Selective cyclized peptide kappa-opioid receptor agonists

Abstract

The present invention relates to a novel cyclic peptide / cyclized dynorphin A analog (dynA analog) which acts a specific ((G-protein-)biased) agonist 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 pain (in particular (chronic) peripheral pain) and inflammation (e.g. inflammatory bowel disease etc.). The present invention also relates to a method of preparing the novel cyclic peptide / cyclized dynA analog, for example by (late-stage) functionalization upon thioether cyclization.

Description

[0001] Selective cyclized peptide K-opioid receptor agonists

[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 as a (specific) ligand of the kappa opioid receptor (K-opioid receptor; KOR). In particular, the cyclic peptide / dynA analog of the invention acts as a (specific) ((G-protein-)biased) agonist 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 cyclized dynA analog of the invention for use in treating, inhibiting or preventing KOR-related diseases / disorders, including pain (in particular (chronic) peripheral pain) and inflammation (e.g. inflammatory bowel disease etc.). The present invention also relates to a method of preparing the cyclized dynA analog, for example by (latestage) functionalization upon thioether cyclization.

[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, loc. cit.) 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 manifest themselves by virtue of KOR activation in the central nervous system and which have precluded clinical development of KOR agonists (White, J Pharmacol Exp Ther 352, 2015, 98-109; Brust, Sci Signal 9, 2016, rall7). 0-arrestins regulate opioid receptor desensitization and internalization in a phosphorylation-dependent fashion (Allouche, Front Pharmacol 2014, 280). Kliewer and colleagues have recently unraveled that MOR, unable to recruit 0-arrestins, displays greater morphine- and fentanyl-promoted analgesia in vivo (Kliewer, Nat Commun 2019, 367). The concept of biased signaling relying on therapeutically relevant G protein and side effect-associated p-arrestin pathways has been applied as a strategy to address the development of KOR-related adverse effects (White, loc. cit.; Brust, loc. cit.). More particular, over recent years, biased opioid ligands selectively activating G protein over p-arrestin signaling pