned above / elsewhere).

[0096] It will be acknowleged by the skilled person that that certain status' / conditions may be mild, and only slightly embarrassing, respectively. Examples of such mild / slightly embarrassing (non- pathological) status' / conditions are anxiety conditions or nervousness conditions (e.g. over only brief periods), like those caused by (temporary) stressful events (e.g. examinations, competitions / tournaments, court hearings etc.). Other examples are mild / slightly embarrassing (non-pathological) phobia-like conditions, like arachnophobia-, ophidiophobia-, glossophobia-, acrophobia-, and social phobia-like conditions (e.g. (non-pathological) shyness), (temporary / non-pathological) melancholy / (mild / temporary / non-pathological) dejection. In principle, the (non-medically / non-pharmaceutically) amelioration of / intervention in such (non-pathological) disorders / status' is also within the invention. This, however, is to be seen differently from the medical / pharmaceutical treatment of, and the medical / pharmaceutical intervention in, diseases / disorders (pathological status') of the above-mentioned predominant aspect of the invention, like the medical / pharmaceutical treatment / intervention of / in the diseases / disorders (pathological status') mentioned herein above / elsewhere. Examples of such diseases / disorders (pathological status') are the diseases / disorders mentioned above (or herein elsewhere), like depression, (depression-related) neuropsychiatric diseases, phobias, anxiety / anxiety disoders, drug addictions etc.

[0097] The cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it, may be used in the context of an acute medication (for example in the context of an acute medication in the treatment, amelioration or prevention / prophylaxis as defined herein above). For example, the cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it, may be used in (treating or ameliorating of) an acute depressive episode. Similarily, the cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it, may be used when a rapid efficacy (e.g. a rapid onset of a desired (biological) function like the (competitive) KOR-antagonizing effect) is desired, e.g. in the context of the herein defined treatment, amelioration or prevention / prophylaxis. For example, the cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it, may be used when an efficacy after <lh, <45 min, <30 min, <15 min or even <10 min upon administration (e.g. s.c.) is desired, e.g. in the context of the herein defined treatment, amelioration or prevention / prophylaxis.

[0098] Furthermore, the cyclized dynA analog according to the invention may, in principle, be shortacting, medium-acting or long-acting. Accordingly, the cyclized dynA analog according to the invention may be a short-acting, medium-acting or long-acting antagonist of the KOR. The cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it, may be used accordingly, e.g. in the context of respective treatment regimens. Short-, medium-, and long-acting refers to the time (e.g. half-life) the compound remains in the body and / or exhibits its (biological) functions in the body (duration / period of action). As used herein "short-acting" may refer to a time (e.g. half-life) of about one to four hours (e.g. about 1.5h, 2. Oh, 2.5h, 3. Oh, 3.5h). As used herein "medium-acting" may refer to a time (e.g. half-life) of >4 to 36 hours (e.g. about lOh, 12h, 15h, 24h, 30h). As used herein "long-acting" may refer to a time (e.g. half-life) of about >36 hours to weeks or months (e.g. about 4 days, 2 weeks, 3 weeks, 1 month, 2 months, 4 months). Acute medication and treatment options based on short-acting efficacy may, for example, be particularly useful for the treatment of acute disorders, like an acute depressive episode, acute anxiety. Treatment options based on medium-acting efficacy may, for example, be particularly useful for the treatment of stress-mediated symptoms in depression and mood disorders. Treatment options based on long-acting efficacy may, for example, be particularly useful for the treatment of chronic / long-term disorders, like depressions, neuropsychiatric diseases, phobias, drug addiction.

[0099] Although, in the context of specific aspects, the cyclized dynA analog of the invention, or the pharmaceutical composition comprising it, may be used in the context of a time-dependent medication (e.g. acute, fast, short- / medium- / long-acting manner; see above), a general (timeindependent) medical use of the cyclized dynA analog of the invention is also within the scope of the invention.

[0100] The cyclized dynA analog, and pharmaceutical composition comprising it, to be medically used in accordance with the invention provides the advantageous property of contributing to (a) less adverse effect(s), and (an) improved profile(s) of (an) adverse effect(s), respectively. In the context of one aspect of the invention, adverse effects, in particular MOR- and / or DOR- dependent adverse effects (centrally-mediated or peripherally-mediated) may be reduced / ameliorated or avoided in accordance with the invention. Adverse effects to be reduced / avoided may be, for example, (adverse effects related to) itch / scratching, cold / heat hypersensitivity, hyperalgesia. In the context of one aspect of the treatment methods and related diseases, defects and / or symptoms described herein, one or more of such adverse effects is / are to be reduced / ameliorated or avoided.

[0101] The present invention further relates to a kit / kit of contents / kit of parts, said kit comprising a cyclized dynA analog of the invention (and optionally, at least one further (active) agent). The present invention further relates to a cyclized dynA analog or pharmaceutical composition of the invention as part of a kit / kit of contents / kit of parts, wherein the cyclized dynA analog or the pharmaceutical composition may (separately and independently from said kit) be for use in accordance with the invention (e.g. in the medical uses described herein elsewhere). The present invention further relates to a kit / kit of contents / kit of parts comprising a cyclized dynA analog or pharmaceutical composition of the invention, wherein said pharmaceutical composition and / or said cyclized dynA analog may (separately and independently from the kit) be for use in accordance with the invention (e.g. in the uses described herein elsewhere). In the context of the kit / kit of contents / kit of parts of the invention, and the respective pharmaceutical compositions / cyclic peptide(s) / cyclized dynA analog(s), but also in the context of the pharmaceutical compositions in general as described herein, the cyclized dynA analog may be contained in a (single) container or vial, and / or (a) further (active) agent(s) may be contained in (a) further container(s) or vial(s). What is said herein elsewhere with respect to the medical uses, treatment methods and pharmaceutical composition also applies to the kit.

[0102] In accordance with the present invention, the disclosed pharmaceutical composition or cyclized dynA analog, can / will be administered in a pharmaceutically / therapeutically effective dose. This means that a pharmaceutically / therapeutically effective amount of the cyclized dynA analog (active ingredient) which is to be administered is reached. Preferably, a pharmaceutically / therapeutically effective dose refers to that amount of the compound administered which, for example, results in amelioration (of symptoms) of and / or curation from the disease, in an improved condition and / or in a prolongation of survival of a subject. This can be determined by the one skilled in the art by routine testing.

[0103] The dosage regimen of the pharmaceutical composition(s) / cyclic peptide(s) / cyclized dynA analog(s) to be administered in accordance with the invention will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, sex, general health etc., the particular compounds to be administered, time and route of administration, and / or other drugs being administered concurrently. A person skilled in the art is aware of, will / can determine and is able to test the relevant doses, routes of administration and administration schemes of the compounds to be medically applied in accordance with the present invention.

[0104] In accordance with the invention, the active agent (cyclized dynA analog) may, for example, be administered at a dose in a range of 0.01 pg / kg BW to 100 mg / kg BW, 0.1 pg / kg BW to 100 mg / kg BW, 1 pg / kg BW to 100 mg / kg BW, 10 pg / kg BW to 100 mg / kg BW, 200 pg / kg BW to 60 mg / kg BW, 400 pg / kg BW to 40 mg / kg BW, 600 pg / kg BW to 20 mg / kg BW, or in a range of 800 pg / kg BW to 10 mg / kg BW. Other dose ranges may be up to 20 mg / kg BW. This may include even low doses (e.g. <0.01 pg / kg BW, <0.1 pg / kg BW, <1 pg / kg, <10 pg / kg).

[0105] Of course, as mentioned, the doses may vary depending on the administration scheme and / or administration route. For example, if administered i.v., the doses are usually lower as compared to a p.o. administration. Non-limiting examples of possible doses in the context of p.o. administration are doses in a range of 10 pg / kg BW to 100 mg / kg BW, preferably in a range of 200 pg / kg BW to 60 mg / kg BW, more preferably in a range of 400 pg / kg BW to 40 mg / kg BW. Non-limiting examples of possible doses in the context of i.v. administration are doses in a range of 600 pg / kg BW to 20 mg / kg BW, preferably in a range of 800 pg / kg BW to 10 mg / kg BW. Doses in the range of up to 20 mg / kg BW may, for example, be appropriate and safe in the context of all three, p.o., i.p. and i.v. administration. This also may include even low doses (e.g. <0.01 pg / kg BW, <0.1 pg / kg BW, <1 pg / kg, <10 pg / kg).

[0106] A dose / doses in accordance with the invention may be administered on a daily, weekly or monthly basis. The cyclized dynA analog may be administered in form of 1 or more single doses (e.g. 1 or more single bolus); in particular, 1, 2, 3, 4 or 5 single doses (e.g. per day, week or month). A particular, however non-limiting, example is the administration of a single dose (of up to) 3 times a week. Continuous administration is also envisaged in the context of the invention.

[0107] The cyclized dynA analog may, for example, be administered subcutaneously, intraperitoneally or orally. Thus, in the context of a non-limiting embodiment, a cyclized dynA analog, or a pharmaceutical composition comprising it, is administered s.c., i.v., i.p. or orally, is formulated for s.c., i.p. or oral administration and / or comprises a pharmaceutically acceptable carrier for s.c., i.p. or oral administration. A non-limiting example of a particular administration scheme are 3 single s.c. or i.p. injections of up to 10 mg / kg BW at weekly intervals, for example in PBS. This may include low doses (e.g. <0.01 pg / kg BW, <0.1 pg / kg BW, <1 pg / kg, <10 pg / kg). Another non-limiting example of a particular administration scheme are 3 single oral administrations of up to 50 mg / kg BW at weekly intervals, for example in PBS. Also this may include low doses (e.g. <0.01 pg / kg BW, <0.1 pg / kg BW, <1 pg / kg, <10 pg / kg). Further possible administration schemes are described herein elsewhere.

[0108] In general, administration of the pharmaceutical composition or the cyclic peptide(s) / cyclized dynA analog(s) in accordance with this invention may be effected by different ways. Such may be, for example, subcutaneous, oral, intravenous, intraarterial, intraperitoneal, intravesical intranodal or administrations, or administration by inhalation as well as transdermal administration. Other examples are parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intranodal, intrathecal, transdermal, transmucosal, transpulmonal subdural administrations, local or topical administrations and administrations via iontophoresis, sublingual administrations, administrations by inhalation spray or aerosol or rectal administrations, and the like. In particular, for particular patients and / or medical uses, particular administration routes like blood infusion (e.g. intravenous infusion), rectal administration (e.g. in form of enemas or suppositories) or topical administration routes may be indicated.

[0109] In one aspect, the cyclized dynA analog, or the pharmaceutical composition comprising it, is (to be) administered parenterally (e.g. s.c.; i.v.; i.m.; i.a.), perorally (e.g. in form of tablets; pills; capsules; powders; solutions; suspensions), rectally (e.g. by suppository) or bronchially (e.g. by inhalation (e.g. of a spray; aerosol; via a nebulizer; via an inhaler)).

[0110] In one aspect, the cyclized dynA analog, or the pharmaceutical composition comprising it, is (to be) administered systemically.

[0111] A preferred, however non-limiting, mode of administration scheme is s.c. administration.

[0112] The cyclized dynA analog may be comprised in the respective pharmaceutical composition or kit as the sole active ingredient or together with (an)other (active) ingredient(s). The herein described pharmaceutical composition or kit may comprise also one or more of the herein disclosed cyclized dynA analogs (e.g. 2, 3, 4, 5 or more). Likewise, one or more of the herein disclosed cyclized dynA analog may be administered in the context of the treatment methods disclosed herein. Specifically, the herein described pharmaceutical composition or kit may comprise at least two, three, four or five of the herein disclosed cyclized dynA analogs, and at least two, three, four or five of the herein disclosed cyclized dynA analogs may be administered, respectively.

[0113] The herein described two or more cyclized dynA analogs, or cyclized dynA analog and one or more further (active) agent(s), may be administered together, i.e. simultaneously, at the same or different administration sites and / or by the same or different administration routes; or they may be administered subsequently, at the same or different administration sites and / or by the same or different administration routes. In the latter case, the cyclized dynA analog(s) may be administered first, followed by a subsequent administration of another agent, or the other agent may be administered first, followed by a subsequent administration of the cyclized dynA analog(s).

[0114] In one specific embodiment, the herein described pharmaceutical composition may further comprise one or more additional (active) agent(s) (in addition to the cyclized dynA analog(s)). Likewise, the herein described (pharmaceutical composition comprising the) cyclized dynA analog(s) may be co-administered with one or more additional (active) agent(s). The herein described cyclized dynA analog(s) may also be used / administered in the context of a combination therapy or co-therapy, e.g. with or without one or more additional (active) agent(s). In another specific embodiment, the additional (active) agent(s) is (are) administered separately (temporary and / or spatially). The herein described (pharmaceutical composition comprising the) cyclized dynA analog(s) may be administered together with one or more additional (active) agent(s), i.e. prior, simultaneously or subsequently with respect to the administration of the additional active agent(s).

[0115] Non-limiting examples of one or more additional active agent(s) may be selected from the group consisting of KOR antagonists (e.g. https: / / www.guidetopharmacology.org / GRAC / ObjectDisplayForward?objectld=318&familyld= 50&familyType=GPCR) and / or (other) depression therapeutics (cf., foe example, WO 2009 / 049233).

[0116] The pharmaceutical composition of the invention may comprise (a) pharmaceutically acceptable carrier(s), excipient(s) or diluent(s). The pharmaceutical composition or cyclized dynA analog of the invention may also be administered together with (a) pharmaceutically acceptable carrier(s), excipient(s) or diluent(s). Respective carriers, excipients or diluents are well known in the art. The skilled person is readily in the position to choose carriers, excipients or diluents which are suitable to be employed in accordance with the present invention.

[0117] Pharmaceutically acceptable carriers / excipients / diluents (that may be used in the formulation of the pharmaceutical compositions comprising the active compounds as defined herein (or a salt thereof, or the like)) may generally comprise carriers, vehicles, diluents, solvents such as monohydric alcohols such as ethanol, isopropanol and polyhydric alcohols such as glycols and edible oils such as soybean oil, coconut oil, olive oil, safflower oil cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate, binders, adjuvants, solubilizers, thickening agents, stabilizers, disintergrants, glidants, lubricating agents, buffering agents, emulsifiers, wetting agents, suspending agents, sweetening agents, colourants, flavours, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers such as calcium phosphate, magnesium state, talc, monosaccharides, disaccharides, starch, gelatine, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-R- cyclodextrin, polyvinylpyrrolidone, low melting waxes, ion exchange resins. These and other suitable pharmaceutically acceptable carriers / excipients are, for example, described in Remington's Pharmaceutical Sciences, 15thEd., Mack Publishing Co., New Jersey (1991). The nature of the pharmaceutically acceptable carrier / excipient / diluent may be so that the active agent(s) are released in a slow or retarded manner.

[0118] In the following, several non-limiting administration schemes, and the use of corresponding suitable pharmaceutically acceptable carriers, are described.

[0119] For an administration of the pharmaceutical composition or the cyclized dynA analog(s) in accordance with this invention via subcutaneous (s.c.) or intravenous (i.v.) / intraarterial (i.a.) injection, (a) cyclized dynA analog(s) may be formulated in aqueous solution, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically saline buffer. For transmucosal, transpulmonal and trans-BBB administration, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art.

[0120] The use of pharmaceutical acceptable carriers to formulate the cyclized dynA analog(s) into dosages or pharmaceutical compositions suitable for systemic, i.e. intravenous / intraarterial, intranodal or subcutaneous administration is within the scope of the present invention. With proper choice of carrier and suitable manufacturing practice, the cyclized dynA analog(s) or compositions of the present invention, in particular those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be readily formulated using pharmaceutically acceptable carriers well known in the art into dosages suitable for subcutaneous or oral administration. Carriers, e.g. for oral administration, enable the compounds according to the present invention to be formulated as tablets, pills, capsules, dragees, liquids, gels, syrups, slurries, suspensions and the like, in particular for oral ingestion by a subject to be treated.

[0121] Cyclized dynA analogs / compounds according to the present invention, or medicaments or pharmaceutical compositions comprising them, intended to be administered intracorporally / intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered near the cell surface. Delivery systems involving liposomes are disclosed in US 4,880,635. The patent provides useful descriptions of techniques for liposome drug delivery.

[0122] Pharmaceutical compositions comprising a cyclized dynA analog / compound according to the present invention for parenteral and / or subcutaneous administration include aqueous solutions of the active cyclized dynA analog(s) / compound(s) in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or castor oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injections suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the cyclized dynA analog / compounds to allow for the preparation of highly concentrated solutions and to allow for a constantly slow release of the substance in the organism.

[0123] In the context of the invention, "treating" / "treatment" is, if not indicated otherwise, generally envisaged to encompass both, therapy and prevention / prophylactic treatment. Therapy may result in amelioration (of (a) symptom(s) of) or even in curing / healing the disease / disorder).

[0124] A "patient" / "subject" in the context of the purposes of the present invention, i.e. to whom (a) pharmaceutical composition(s) or cyclized dynA analog(s) according to the present invention is to be administered and / or who suffers from the disease or disorder and / or symptom(s), and / or condition, as defined and described herein, in general includes both, humans and animals, as well as other organisms. Thus, the compositions and methods of this invention are applicable to, or in connection with, both, human treatments and veterinary applications, including treating and preventing procedures and methods. Preferably, the patient / subject is a mammal, and most preferably, the patient / subject is a human.

[0125] The pharmaceutical composition, cyclized dynA analog(s) / active ingredient(s), kit / kit of contents / kit of parts etc. of the invention may be comprised in, may be a part of and / or may be a medical device, a medicinal product packaging or pharmaceutical composition packaging. Such device or packaging may, for example, comprise or be (a) vial(s) / (a)container(s), (a) syringe(s) or (a) blister pack(s). The active ingredient(s), and pharmaceutical composition(s), respectively, may be comprised in the device or packaging separately and independently (e.g. in different vials / containers or syringes or blisters), or they may be comprised in combination (e.g. in the same via l(s) / contai ner(s) or syringe(s) or blister(s)). Any of the pharmaceutical compositions, cyclized dynA analogs / active ingredients, kits etc. of the invention may be provided together with an instruction manual or instruction leaflet. The instruction manual / leaflet may comprise guidance for the skilled person / attending physician (on how) to treat or prevent a disease, disorder or symptom as described herein in accordance with the invention, for example depression (major depressive disorder (MDD)) and / or a (depression-related) neuropsychiatric disease, a stress related disorder (e.g. post-traumatic stress disorder and / or phobia (e.g. social phobia, agoraphobia, specific phobia), anxiety and / or drug addiction (e.g. opiate, methamphetamine, alcohol, nicotine, ecstasy and / or cocaine / cocaine derivative addiction). In particular, the instruction manual / leaflet may comprise guidance as to the herein described mode of administration / administration regimen (for example route of administration, dosage regimen, time of administration, frequency of administration, etc.). In principle, what has been said herein elsewhere with respect to the mode of administration / administration regimen may be comprised as (an) instruction(s) in the instruction manual / leaflet.

[0126] The present invention further relates to a method of producing a pharmaceutical composition, e.g. for the treatments / uses described herein. Said method may comprise the step of mixing (a) cyclized dynA analog(s) as defined herein (and optionally (a) further active agent(s)) with a pharmaceutically acceptable carrier, for example a pharmaceutically acceptable carrier, excipient or diluent as defined herein elsewhere. The method for producing a pharmaceutical composition may comprise the step of mixing (a) cyclized dynA analog(s) as defined herein, and a pharmaceutically acceptable carrier, for example a pharmaceutically acceptable carrier / excipient / diluent as defined herein elsewhere.

[0127] The present invention further relates to a method of preparing the cyclized dynA analog of the invention.

[0128] The method of preparing the cyclized dynA analog of the invention may comprise (e.g. as a step prior to a step of cyclization) a step of generating the (linear) peptide backbone ((linear) peptide precursor). Means and methods of generating a (linear) peptide are well known in the art and are, for example, described in Cheneval (J. Org. Chem. 79, 2014, 5538-44) and herein elsewhere (see also Example 1, "Peptide synthesis").

[0129] The (linear) peptide backbones of the dynA analogs to be produced may be produced by recombinant engineering techniques. Such techniques are well known in the art (e.g. Sambrook, supra). By this kind of production of the (linear) peptide backbones, nucleic acid molecules and / or vectors encoding the (linear) peptide backbones, and / or host cells comprising the nucleic acid molecules and / or vectors, may be used. The present invention also relates to respective (linear) peptide backbones, nucleic acid molecules, vectors and host cells.

[0130] The (linear) peptide backbones of the cyclic peptide(s) / cyclized dynA analog(s) to be produced may also conveniently be produced by non-recombinant (chemical) peptide synthesis techniques. Several approaches of such peptide synthesis are known in the art. (e.g. Williams, Chemical Approaches to the Synthesis of Peptides, CRC-Press 1997; Benoiton: Chemistry of Peptide Synthesis. CRC-Press, 2005). The (linear) peptide backbones and (cyclic) peptides disclosed herein, more particular the cysteine-stapled peptides like the particular CH2-stapled peptides may, for example, be generated / synthesized as described in Example 1, "Peptide synthesis", below).

[0131] The skilled person is readily in the position to apply the prior art knowledge to the disclosed methods for preparing the cyclized dynA analog of the invention, respectively, e.g. based on the herein provided teaching.

[0132] In particular, the method for preparing the cyclized dynA analog of the invention may comprise at least one, preferably both of, the following steps (most preferred in this order):

[0133] (i) preparing a linear peptide (peptide backbone / linear peptide precursor) having the structure HS-L3-CH2C(=O)-SQ1-Z-SQ2-NRH, wherein L3, R, SQ1and SQ2are as defined as herein elsewhere and Z is -NH-C(H)(-L1-SH)-C(=O)-, wherein L1is defined as herein elsewhere, preferably Z is a cysteine residue or a homocysteine residue, more preferably a cysteine residue;

[0134] (ii) reacting the linear peptide (peptide backbone / linear peptide precursor) of step (i) with an alkylene dihalide, e.g. a Ci-4-diiodoalkylene (preferred) or Ci-4-dibromoalkylene (such as Br- CH2-Br) or Ci-4-dichloroalkylene (such as CI-CH2-CI), or Ci-4-iodobromoalkylene or C1-4- iodochloroalkylene or Ci-4-bromochloroalkylene, which may be represented by Ha l-L2-Hal, wherein each Hal is independently selected from Cl, Br and I and L2is defined as in claim 1, preferably l-L2-l, wherein L2is defined as in claim 1; l-L2-l is preferably selected from I-CH2- I, l-CH(CH3)-l, l-C(CH3)2-l, I-CH2-CH2-I, l-CH(CH3)-CH2-l, l-CH2-CH(CH3)-l and l-CH(CH3)- CH(CH3)-I, more preferably I-CH2-I, wherein the peptide comprising SQ1and SQ2is optionally attached to a solid phase and the side chains of the amino acids in SQ1and SQ2are optionally protected. As used herein, the term "(di)halide" refers to (di)fluoro ((F— )...- F), (di)chloro ((Cl-).. -Cl), (di)bromo (( Br— )...— Br), or iodo ((I-)...-I).

[0135] As used herein, the term "alkylene" refers to a group which corresponds to an "alkyl" group in which (conceptually) a hydrogen radical has been removed, whereby a divalent group is obtained. In other words, the term "alkylene" refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched and which has two carbon atoms which can be bound to the rest of the molecule containing the "alkylene". Preferred examples of "alkylene" include methylene (-CH2-), ethylene (-CH2-CH2-) and propylene (-CH2- CH2-CH2- or -CH(CH3)-CH2-), of which methylene (-CH2-) and ethylene (-CH2-CH2-) are preferred.

[0136] In the context of the method of preparing the cyclized dynA analog of the invention, step (i) may comprise solid phase peptide synthesis (SPPS), such as Fmoc solid phase peptide synthesis (SPPS), e.g. by using a rink amide resin, PAL Resin, or a Sieber amide resin, or Boc solid phase peptide synthesis (SPPS), e.g. by using a MBHA resin.

[0137] Further, in the context of the method of preparing the cyclized dynA analog of the invention, step (ii) may be performed in an aqueous solvent, preferably in the presence of a base and preferably in the presence of a reducing agent. The aqueous solvent may be (preferably) a mixture of water and tetra hydrofuran; such as a 5:1 v / v mixture of water and tetrahydrofuran. The base may (preferably) be selected from a carbonate and an amine. More preferably, the base may include a carbonate and an amine. The carbonate may preferably be potassium carbonate and( / or) the amine may preferably be triethyl amine. The reducing agent may (preferably) be a phosphine, more preferably tris-(2-carboxyethyl)-phosphine.

[0138] In the context of the method of preparing the cyclized dynA analog of the invention, step (ii) may be performed by dissolving the linear peptide of step (i) in the aqueous solvent and adding the optional carbonate, the optional reducing agent, the amine and the alkylene dihalide, e.g. the Ci-4-diiodoalkylene, preferably in this order.

[0139] In the context of the method of preparing the cyclized dynA analog of the invention, in step (ii), relative to 1 molar equivalent, the amount of the aqueous solvent may preferably be 1000 to 3000 I, more preferably 1700 to 2300 I; the amount of the carbonate may preferably be 1 to 2, more preferably 1.4 to 1.6, molar equivalents; the amount of the reducing agent may preferably be 2 to 4, more preferably 2.5 to 3.5, molar equivalents; the amount of the amine may preferably be 5 to 15, more preferably 8 to 12 molar equivalents; a nd ( / or) the amount of the alkylene dihalide, e.g. of the Ci-4-diiodoalkylene, may preferably be 4 to 12, more preferably 6 to 10, molar equivalents.

[0140] In principle, the method of preparing the cyclized dynA analog of the invention can be performed by omitting the carbonate and / or reducing agent. Merely the amine and alkyl dihalide can also be used. However, the carbonate and / or reducing agent improve(s) the reaction yields (by preventing disulfide bond formation).

[0141] Non-limiting examples of protecting groups that may be used for the amino acid residues in the peptide backbone (linear peptide precursor), in SQ1and in SQ2of the cyclized dynA analog to be prepared are: Fmoc-Tyr(tBu)-OH (for Y), Fmoc-Gly-OH (for G), Fmoc-Phe-OH (for F), Fmoc- Leu-OH (for L), Fmoc-Arg(Pbf)-OH (for R), Fmoc-lle-OH (for I), Fmoc-Pro-OH (for P), Fmoc- Lys(Boc)-OH (for K), Fmoc-Cys(Trt)-OH.

[0142] Examples of variations of the side chain protecting groups that may be used are:

[0143] Tyr: Bn (benzyl), Deb (2,6-dichlorobenzyl), BrBn (2-bromobenzyl), Z (benzyloxycarbonyl), BrZ (2- bromobanzyloxycarbonyl), Pen (3-pentyl), Boc (tert-butyloxycarbonyl), Trt (trityl), 2-CI-Trt (2- chlorotrityl), TBDMS (tert-butyldimethylsilyl), Al (allyl), oNB (o-nitrobenzyl).

[0144] Arg: Tos (p-toluenesulfonyl), Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl), Mts (mesityl-2- sulfonyl), Mrt (4-methoxy-2,3,6-trimethylphenylsulfonyl), Mis (l,2-dimethyllindole-3-sulfonyl), bis-Boc (bis-tert-butyloxycarbonyl), Suben (5-dibenzosuberenyl), NO2, (nitro), Alloc (bisallyloxycarbonyl).

[0145] Lys: Z (benzyloxycarbonyl), Alloc (allyloxycarbonyl), oNBS (o-nitrobenzenesulfonyl), dNBS (2,4- dinitrobenesulfonyl), Dde (l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-3-ethyl), Phdec (phenyldisulphanylethyloxycarbonyl), Pydec (2-pyridyldisulphanylethyloxycarbonyl), Bts (benzothiazole-2-sulfonyl), Troc (2,2,2-trichloroethyloxycarbonyl), Dts (dithiasuccinoyl), pNZ (p- nitrobenzyloxycarbonyl), Poc (ropargyloxycarbonyl), Nvoc (4-nitroveratryloxycarbonyl), Azoc (azidomethoxycarbonyl).

[0146] Cys: Meb (p-methylbenzyl), Mob (p-methoxybenzyl), tBu (tert-butyl), Bn (benzyl), Fm (9- fluorenylmethyl), Dnpe (2-(2,4-dinitrophenyl)ethyl) and Fmoc (9-Fluorenylmethoxycarbonyl). In general, by applying the particular method of preparing the cyclized dynA analog of the invention (e.g. as described in the appended examples), the cyclized dynA analogs of the invention can be produced conveniently and efficiently; in particular if the late-stage functionalization as described herein is applied (cf., for example, Figure 1).

[0147] The method of preparing the cyclized dynA analog of the invention may be performed in accordance with / in analogy to the scheme as depicted in Figure 1.

[0148] The present invention also relates to a cyclized dynA analog as obtainable or obtained by the herein described / exemplified method(s) of preparing in accordance with the herein provided disclosure. What has been said herein elsewhere with resepect to the cyclized dynA analog of the invention and its components, e.g. with respect to Formula (I), Formula (II), Formula (II'), the peptide backbone (linear peptide precursor), SQ1, SQ2, L1, L2, and / or L3(in particular L1, and L3), as well as with respect to the cyctein-stapling options, applies also here, mutatis mutandis.

[0149] The present invention also relates to a cyclized dynA analog as disclosed herein, e.g. to a particular cyclized dynA analog as depicted in Figure 1. The present invention also relates to a C-terminally hydroxylated form of a cyclized dynA analog as disclosed herein, e.g. to a C- terminally hydroxylated form of a particular cyclized dynA analog as depicted in Figure 1 (in particular of CSD-CH2(i,8)-NH2 and CSD-CH2(i,io)-NH2; e.g. CSD-CH2(i,8)-OH and CSD- CH2(i,io)-OH). The present invention also relates to a cyclized dynA analog as defined above by reference to Formula (I), Formula (II) or Formula (IK), wherein the C-terminal -NRH group (e.g. -NH2 group is replaced by an -OH group. What has been said herein elsewhere with resepect to the cyclized dynA analog of the invention and its components, e.g. with respect to Formula (I), Formula (II), Formula (II'), the peptide backbone (linear peptide precursor), SQ1, SQ2, L1, L2, and / or L3(in particular L1, and L3), as well as with respect to the cyctein-stapling options, applies also here, mutatis mutandis.

[0150] The present invention further relates to the following items:

[0151] 1. A cyclic peptide / cyclized dynorphin A analog (dynA analog) of Formula (I), below:

[0152] Formula (I), wherein

[0153] (i) SQ1consists of 5 to 8 amino acid residues and comprises

[0154] (I) at its N-terminus, the amino acid sequence YGGF (SEQ ID NO.l; most preferred), or the amino acid sequence YGGF having 1, 2, 3 or 4 conservative amino acid substitutions (for example, the amino acid sequence YGGF having Y1 substituted by / Va-AcTyrl, DTyrl, Phel or Phe(p-Br)l and / or G2 substituted by DAIa2); and

[0155] (II) at its C-terminus, 1, 2, 3 (preferred) or 4 (consecutive) amino acid residue(s) of the amino acid sequence LRR (most preferred) or LRRI (SEQ ID NO.2), or 1, 2, 3 (preferred) or 4 (consecutive) amino acid residue(s) of the amino acid sequence LRR or LRRI having 1, 2, 3 or 4 conservative amino acid substitution(s);

[0156] (ii) SQ2consists of 4 to 6 amino acid residues and comprises 1, 2, 3, 4 or 5 (preferred) (consecutive) amino acid residue(s) of the amino acid sequence RPKLK (SEQ ID NO.3) (preferred) or IRPKLK (SEQ ID NO.4), or comprises 1, 2, 3, 4 or 5 (preferred) (consecutive) amino acid residue(s) of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s);

[0157] (iii) each of L1, L2and L3is independently selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2- CH2-, -CH(CH3)-CH2-, -CH2-CH(CH3)- and -CH(CH3)-CH(CH3)-, preferably -CH2-; and

[0158] (iv) R is selected from H, methyl and ethyl, preferably selected from H and methyl, more preferably R is H. The cyclic peptide / cyclized dynA analog according to item 1, wherein SQ1consists of 7 amino acid residues. The cyclic peptide / cyclized dynA analog according to item 1 or 2, wherein SQ2consists of 5 amino acid residues. The cyclic peptide / cyclized dynA analog according to any one of items 1 to 3, wherein SQ1consists of the amino acid sequence YGGFLRR (SEQ ID NO.5) (most preferred) orYGGFLRRI (SEQ ID NO.6), or of the amino acid sequence YGGFLRR or YGGFLRRI having 1, 2, 3, 4 ,5, 6, 7 or 8 conservative amino acid substitution(s). The cyclic peptide / cyclized dynA analog according to any one of items 1 to 4, wherein SQ2consists of the amino acid sequence RPKLK (most preferred) or IRPKLK, or of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s). The cyclic peptide / cyclized dynA analog according to any one of items 1 to 5, wherein said cyclic peptide / cyclized dynA analog

[0159] (a) is an antagonist of the kappa opioid receptor (KOR); e.g. with a KOR antagonist potency corresponding to an EC50 of < / about 30 nM in an adenylyl cyclase assay quantifying the reduction of cyclic AMP (cAMP) (e.g. as described in https: / / doi.org / 10.1021 / jm050105i), an EC50 of < / about 560 nM in a FMP blue assay (e.g. as described in https: / / doi.Org / 10.1016 / i.biopha.2021.112173), and / or an ECso of < / about 340 nM in a [35S]GTPgS assay (e.g. as described in https: / / doi.org / 10.1021 / im501827k):

[0160] (b) modulates KOR function within the CNS and / or crosses the blood-brain barrier (BBB) and enters the central nervous system (CNS), respectively;

[0161] (c) specifically binds to KOR (but not to MOR and DOR; e.g. with a >400-fold selectivity for KOR over MOR and DOR);

[0162] (d) binds to KOR with an affinity corresponding to a Ki of <10-8M, preferably <8. OxlO-9M; and / or

[0163] (e) is stable in (human) serum for >0.5 h (e.g. >50% (preferably >75%) of the cyclic peptide / cyclized dynA analog remains in the (human) serum after (about) 0.5 h; >25% (preferably >50%) of the cyclic peptide / cyclized dynA analog remains in the (human) serum after (about) 1.0 h; >20% (preferably >30%) of the cyclic peptide / cyclized dynA analog remains in the (human) serum after (about) 1.5 h; and / or >15% (preferably >25%) of the cyclic peptide / cyclized dynA analog remains in the (human) serum after (about) 2.0 h). The cyclic peptide / cyclized dynA analog according to any one of items 1 to 6, wherein

[0164] (i) SQ1is YGGFLRR (SEQ ID NO.5);

[0165] (ii) SQ2is RPKLK (SEQ ID NO.3);

[0166] (iii) each of L1, L2and L3is -CH2-; and

[0167] (iv) R is H. A pharmaceutical composition comprising the cyclic peptide / cyclized dynA analog according to any one of items 1 to 7, and optionally a pharmaceutical carrier. A pharmaceutical composition comprising the cyclic peptide / cyclized dynA analog according to any one of items 1 to 7 for use in treating, inhibiting or preventing (i) depression (major depressive disorder (MDD)) and / or a (depression-related) neuropsychiatric disease (e.g. stress-mediated symptoms in depression and mood disorders);

[0168] (ii) adverse sequelae arising from stress during depression, impairment in reward- related function in patients with mood- and anxiety spectrum disorders as well as schizophrenia (e.g. anhedonia);

[0169] (iii) stress related disorders (e.g. post-traumatic stress disorder and / or phobias (e.g. social phobias, agoraphobias, specific phobias, social anxiety disorders));

[0170] (iv) anxiety; and / or

[0171] (v) drug addiction (e.g. opiate, methamphetamine, alcohol, nicotine, ecstasy and / or cocaine / cocaine derivative addiction). The pharmaceutical composition according to item 8 or the pharmaceutical composition for use according to item 9, wherein said cyclic peptide / cyclized dynA analog or said pharmaceutical composition is (to be) administered systemically. The pharmaceutical composition according to item 8 or 10 or the pharmaceutical composition for use according to item 9 or 10, wherein said cyclic peptide / cyclized dynA analog or said pharmaceutical composition is (to be) administered parenterally (e.g. s.c.; i.v.; i.m.; i.a.), perorally (e.g. in form of tablets; pills; capsules; powders; solutions; suspensions), rectally (e.g. by suppository) or bronchially (e.g. by inhalation (e.g. of a spray; aerosol; via a nebulizer; via an inhaler)). A method of preparing the cyclic peptide / cyclized dynA analog of any one of items 1 to 7, said method comprising the steps of

[0172] (i) preparing a linear peptide having the structure HS-L3-CH2C(=O)-SQ1-Z-SQ2-NRH, wherein L3, R, SQ1and SQ2are as defined in any one of items 1 to 3 and Z is -NH- C(H)(-L1-SH)-C(=O)-, wherein L1is defined as in item 1, preferably Z is a cysteine residue or a homocysteine residue, more preferably a cysteine residue;

[0173] (ii) reacting the linear peptide of step (i) with an alkylene dihalide, e.g. a C1-4- diiodoalkylene (preferred) or Ci-4-dibromoalkylene (such as Br-CH2-Br) or C1-4- dichloroalkylene (such as CI-CH2-CI), or Ci-4-iodobromoalkylene or C1-4- iodochloroalkylene or Ci-4-bromochloroalkylene, which may be represented by Ha l-L2-Ha I, wherein each Hal is independently selected from Cl, Br and I and L2is defined as in item 1, preferably l-L2-l, wherein L2is defined as in item 1; l-L2-l is preferably selected from I-CH2-I, l-CH(CH3)-l, l-C(CH3)2-l, I-CH2-CH2-I, l-CH(CH3)- CH2-I, l-CH2-CH(CH3)-l and l-CH(CH3)-CH(CH3)-l, more preferably I-CH2-I, wherein the peptide comprising SQ1and SQ2is optionally attached to a solid phase and the side chains of the amino acids in SQ1and SQ2are optionally protected.

[0174] 13. The method of item 12, wherein step (i) of said method comprises solid phase peptide synthesis (SPPS), such as Fmoc solid phase peptide synthesis (SPPS), e.g. by using a rink amide resin, PAL Resin, or a Sieber amide resin, or Boc solid phase peptide synthesis (SPPS), e.g. by using a MBHA resin.

[0175] 14. The method of item 12 or 13, wherein step (ii) is performed in an aqueous solvent, preferably in the presence of a base and preferably in the presence of a reducing agent, wherein the aqueous solvent is preferably a mixture of water and tetrahydrofuran, such as a 5:1 v / v mixture of water and tetrahydrofuran; the base is preferably selected from a carbonate and an amine, more preferably the base includes a carbonate and an amine, where the carbonate is preferably potassium carbonate and the amine is preferably triethyl amine; and the reducing agent is preferably a phosphine, more preferably tris-(2-carboxyethyl)- phosphine.

[0176] 15. The method of item 14, wherein step (ii) is performed by dissolving the linear peptide of step (i) in the aqueous solvent and adding the optional carbonate, the optional reducing agent, the amine and the alkylene dihalide, e.g. the Ci-4-diiodoalkylene, preferably in this order.

[0177] 16. The method of item 14 or 15, wherein in step (ii), relative to 1 molar equivalent, the amount of the aqueous solvent is preferably 1000 to 3000 I, more preferably 1700 to 2300 I, the amount of the carbonate is preferably 1 to 2, more preferably 1.4 to 1.6, molar equivalents, the amount of the reducing agent is preferably 2 to 4, more preferably 2.5 to 3.5, molar equivalents, the amount of the amine is preferably 5 to 15, more preferably 8 to 12 molar equivalents, and the amount of the alkylene dihalide, e.g. of the Ci-4-diiodoalkylene is preferably 4 to 12, more preferably 6 to 10, molar equivalents.

[0178] 17. The method of any one of items 12 to 16, wherein said method is performed in accordance with the scheme as depicted in Figure 1.

[0179] As used herein, in particular with respect to the structures described and the method of preparing the cyclized dynA analog of the invention, the terms "optional", "optionally" and "may" denote that the indicated feature may be present but can also be absent. Whenever the term "optional", "optionally" or "may" is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression "X is optionally substituted with Y" (or "X may be substituted with Y") means that X is either substituted with Y or is unsubstituted. Likewise, if a component of a composition is indicated to be "optional", the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition. It is to be understood that where a list of groups is preceded by the expression "optionally substituted", the expression "optionally substituted" applies to each one of the respective groups in that list, not just to the first item in the list.

[0180] As used herein, unless explicitly indicated otherwise or contradicted by context, the terms "a", "an" and "the" are used interchangeably with "one or more" and "at least one". Thus, for example, a composition comprising "a" compound of the present invention can be interpreted as referring to a composition comprising "one or more" compounds of the present invention.

[0181] As used herein, the term "comprising" (or "comprise", "comprises", "contain", "contains", or "containing"), unless explicitly indicated otherwise or contradicted by context, has the meaning of "containing, inter alia", i.e., "containing, among further optional elements, ...". In addition thereto, this term also includes the narrower meanings of "consisting essentially of" and "consisting of". For example, the term "A comprising B and C" has the meaning of "A containing, inter alia, B and C", wherein A may contain further optional elements (e.g., "A containing B, C and D" would also be encompassed), but this term also includes the meaning of "A consisting essentially of B and C" and the meaning of "A consisting of B and C" (i.e., no other components than B and C are comprised in A).

[0182] As used herein, the term "about", unless explicitly indicated otherwise or contradicted by context, has the meaning of ± <20%, ± <10%, ± <5%, ± <3%, ± <2% or ± <1%. In principle, the lower values are preferred in this respect.

[0183] The present invention is further described by reference to the following non-limiting figures and examples.

[0184] The Figures show: Figure 1. Overview of the synthetic strategy used to generate oxidized and stapled dynorphin analogs. 2CTC: 2-chlorotrityl chloride; HATU: 2-(7-aza-lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate; HCTU; 2-(6-chloro-lH-benzotriazol-l-yl)-l, 1,3,3- tetramethylaminium hexafluorophosphate; DIPEA: N,N-diisopropylethylamine; TFA: trifluoroacetic acid; TIPS: triisopropylsilane. For details to synthesis conditions see also Materials and Methods section.

[0185] Figure 2. Receptor pharmacology of C-terminally amidated CSD ligands at the KOR. (A) The affinity of CSD-CH2(i,8)-NH2 (n=3, solid line, open squares), CSD-CH2(i,io)-NH2 (n=3, dashed line, triangles) and CSD-ace(i,8)-NH2 (n=3, dashed line, inverted triangles) was measured in in radioligand binding studies on HEK293 cell membranes stably expressing mouse KOR. Specific binding was obtained via subtraction of the total from the non-specific binding. Graph of dynAi- 13-NH2 (black solid line, black circles) is included for comparison. Data are presented as the mean ± SD and are normalized to the percentage of the maximum binding detected in the presence of 1 nM of [3H]-diprenorphine (DPN) and absence of competing ligands. (B) Gai-mediated cAMP inhibition of mouse KOR was measured in HEK293 cells stably expressing mouse K-opioid receptor (KOR) after the cell treatment with CSD-CH2(i,8)-NH2 (n=3, solid line, open squares), CSD-CH2(i,io)-NH2 (dashed line, triangles) and CSD-ace(i,8)-NH2 (dashed line, inverted triangles). DynAi-i3-NH2 was used as a positive control (n=3, solid black line, black circles; same as in Figure 1). Data are shown as mean ± SD. Normalization of data was performed according to the percentage of the maximum activation of dynAi-i3-NH2. (C) Bioluminescence resonance energy transfer (BRET) assay was employed to measure the transient interaction of K-opioid receptor (KOR) with p-arrestin-2 in HEK293 cells over time following cell treatment with 10 pM of CSD- CH2(i,8)-NH2(n=3, 2nd, open bar), CSD-CH2(i,io)-NH2(n=3, 3rdbar) and CSD-ace(i,8)-NH2(n=3, 4thbar). DynAi-i3-NH2 (black filled, 1stbar) was used a positive control ( n=3, black filled, 1stbar) and furimazine as a substrate for luciferase which was incubated for 5 min prior to the establishment of baseline and ligand treatment.

[0186] Figure 3. Subtype selectivity and stability of CSD-NH2 peptides. CSD-NH2 peptide ligands were measured for displacing 1 nM of tritiated diprenorphine ([3H]-DPN, black filled, 1stbar) in radioligand binding studies in HEK293 cells stably expressing (A) mouse p-opioid receptor (MOR) and (B) mouse 6-opioid receptor (DOR) (n=2). DynAi-i3-NH2 (2nd, open black bars) served as positive control (10 pM, n=2). To determine specific binding, total from non-specific binding was subtracted. CSD-CH2(i,8) peptides are shown in 3rdand 6th, empty bars, CSD-CH2(i,io)-NH2 ligands in 4thand 7thbars and CSD-ace(i,8)-NH2 peptides are displayed in 5thand 8thbars. (C) Stability of dynAi-i3-NH2 (n=3, solid black line, black circles), CSD-CH2(i,8)-NH2 (n=3, solid line, empty squares), CSD-CH2(i,io)-NH2 (n=3, dashed line, triangles) and CSD-ace(i,8)-NH2 (dashed line, inverted triangles) was measured in human plasma.

[0187] Figure 4. Competitive antagonism of CSD-CH2(i,s)-NH2 at the KOR and its inhibition of calcium mobilization in DRGs. (A) Concentration-response curves of U50,488 (solid black line, black circles) in the absence and presence of 0.3 pM (solid line, diamonds), 1 pM (solid line, inverted triangles), 3 pM (solid line, triangles) and 10 pM (dashed line, squares) of CSD-CH2(i,8)-NH2. Gai- mediated cAMP inhibition of mouse KOR was measured in HEK293 cells stably expressing mouse K-opioid receptor (KOR). Data were normalized to the percentage of maximum activation of U50,488 (n=4). (B) Schild regression analysis. A denotes ECso of U50,488 in presence of CSD-CH2(i,8)-NH2; A' is ECso of U50,488 and B indicates logarithm of concentration of CSD-CH2(i,8)-NH2. The dotted line with a slope of 1 (n=4) is used for comparison. (C) Representative traces of calcium signals in dorsal root ganglion (DRG) cultures without stimulation (CTRL, black traces, n=648), or stimulated by U50,488 (U50 10 pM, blue traces, n=1467), U50,488 (10 pM) in combination with nor-BNI (0.5 pM, red traces, n=537), U50,488 (10 pM) in combination with CSD-CH2(i,8)-NH2 (1 pM, violet traces, n=1126) and U50,488 (10 pM) in combination with CSD-CH2(i,8)-NH2 (10 pM, cyan traces, n=720). The antagonists nor-BNI and CSD-CH2(i,8)-NH2were pre-applied followed by addition of the agonist. Carvacrol (100 pM) and capsaicin (1 pM) served as controls for TRPA1 and TRPVl, respectively. CTRL was an external solution. A potassium chloride (KCI) solution (60 mM) added at the end of each recording was used as positive control for detecting viable cells and generated similar response amplitudes. (D) Quantification by area under the curve (AUC) of control (CTRL, black, 1stcircle), U50,488 (U50 10 pM, 2ndcircle), (nor-BNI, 0.5 pM, 3rdcircle), U50 (10 pM) in combination with CSD-CH2(i,8)-NH2(1 pM, 4thcircle) and U50 (10 pM) in combination with CSD-CH2(i,8)-NH2(10 pM, 5thcircle) for the interval 120-180 s. Data are shown as mean ± 95% C.L from two independent experimental days. One-way ANOVA with Tukey's multiple comparisons test. Two asterisks indicate significance between U50,488 (10 pM) and U50,488 (10 pM) + nor-BNI (0.5 pM) while three asterisks represent significance between U50,488 (10 pM) and U50,488 (10 pM) + CSD-CH2(i,8)-NH2(1 pM) as well as U50,488 (10 pM) and U50 + CSD-CH2(i,8)-NH2(10 pM) (***p < 0.001). Four asterisks indicate significance between U50,488 (10 pM) and CTRL (****p < 0.0001).

[0188] Figure 5. In vivo KOR antagonist activity and site of action of CSD-CH2(i,s)-NH2. (A) Antagonism of U50,488-induced antinociception by CSD-CH2(i,8)-NH2 and nor-BNI after s.c. administration in the radiant heat tail-flick assay in mice. (B) Antagonism by CSD-CH2(i,8)-NH2 and nor-BNI on the effect of U50,488 in the rotarod test in mice after s.c. administration. Groups of mice were s.c. treated with CSD-CH2(i,8)-NH2 (20 mg / kg) or nor-BNI (10 mg / kg), 15 min or 24 h, respectively, prior to administration of U50,488 (5 mg / kg), and (A) tail-flick latencies (at 30 and 60 min) or (B) latencies to fall from the rotarod (at 30 min) were determined. Values represent means ± SEM (n = 5-6 mice per group). ***p < 0.001 vs. saline group, two-way ANOVA with Bonferroni post-hoc test;##P<0.01 and###P < 0.001 vs. U50,488-treated group, one-way ANOVA with Tu key's post hoc-test.

[0189] Saline: 1stbar; U50,488: 2ndbar; U50,488 + CSD-CH2(i,8)-NH2: 3rdbar; U50,488 + nor-BNI: 4thbar, at 30min, 60 min and in B, respectively.

[0190] Figure 6. Kinetic bioluminescence resonance energy transfer (BRET) reported by nanoLuc. Substances at a concentration of 10 pM were applied after 300 s to measure p-arrestin recruitment at the KOR of (A) CSD-ox(i,s)-OH (n=3, empty squares), (B) CSD-ox(i,io)-OH (n=3, squares), (C) CSD-CH2(i,8)-OH (n=3, empty triangles), (D) CSD-CH2(i,io)-OH (n=3, triangles), (E) CSD-mXYL(i,8)-OH (n=3, empty inverted triangles), (F) CSD-mXYL(i,io)-OH (n=3, inverted triangles), (G) CSD-tet(i,s)-OH (n=3, empty diamonds), (H) CSD-tet(i,io)-OH (n=3, diamonds), (I) CSD-ace(i,8)-OH (n=3, empty hexagons) and (J) CSD-ace(i,io)-OH (n=3, hexagons), CSD-CH2(i,8)- NH2 (n=3, empty squares), CSD-CH2(i,io)-NH2 (n=3, squares), CSD-ace(i,8)-NH2(n=3, inverted triangles). Dynorphin (dyn) A1-13-NH2 served as control (n=3, filled black circles in all panels).

[0191] Figure 7.XH NMR spectrum of CSD-CH2(I,8)-NH2at 600 MHz in H2O / D2O (9:1, v / v). NMR was performed as described in Example 1.

[0192] In the foregoing detailed description of the invention, a number of individual elements, characterizing features, techniques and / or steps are disclosed. It is readily recognized that each of these has benefit not only individually when considered or used alone, but also when considered and used in combination with one another. Accordingly, to avoid exceedingly repetitious and redundant passages, this description has refrained from reiterating every possible combination and permutation. Nevertheless, whether expressly recited or not, it is understood that such combinations are entirely within the scope of the presently disclosed subject matter.

[0193] All technical and scientific terms used herein, unless otherwise defined, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0194] Further, reference is made herein to the following amino acid sequences (and also to the respectiove coding nucleotide sequences):

[0195] SEQ ID NO.l (N-terminal "message" motif of dynAi-13 and dynAi-17)

[0196] YGGF

[0197] SEQ ID NO.2 (N-terminal part of C-terminal "address" sequence of dynAi-13 and dynAi-17)

[0198] LRRI

[0199] SEQ ID NO.3 (C-terminal part of C-terminal "address" sequence of dynAi-13; most preferred example of SQ2)

[0200] RPKLK

[0201] SEQ ID NO.4 (C-terminal part of C-terminal "address" sequence of dynAi-13; example of SQ2) IRPKLK

[0202] SEQ ID NO.5 (N-terminal part of dynAi-13 and dynAi-17; most preferred example of SQ1) YGGFLRR

[0203] SEQ ID NO.6 (N-terminal part of dynAi-13 and dynAi-17; example of SQ1)

[0204] YGGFLRRI

[0205] SEQ ID NO.7 (Dynorphin A1-13 (dynAi-13))

[0206] YGGFLRRIRPKLK

[0207] SEQ ID NO.8 (C-terminal "address" sequence of dynAi-13) LRRIRPKLK

[0208] SEQ ID NO.9 (Dynorphin A1-17 (dynAi-17))

[0209] YGGFLRRIRPKLKWDNQ SEQ ID NO.10 (C-terminal "address" sequence of dynAi-17) LRRIRPKLKWDNQ

[0210] SEQ ID NO.11 (most preferred example of the peptide backbone of the cyclized dynA analog of the invention) YGGFLRRCRPKLK

[0211] SEQ ID NO.12 (example of the peptide backbone of the cyclized dynA analog of the invention) YGGFLRRI CPKLK

[0212] SEQ ID NO.13 (example of the peptide backbone of the cyclized dynA analog of the invention) YGGFLRCIRPKLK

[0213] Tripeptide (no SEQ ID NO. assigned; N-terminal part of C-terminal "address" sequence of dynAi-13 and dynAi-17) LRR

[0214] The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.

[0215] Example 1: Materials and Methods

[0216] Materials

[0217] Dynorphin (dyn) A1-13 amide trifluoroacetate salt was purchased from Bachem (Austria). Naloxone and (±)-trans-U50,488 methanesulfonate salt were obtained from Sigma (Austria). [3H]-diprenorphine (DPN) was ordered from Perkin Elmer (Austria). cAMP Gi kit was from Cisbio (France) and jetPRIME transfection reagent from Polyplus (Austria). Bromoacetic acid was ordered from Chem-Supply (Australia). Carvacrol and capsaicin were obtained from Sigma (Austria). U50,488 and nor-BNI were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA).

[0218] Peptide Synthesis

[0219] Peptides were assembled using automated Fmoc solid phase peptide synthesis (SPPS) using either rink amide or 2-chlorotrityl chloride resins on a 0.125 mmol scale. Amino acid couplings were performed with 2 eq. of amino acid, 2 eq. of HCTU and 4 eq. of DIPEA in DMF (4 mL) for 15 min and repeated twice for each residue. Fmoc was deprotected using 30% piperidine in DMF for 5 min, repeated twice. CSD peptides were assembled using an N-terminal 3- (tritylthio)propanoic acid, manually coupled using 4 eq. of HATU, 8 eq. DIPEA in 4 mL DMF for 30 min. Each peptide was cleaved from the resin using a cleavage cocktail of TFA / TIPS / H2O (95:2.5:2.5) for 2 h and the crude peptides were precipitated with cold Et20. The precipitated peptides were then redissolved in FhO / MeCN (1:1) and lyophilized to yield a white powder. Each peptide was purified by reversed-phase high performance liquid chromatography (RP- HPLC) on a Shimadzu Prominence HPLC system prior to oxidation and stapling.

[0220] Disulfide bonded peptides (ox) were synthesized by stirring the reduced peptide precursor (5.2 pmol) in 50 mM ammonium bicarbonate (pH 8.5) at 1 mg / mL in an open reaction vessel. After 18 h the mixture was acidified with TFA and purified by RP-HPLC.

[0221] Acetone ("ace"; sometimes also termed "dca" (dichloroacetone)) stapled peptides were prepared by dissolving the reduced peptide precursor (6.5 pmol) in 15 mL of 50 mM ammonium bicarbonate (pH 9.5) with 1 eq. of TCEP. 1,3-dichloroacetone was (2 eq.) added from a 40mM stock solution in MeCN. After stirring for 2 h the reaction was acidified with TFA and purified by RP-HPLC.

[0222] Xylene (mXYL) stapled analogs were prepared by dissolving the reduced peptide precursor (5.2 pmol) in 8 mL of 50 mM ammonium bicarbonate (pH 9.5) with 20% MeCN and 1 eq. of TCEP. l,3-bis(bromomethy)benzene (1.5 eq.) was added dropwise from a 40 mM stock solution in DMF and mixed for 30 min before acidification with TFA and purification by RP-HPLC.

[0223] Tetrazine (tet) stapled peptides were prepared by dissolving the reduced peptide precursor (6.2 pmol) in 12 mL of 50 mM ammonium bicarbonate (pH 5.5). The mixture was added to a 50 mL falcon tube containing 3 eq. of 3,6-dichloro-l,2,4,5-tetrazine in 12 mL of CHCI3 and vortexed for 5 min. The organic and aqueous layers were separated by centrifuge and the aqueous layer isolated. The organic layer was further washed with 50 mM ammonium bicarbonate and aqueous isolates were combined, acidified by TFA and purified by RP-HPLC.

[0224] Methylene (CH2) stapled peptides were prepared by dissolving the reduced peptide precursor (6.2 pmol) in 12 mL of H2O / THF (5:1, v / v) with 1.5 eq of K2CO3, 3 eq. of TCEP, 10 eq. of triethylamine and 8 eq. of diiodomethane added sequentially. The mixture was stirred for 6 h before acidification with TFA and purification by RP-HPLC.

[0225] Peptide Purification

[0226] Peptides were purified by RP-HPLC on preparative or semipreparative Phenomenex (Germany) Jupiter Cis columns (5 pm, 300 A, 250 x 21.2 mm or 250 x 10 mm) applying a linear gradient from 5% to 65% solvent B (90% MeCN, 10% H2O, 0.05% TFA) and flow rates of 8 mL / min or 3 mL / min, respectively. Automatically collected fractions were analyzed by electrospray ionization mass spectrometry (ESI-MS) using a Shimadzu LCMS 2010 system and analytical RP- HPLC on a Phenomenex Jupiter Cis column (5 pm, 300 A, 150 x 2 mm). Mass and purity of all peptides was confirmed by matrix-assisted laser desorption / ionization (MALDI) time-of-flight (TOF) MS using an AB Sciex 5800 spectrometer, and analytical RP-HPLC using a Shimadzu UPLC system (Table 2). MALDI-TOF MS was performed by mixing 3 pL of a-cyano-hydroxycinnamic acid (CHCA) and 0.5 pL of peptide and spotting 0.5 pL of the mixture onto the MALDI target plate. All peptides were purified to >95% purity.

[0227] NMR

[0228] NMR was performed for each cyclized dynA analog exemplified herein by using a Bruker AVANCE III 600 MHz spectrometer equipped with a cryogenically cooled probe. Each peptide was prepared in a ~2 mM solution of H2O / D2O (9:1, v / v) and experiments were run at 298 K with referencing to H2O at 4.70 ppm.1H, TOCSY and NOESY experiments were run for each sample. An exemplaryTH NMR spectrum is provided herein as Figure 7.

[0229] Stability Assay

[0230] Stability of peptide ligands was determined with human serum from male AB plasma (Sigma) centrifuged at 13,300 x g for 10 min to remove lipid content. The supernatant was preincubated at 37°C for 5 min before 10 pL of a peptide stock solution (3 mg / mL in H2O) was added to 300 pL of serum. A reference solution was prepared in phosphate buffered saline (PBS) buffer. The serum was incubated at 37°C and analyzed over a time course of 5 hours with 40 pl aliquots removed at time points T = 0, 15, 30, 60, 90, 120, 240 and 300 min. Each aliquot was quenched with 40 pL of 5 M urea and incubated on ice for 10 min. The proteins were then precipitated with the addition of 40 pL of 20% (w / v) trichloroacetic acid and further incubated on ice for 10 min. The samples were centrifuged for 10 min at 15,000 x g and the supernatant was analyzed by analytical RP-HPLC using a Phenomenex Jupiter Cis column (5 pm, 300 A, 150 x 2 mm) using a linear gradient from 0 to 50% solvent B (90% acetonitrile, 10% H20, 0.05% trifluoroacetic acid) and a flow rate of 1 mL / min. The percentage of peptide remaining was determined by the integration of the analyte in comparison to T = 0 min. Peptides were analyzed in triplicate and data were fitted to an exponential decay curve using GraphPad Prism software.

[0231] Cell culture and Transfection

[0232] HEK293 cells were grown at 37°C and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 50 U / mL penicillin as well as streptomycin. Cell transfection was performed with jetPRIME transfection reagent as per the manufacturer's protocol (Polyplus, France) using 2 pg of plasmids encoding mouse KOR-EGFP and / or human |3- arrestin-2-nano luciferase. Cloning of mouse KOR into pEGFP-Nl vector and generation of a HEK293 cell line stably expressing mouse KOR-EFGP were performed as previously described (Muratspahic, J Med Chem 64, loc. cit.).

[0233] Radioligand Displacement Binding Assays

[0234] HEK293 cell membranes stably expressing KOR, MOR and DOR were prepared as described in Muratspahic et al. (Muratspahic, J Med Chem 64, loc. cit.). The dissociation constant Ka values for the mouse KOR was previously determined in a saturation binding assay (Muratspahic, J Nat Prod 84, 2021, 2238-48). Non-specific binding was examined in the presence of 10 pM of naloxone. Displacement binding was performed in a final volume of 300 pL containing [3H]- diprenorphine (1 nM final), peptide solution (4X), membranes (3.5-7 pg / assay) and standard binding buffer (50 mM Tris-HCI (pH 7.4), 10 mM MgCl2 and 0.1% BSA). Equilibrium was achieved by incubating displacement binding reactions at 37°C for 1 h. The Skatron cell harvester device was utilized to terminate reactions by rapid filtration onto GF / C filters pre-soaked with 0.1% polyethlyenimine. Displacement binding for MOR and DOR was conducted with 50-100 pg and 20 pg of HEK293 stable cell membranes, respectively. cAMP Assay

[0235] Peptide ligands were assayed for cAMP inhibition in HEK293 stable mouse KOR cell line in a 384- well format and triplicates according to the Cisbio protocol. Briefly, 5 pL containing 2 000 cells per well were incubated with 5 pL of peptide solutions prepared 2X in IX stimulation buffer and forskolin (10 pM final). Following an incubation of peptide ligands at 37°C for 30 min cryptate- labeled cAMP and cAMP d2-labeled antibody (5 pL of each) were added to the mixture and further incubated for at least 1 h at room temperature. Subsequently, cAMP levels were quantified on a Flexstation 3 (Molecular Devices, USA) using homogenous time-resolved fluorescence resonance energy transfer (HTRF) and the ratio 665 / 620 nm. Antagonism of CSD- CH2(i,8)-NH2 at KOR was evaluated by Schild regression analysis as previously described (Duerrauer, Sci Rep 9, 2019, 19295). Briefly, concentration-response curves of U50,488 were measured in the presence and absence of CSD-CH2(i,8)-NH2. The cells were pretreated with CSD- CH2(i,8)-NH2 (0.3, 1, 3 and 10 pM) for 30 min at 37°C followed by co-incubation with U50,488 for additional 30 min at 37°C. The reaction was terminated by the addition of 5 pL of cAMP-d2 antibody and 5 pL of cAMP Eu Cryptate. After 1 h of incubation at 37°C fluorescence was measured on a Flexstation 3 and quantified using the 665 / 620nm ratio. Samples were measured in technical triplicates.

[0236] Bioluminescence Resonance Energy Transfer (BRET) Assay BRET assay was performed as previously described (Duerrauer, Sci Rep 9, 2019, 19295). Briefly, human p-arrestin-2-nano luciferase (NIuc) and mouse KOR-EGFP were transiently expressed in HEK293 cells following co-transfection in a 1:10 ratio. 16 h post -transfection, 50,000 cells in 100 pL of phenol red-free DMEM with 10% FBS per well were transferred into white clear bottom 96-well plates and incubated overnight. On the day of assay, cells were incubated for 1 h at 37°C in phenol red- and serum-free DMEM. Furimazine (Promega, USA) and peptide ligands were diluted 4X and 1:50 in Hank's balanced salt solution (HBSS), respectively. Prior to establishing the baseline and measuring kinetic BRET furimazine was incubated for 5 min at 37°C. The ability of peptide ligands to induce p-a rresti n recruitment was assessed by measuring light emissions for EGFP (510 nm) and NIuc (460 nm) over 35 min using a Flexstation 3. The BRET ratio was calculated as emission EGFP (510 nm) / emission NIuc (460 nm). The ligand-induced BRET was calculated by subtracting the BRET ratio of the ligand from the BRET ratio of the HBSS. Relative BRET quantification was determined by calculating the mean value of ligand-induced BRET for each peptide ligand from 311 to 2,411 sec (35 min) which was then divided by the mean value of ligand-induced BRET of dynAi-i3-NH2 set as 100.

[0237] Animals and Drug Administration

[0238] Breeding, euthanasia and all procedures of animal handling were performed according to regulations of animal care and welfare. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86 / 609 / EEC). Behavioral experiments were performed in male CD1 mice (8-10 weeks old) purchased from Janvier Labs (Le Genest-Saint-lsle, France). All animal care and experimental procedures were in accordance with the ethical guidelines for the animal welfare standards of the European Communities Council Directive (2010 / 63 / EU), and were approved by the Committee of Animal Care of the Austrian Federal Ministry of Science and Research. Mice were group-housed in a temperature- controlled specific pathogen free room with a 12 h light / dark cycle and with free access to food and water. U50,488, nor-BNI and CSD-CH2(i,8)-NH2 were prepared in sterile physiological 0.9% saline solution. Test compounds or vehicle (saline) were s.c. administered in a volume of 10 pL / g body weight.

[0239] Isolation of Dorsal Root Ganglion (DRG) Neurons

[0240] Wild type C57BL / 6 mice (13-17 weeks of age) were euthanized by cervical dislocation, preceded by anesthesia by an exposure to isoflurane. Dorsal root ganglia (DRGs) from all spinal levels were excised and transferred to DMEM containing 1% streptomycin / penicillin and 1% L- glutamine treated with 1 mg / mL collagenase (Sigma, Austria) and 3 mg / mL dispase II (Roche, Austria) for 60 min at 37°C. Digested DRGs were then mechanically dissociated with a Pasteur pipette, centrifuged at 1000 rpm for 5 min and plated onto 12 mm glass coverslips previously coated with 100 mg / mL poly-D-lysine (Sigma). DRG neurons were cultured in DMEM supplemented with 100 mg / mL streptomycin / penicillin, 1% L-glutamine and 100 ng / mL mouse nerve growth factor (Alomone Labs, Israel). Neurons were cultured at 37°C and 5% CO2 for 15 to 30 h.

[0241] Single-cell Calcium Microfluorimetry

[0242] The coverslips were incubated with Fura-2 AM ester (Biotium, USA) for 30 min at 37°C and 5% CO2, before placement in glass-bottom 35 mm dishes in extracellular solution (in mM: 145 NaCI, 5 KCI, 10 glucose, 10 HEPES, 1.25 CaCl2, 1 MgCb, pH 7.4, 300 mOsm). After a recovery period of 10 min, dishes were mounted onto an Olympus IX73-inverted microscope (Olympus, Tokyo, Japan) and imaged using a lOx objective. Cells were permanently superfused with extracellular solution using a software-controlled 8-channel, gravity-driven, common-outlet system (ALA Scientific Instruments Inc, USA). This superfusion was switched to different solutions after 2 min of baseline recording. A positive control detecting viable cells was added at the end of each recording, using depolarization by an extracellular solution with 60 mM KCI (isotonic replacement of NaCI). Fura-2 was alternatingly excited for 30 msec by a 340-nm LED (50 mW, used at 100%) and by a 385-nm LED (1435 mW, used a 5%) using an Omicron LEDHub (Laserage- Laserprodukte GmbH, Germany). Fluorescence emission was long-pass filtered at 495 nm, and pairs of images were acquired at a rate of 1 Hz with a 4.2-megapixel 16-bit CCD camera (6.5 pm pixel edge length, 18.8 mm sensor diameter, Prime BSI; Teledyne Photometries, USA). The hardware was controlled by the pManager 1.4 plugin in ImageJ. (Schneider, Nat Methods 9, 2012, 671-675) The background intensity was subtracted before calculating the ratio between the fluorescence emitted when the dye was excited at 340 nm and at 385 nm (F340 / F385 nm). The time course of this ratio was analyzed for regions of interest adapted to individual cells.

[0243] Tail-flick Assay

[0244] The radiant heat tail-flick test was performed using an UB 37360 Ugo Basile analgesiometer (Ugo Basile s.r.l., Varese, Italy) as described previously (Dumitascuta, Sci Rep 10, 2020, 5653). The reaction time required by the mouse to remove its tail after application of the radiant heat was measured and defined as the tail-flick latency (in seconds). Tail-flick latencies were measured before and after s.c. administration of saline (control) or U50,488 (5 mg / kg) (i.e. 30 and 60 min) (test latency, TL). In antagonist studies, CSD-CH2(i,8)-NH2 (20 mg / kg) or nor-BNI (10 mg / kg) were s.c. administered 15 min or 24 h, respectively, before U50,488 (5 mg / kg). A cut-of time of 10 sec was used in order to minimize tissue damage. Each experimental group included 5-6 mice. The antinociceptive effect (as percentage of maximum possible effect, % MPE) was calculated according to the formula = [(TL - BL) / (cutoff time - BL)] x 100 where TL represents the test latency and BL is the basal latency.

[0245] Rotarod Test

[0246] The rotarod test was performed using the accelerating rotarod treadmill (Acceler Rota-Rod 7650, Ugo Basile s.r.L, Varese, Italy) for mice (diameter 3.5 cm) as described previously. (Erli, J Med Chem 60, 2017, 7579-90) Animals were habituated to the equipment in two training sessions (30 min apart) one day before testing. On the experimental day, mice were placed on the rotarod, and treadmill was accelerated from 4 to 40 rpm over a period of 5 min. The time spent on the drum was recorded for each mouse before (baseline) and at 30 min afer s.c. administration of saline (control) or U50,488 (5 mg / kg). In antagonist studies, CSD-CH2(i,8)-NH2 (20 mg / kg) or nor-BNI (10 mg / kg) were s.c. administered 15 min or 24 h, respectively, before U50,488 (5 mg / kg). Decreased latencies to fall in the rotarod test indicate impaired motor performance. A 300 sec cut-of time was used. Each experimental group included 5-6 mice. The rotarod data are expressed as percentage (%) changes from the rotarod latencies obtained before (baseline, BL) and after drug administration (test, TL) were calculated as 100 x (TL / BL).

[0247] Data and Statistical Analysis

[0248] Data analysis was performed with GraphPad Prism (GraphPad Software, USA). The potency (EC50) and maximum efficacy (Emax) values of peptide ligands were derived from functional data fitted to three-parameter non-linear regression curves constrained to a bottom of zero and a hill slope of one. Graphs were normalized to 100%, i.e., the highest concentration (10 pM) of dynAi-i3-NH2. For Schild plot analysis, data were normalized to 100%, i.e., the highest concentration (10 pM) of U50,488 and fitted to three-parameter non-linear regression curves constrained to a bottom of zero. The logarithm of the concentration-ratio (A' / A-l) was plotted against the logarithm of the respective concentration of antagonist CSD-CH2(i,8)-NH2 (B) to derive the pA2 value (functional inhibitory affinity).

[0249] Data from radioligand binding studies were fitted using the three-parameter one-site competition binding equation with constrained Kd and concentration of [3H]-diprenorphine used in the assay (1 nM) to obtain IC50 values. Calculated IC50 values were then used to obtain inhibition constants K) using the Cheng and Prusoff equation. Data were normalized to specific binding of [3H]-diprenorphine without competing ligands and are reported as maximum percentage (100%), which denotes an average of 7000 - 8000 fmol / mg for KOR, MOR and DOR. In vivo data were statistically evaluated using one-way ANOVA with Tukey's multiple comparison post hoc test, or two-way ANOVA with Bonferroni post-hoc test with significance set at P < 0.05. Example 2: Design and Synthesis of CSD Analogs

[0250] To design constrained dynA ligands to target the KOR, a strategy was utilized that could be implemented at a late stage, on unprotected peptide scaffolds and that would be amenable to diversification. Lactam bridges, disulfide linkages, and carbon-carbon crosslinks have previously been explored to constrain dynA analogs with mixed success (Schiller, loc. cit., Kawasaki, loc. cit.; Arttamangkul, J Med Chem 38, loc. cit.; Arttamangkul, J Med Chem 40, loc. cit.; Fang, loc. cit.). Further, these strategies do not provide synthetic flexibility to rapidly diversify analogs with varying crosslinker functionality. Cysteine staples have emerged as a late-stage functionalization strategy that can be used to modulate GPCR pharmacology (White, J Med Chem 65, loc. cit.), but hitherto have not been investigated in the design of KOR ligands. Applied herein was a cysteine stapling strategy to constrain a dynAi-13 peptide template bearing a native C-terminal carboxylic acid (cf. Goldstein, Proc Natl Acad Sci U 5 A 76, 1979, 6666-70). A cysteine residue was introduced by substituting either isoleucine at position 8, or proline at position 10, and a second thiol was manually coupled onto the N-terminus using 3-(tritylthio)propionic acid. This generated two scaffolds (CSD(i-s) and CSD(i-io)) that could undergo macrocyclization via disulfide formation or using cysteine stapling strategies (Figure 1). Four cysteine stapling strategies were used to modify the unprotected peptide scaffolds, leading to the formation of methylene (CH2), acetone (ace or dca), m-xylene (mXYL) and tetrazine (tet) stapled motifs, to go alongside disulfide bond constrained (ox) analogs. Overall, this strategy yielded ten cysteine stapled dynAi-13 analogs with diverse crosslinkers, which was named Cysteine Stapled Dyn (CSD) analogs.

[0251] Example 3: Pharmacological Properties of C-terminally Amidated CSD Ligands at the KOR

[0252] A C-terminal amidation was introduced to the most promising peptides, i.e. CSD-CH2(i,8)-OH, CSD-CH2(i,io)-OH and CSD-ace(i,s)-OH in order to further improve their desired pharmacological properties at the KOR. Peptides containing an amidated C-terminus were synthesized analogous to peptides with the carboxyl group at the C-terminus (Figure 1). Following synthesis, peptides were subject to pharmacological characterization. The affinity of C-terminally amidated peptides was first assessed in radioligand binding studies. The affinity of CSD-CH2(i,8)-NH2 (Ki = 7 nM), CSD-CH2(i,io)-NH2 (6 nM) and CSD-ace(i,8)-NH2 (32 nM) improved by approximately 12-, 6- and 3-fold, respectively (Figure 2A, Table 1). cAMP were assays were then conducted to explore the potency and efficacy of newly synthesized ligands to modulate G protein signaling. Intriguingly, compared to their counterparts with a carboxyl group at the C-terminus, the C- terminal amidated peptides displayed disparate potencies and efficacies (Figure 2B, Table 1). CSD-CH2(i,8)-NH2was inactive at the KOR on its own compared to CSD-CH2(i,io)-NH2, which acted as a partial agonist following C-terminal amidation with an EC50 and Emax of 540 nM and 49%, respectively (Figure 2B, Table 1). On the other hand, C-terminal amidation of CSD-ace(i,8)-NH2 decreased its potency to EC50 = 740 nM (Figure 2B, Table 1). Finally, peptides were screened in the kinetic p-arrestin-2 recruitment assay. Like previously characterized analogs, all three peptides did not or only partially recruit p-arrestin-2 at concentrations of 10 pM (Figure 2C, Figure 6A-C).

[0253] Example 4: Increased Selectivity and Stability of KOR CSD Ligands

[0254] Next, the selectivity profile of the three peptides comprising an NH2 at the C-terminus was examined by measuring their binding affinity in HEK293 cell membrane preparations stably expressing mouse MOR and DOR. A concentration of 10 pM was tested for each peptide ligand and demonstrated that all three peptides are KOR selective as they did not significantly displace radioligand from MOR and DOR at this concentration (Figure 3A and B). Similar findings were observed for the three peptides with a C-terminal COOH (Figure 3A and B). The stability of three peptides with the C-terminal COOH was first mesured in human serum by reversed-phase high performance liquid chromatography (RP-HPLC). Interestingly, these peptides degraded within 15 min, similar to the native dynAi-i3-NH2 (data not shown). Since C-terminal amidation improved binding affinity of CSD-CH2(i,8)-NH2, CSD-CH2(i,io)-NH2 and CSD-ace(i,8)-NH2 the stability of these peptides in human serum was then determined by RP-HPLC. In fact, C-terminal amidation of CSD peptides improved their stability compared to the linear dynAi-i3-NH2 with CSD-CH2(i,8)-NH2being the most stable peptide ligand (Figure 3C).

[0255] Example 5: Competitive Antagonism of CSD-CH2(i,s)-NH2 and its Inhibitory Action on Calcium Mobilization in DRGs

[0256] To determine the mechanism of antagonism of CSD-CH2(i,8)-NH2, adenylyl cyclase-mediated cAMP inhibition at KOR was measured using Schild regression analysis. KOR expressed in HEK293 cells was activated by the small molecule agonist U50,488 in the absence and presence of increasing concentrations of CSD-CH2(i,8)-NH2. A rightward shift of the concentrationresponse curves of U50,488 was observed, with no change in the Emax typical for competitive antagonism (Figure 4A). Detailed Schild analysis of CSD-CH2(i,8)-NH2, exhibited a linear regression slope of 1.03 and a pA2 value of 7.5, which corresponds to an average functional affinity of 32 nM, thus demonstrating the competitive mechanism of the CSD-CH2(i,8)-NH2 antagonist (Figure 4B). Importantly, the antagonistic activity of CSD-CH2(i,8)-NH2 was verified in a physiological context by exploring its ability to modulate calcium mobilization in peripheral DRGs known to express KOR (Scherer, Proc Natl Acad Sci U S A, 114, 2017, 13561-6; Dembla, Elife, 6, 2017, e26280; Snyder, Neuron 99, 2018, 1274-1288 e6). Using single-cell calcium microfluorimetry on neuronal populations described by their response to TRPA1 agonist carvacrol and TRPVl agonist capsaicin, CSD-CH2(i,8)-NH2 in combination with the KOR agonist U50,488 was applied (Figure 4C and D). To confirm antagonism of CSD-CH2(i,8)-NH2, it was coapplied with U50,488 and it was observed that CSD-CH2(i,8)-NH2 significantly blocks the action of U50,488 (Figure 4C and D). This effect was further supported by KOR selective antagonist nor-binaltorphimine (nor-BNI) which inhibited the activity of U50,488 (Figure 4C and D). Taken together, these findings suggest that CSD-CH2(i,8)-NH2 inhibits the action of U50,488 in both KOR-transfected HEK293 cells and DRG neurons, which endogenously express KOR.

[0257] Example 6: In vivo KOR Antagonism and Site of Action of CSD-CH2(i,s)-NH2 after s.c. Administration in Mice

[0258] The in vivo KOR antagonist activity of CSD-CH2(i,8)-NH2, and its site of action was evaluated in mice after subcutaneous (s.c.) administration by assessing CSD-CH2(i,8)-NH2 effect on U50,488- induced antinociception in a mouse model of acute thermal nociception, the radiant heat tailflick assay. U50,488 was used as a prototypical centrally acting KOR agonist. Pretreatment of mice with CSD-CH2(i,8)-NH2 (20 mg / kg) significantly antagonized the antinociceptive effect of U50,488 (Figure 5A). Similarly, s.c. administration of the standard KOR antagonist nor-BNI (10 mg / kg) also blocked the antinociceptive response of U50,488 (Figure 5A). Activation of central KOR is well-recognized to induce CNS effects, such as sedative effects that can be readily observed in animals by a marked decrease in the locomotor activity. As shown in Figure 5B, U50,488, in an antinociceptive dose of 5 mg / kg, significantly affected motor coordination in the rotarod test in mice after s.c. administration. To evaluate if CSD-CH2(i,8)-NH2 reverses the motor impairment caused by U50,488 through the central KOR, mice were pretreated with CSD- CH2(i,8)-NH2(20 mg / kg) prior to U50,488. Administration of CSD-CH2(i,8)-NH2 to mice significantly antagonized the effect of U50,488 in the rotarod test, comparable to the effect produced by nor-BNI (Figure 5B). These behavioral data show that CSD-CH2(i,8)-NH2 has in vivo a KOR- mediated antagonist activity by acting on central KOR.

[0259] Reference is further made herein to the following table(s):

[0260] Table 1: Pharmacological properties of CSD peptide leads at the KOR

[0261]

[0262] Data are from two to three independent experiments; dyn, dynorphin; n.a., not active 'Functional affinity of 32 nM was derived from Schild regression analysis

[0263] Table 2: Characterization and purity analysis of CSD peptide ligands determined by MALDI-TOF mass spectrometry and RP-HPLC

[0264] Monoisotopic masses of peptides obtained by ESI- and / or MALDI-MS are shown. RP-HPLC purity (%) was determined by integration (area under the curve) of the analytical traces at 214 nm.

[0265] If not indicated differently, the following abbreviations are used herein:

[0266] BRET, bioluminescence resonance energy transfer; cAMP, cyclic adenosine monophosphate; CSD, cysteine stappled dynorphin; DOR, 6-opioid receptor; DRG, dorsal root ganglion; dynA, dynorphin A; EGFP, enhanced green fluorescence protein; ESI-MS, electrospray ionization mass spectrometry; GPCR, G protein-coupled receptor; KOR, K-opioid receptor; MALDI-MS, matrix- assisted laser desorption ionization mass spectrometry; MOR, p-opioid receptor; nor-BNI, nor- binaltorphimine; RP-HPLC, reversed-phase high performance liquid chromatography; s.c., subcutaneous.

Claims

CLAIMS1. A cyclic peptide / cyclized dynorphin A analog (dynA analog) of Formula (I), below:Formula (I), wherein(i) SQ1consists of 5 to 8 amino acid residues and comprises(I) at its N-terminus, the amino acid sequence YGGF (SEQ ID NO.1), or the amino acid sequence YGGF having 1, 2, 3 or 4 conservative amino acid substitutions; and(II) at its C-terminus, 1, 2, 3 or 4 amino acid residue(s) of the amino acid sequence LRR or LRRI (SEQ ID NO.2), or 1, 2, 3 or 4 amino acid residue(s) of the amino acid sequence LRR or LRRI having 1, 2, 3 or 4 conservative amino acid substitution(s);(ii) SQ2consists of 4 to 6 amino acid residues and comprises 1, 2, 3, 4 or 5 amino acid residue(s) of the amino acid sequence RPKLK (SEQ ID NO.3) or IRPKLK (SEQ ID NO.4), or comprises 1, 2, 3, 4 or 5 amino acid residue(s) of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s);(iii) each of L1, L2and L3is independently selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2- CH2-, -CH(CH3)-CH2-, -CH2-CH(CH3)- and -CH(CH3)-CH(CH3)-; and(iv) R is selected from H, methyl and ethyl.

2. The cyclic peptide / cyclized dynA analog according to claim 1, wherein SQ1consists of 7 amino acid residues.

3. The cyclic peptide / cyclized dynA analog according to claim 1 or 2, wherein SQ2consists of 5 amino acid residues.

4. The cyclic peptide / cyclized dynA analog according to any one of claims 1 to 3, wherein SQ1consists of the amino acid sequence YGGFLRR (SEQ ID NO.5) or YGGFLRRI (SEQ ID NO.6), or of the amino acid sequence YGGFLRR or YGGFLRRI having 1, 2, 3, 4 ,5, 6, 7 or 8 conservative amino acid substitution(s).

5. The cyclic peptide / cyclized dynA analog according to any one of claims 1 to 4, wherein SQ2consists of the amino acid sequence RPKLK or IRPKLK, or of the amino acid sequence RPKLK or IRPKLK having 1, 2, 3, 4, 5 or 6 conservative amino acid substitution(s).

6. The cyclic peptide / cyclized dynA analog according to any one of claims 1 to 5, wherein, in said amino acid sequence YGGF, Y1 is substituted by / Va-AcTyrl, DTyrl, Phel or Phe(p- Br)l; and / or G2 is substituted by DAIa2.

7. The cyclic peptide / cyclized dynA analog according to any one of claims 1 to 6, wherein said cyclic peptide / cyclized dynA analog(a) is an antagonist of the kappa opioid receptor (KOR);(b) modulates KOR function within the CNS and / or crosses the blood-brain barrier (BBB) and enters the central nervous system (CNS), respectively;(c) specifically binds to KOR;(d) binds to KOR with an affinity corresponding to a Ki of <10-8M; and / or(e) is stable in (human) serum for >0.5 h.

8. The cyclic peptide / cyclized dynA analog according to any one of claims 1 to 7, wherein said cyclic peptide / cyclized dynA analog(a) is an antagonist of the KOR with a KOR antagonist potency corresponding to an EC50 of < 30 nM in an adenylyl cyclase assay quantifying the reduction of cyclic AMP (cAMP), an EC50 of < 560 nM in a FMP blue assay, and / or an EC50 of < 340 nM in a [35S]GTPgS assay;(b) specifically binds to KOR with a >400-fold selectivity for KOR over MOR and DOR;(c) binds to KOR with an affinity corresponding to a Ki of <8.0xl0-9M; and / or(d) is stable in human serum so that >50% of the cyclic peptide / cyclized dynA analog remains in the human serum after 0.5 h; >25% of the cyclic peptide / cyclized dynA analog remains in the human serum after 1.0 h; >20% of the cyclic peptide / cyclized dynA analog remains in the human serum after 1.5 h; and / or >15% of the cyclic peptide / cyclized dynA analog remains in the human serum after 2.0 h.

9. The cyclic peptide / cyclized dynA analog according to any one of claims 1 to 8, wherein said cyclic peptide / cyclized dynA analog is stable in human serum so that >75% of the cyclic peptide / cyclized dynA analog remains in the human serum after 0.5 h; >50% of the cyclic peptide / cyclized dynA analog remains in the human serum after 1.0 h; >30%) of the cyclic peptide / cyclized dynA analog remains in the human serum after 1.5 h; and / or >25% of the cyclic peptide / cyclized dynA analog remains in the human serum after 2.0 h.

10. The cyclic peptide / cyclized dynA analog according to any one of claims 1 to 9, wherein(i) SQ1is YGGFLRR (SEQ ID NO.5);(ii) SQ2is RPKLK (SEQ ID NO.3);(iii) each of L1, L2and L3is -CH2-; and(iv) R is H.

11. A pharmaceutical composition comprising the cyclic peptide / cyclized dynA analog according to any one of claims 1 to 10, and optionally a pharmaceutical carrier.

12. A pharmaceutical composition comprising the cyclic peptide / cyclized dynA analog according to any one of claims 1 to 10 for use in treating(i) depression and / or a neuropsychiatric disease;(ii) adverse sequelae arising from stress during depression, impairment in reward- related function in patients with mood- and anxiety spectrum disorders or schizophrenia;(iii) stress related disorders;(iv) anxiety / anxiety disoders; and / or(v) drug addiction.

13. The pharmaceutical composition according to claim 11, or the pharmaceutical composition for use according to claim 12, for use in treating(i) a major depressive disorder (MDD) and / or a depression-related neuropsychiatric disease;(ii) an anhedonia and / or an adverse sequela arising therefrom;(iii) post-traumatic stress disorders (PTSDs) and / or phobias;(iv) panic disoders, obsessive-compulsive disorder (OCD), PTSD; and / or generalized anxiety disorder (GAD)); and / or(v) an opiate addiction, a methamphetamine addiction, an alcohol addiction, a nicotine, an ecstasy addiction and / or a cocaine / cocaine derivative addiction.

14. The pharmaceutical composition according to claim 11 or 13, or the pharmaceutical composition for use according to claim 12 or 13, for use in treating(i) stress-mediated symptoms in depression and mood disorders; and / or(ii) social phobias, agoraphobias, specific phobias and / or social anxiety disorders.

15. The pharmaceutical composition according to any one of claims 11, 13 or 14, or the pharmaceutical composition for use according to any one of claims 12 to 14, which is for use in an acute manner / acute treatment.

16. The pharmaceutical composition according to any one of claims 11 andl3 to 15, or the pharmaceutical composition for use according to any one of claims 12 to 15, wherein said cyclic peptide / cyclized dynA analog and / or said pharmaceutical composition is for use in a short-acting, medium-acting or long-acting manner.

17. The pharmaceutical composition according to any one of claims 11 and 13 to 16 or the pharmaceutical composition for use according to any one of claims 12 to 16, wherein said cyclic peptide / cyclized dynA analog or said pharmaceutical composition is administered systemically.

18. The pharmaceutical composition according to any one of claims 11 and 13 to 17 or the pharmaceutical composition for use according to any one of claims 12 to 17, wherein said cyclic peptide / cyclized dynA analog or said pharmaceutical composition is administered parenterally, perorally, rectally or bronchially.

19. A method of preparing the cyclic peptide / cyclized dynA analog of any one of claims 1 to 10, said method comprising the steps of(i) preparing a linear peptide having the structure HS-L3-CH2C(=O)-SQ1-Z-SQ2-NRH, wherein L3, R, SQ1and SQ2are as defined in any one of claims 1 to 3 and Z is -NH- C(H)(-L1-SH)-C(=O)-, wherein L1is defined as in claim 1, preferably Z is a cysteine residue or a homocysteine residue, more preferably a cysteine residue; and(ii) reacting the linear peptide of step (i) with an alkylene dihalide, e.g. a C1-4- diiodoalkylene or Ci-4-dibromoalkylene (such as Br-CH2-Br) or C1-4-dichloroalkylene (such as CI-CH2-CI), or Ci-4-iodobromoalkylene or C1-4- iodochloroalkylene or Ci-4-bromochloroalkylene, which may be represented by Ha l-L2-Ha I, wherein each Hal is independently selected from Cl, Br and I and L2is defined as in claim 1, preferably l-L2-l, wherein L2is defined as in claim 1; l-L2-l is preferably selected from I-CH2-I, l-CH(CH3)-l, l-C(CH3)2-l, I-CH2-CH2-I, l-CH(CH3)- CH2-I, l-CH2-CH(CH3)-l and l-CH(CH3)-CH(CH3)-l, more preferably I-CH2-I, wherein the peptide comprising SQ1and SQ2is optionally attached to a solid phase and the side chains of the amino acids in SQ1and SQ2are optionally protected.

20. The method of claim 19, wherein step (i) of said method comprises solid phase peptide synthesis (SPPS).

21. The method of claim 20, wherein said SPPS is Fmoc SPPS or Boc SPPS.

22. The method of claim 20, wherein said SPPS is Fmoc SPPS by using a rink amide resin, PAL Resin, or a Sieber amide resin, or wherein said SPPS is Boc SPPS by using a MBHA resin.

23. The method of any one of claims 19 to 22, wherein step (ii) is performed in an aqueous solvent.

24. The method of any one of claims 19 to 23, wherein step (ii) is performed in an aqueous solvent in the presence of a base.

25. The method of any one of claims 19 to 24, wherein step (ii) is performed in an aqueous solvent in the presence of a reducing agent.

26. The method of any one of claims 23 to 25, wherein the aqueous solvent is a mixture of water and tetrahydrofuran.

27. The method of any one of claims 23 to 26, wherein the aqueous solvent is a 5:1 v / v mixture of water and tetra hydrofuran.

28. The method of any one of claims 24 to 27, wherein the base is selected from a carbonate and an amine.

29. The method of any one of claims 24 to 28, wherein the base includes a carbonate and an amine.

30. The method of claim 28 or 29, wherein the carbonate is potassium carbonate and / or the amine is triethyl amine.

31. The method of any one of claims 25 to 30, wherein the reducing agent is a phosphine.

32. The method of any one of claims 25 to 31, wherein the reducing agent is tris-(2- carboxyethyl)-phosphine.

33. The method of any one of claims 23 to 32, wherein step (ii) is performed by dissolving the linear peptide of step (i) in the aqueous solvent and adding the carbonate, the reducing agent, the amine and / or the alkylene dihalide.

34. The method of claim 33, wherein the carbonate, the reducing agent, the amine and / or the alkylene dihalide are added in the order as mentioned.

35. The method of claim 33 or 34, wherein the alkylene dihalide isCi-4-diiodoalkylene.

36. The method of any one of claims 23 to 35, wherein in step (ii), relative to 1 molar equivalent, the amount of the aqueous solvent is 1000 to 3000 I; the amount of the carbonate is 1 to 2 molar equivalents; the amount of the reducing agent is 2 to 4 molar equivalents; the amount of the amine is 5 to 15 molar equivalents; and / or the amount of the alkylene dihalide is 4 to 12 molar equivalents.

37. The method of claim 23 to 36, wherein in step (ii), relative to 1 molar equivalent, the amount of the aqueous solvent is 1700 to 2300 I; the amount of the carbonate is 1.4 to 1.6 molar equivalents; the amount of the reducing agent is 2.5 to 3.5 molar equivalents; the amount of the amine is 8 to 12 molar equivalents; and / or the amount of the alkylene dihalide is 6 to 10 molar equivalents.

38. The method of any one of claims 19 to 37, wherein said method is performed in accordance with the scheme as depicted in Figure 1.

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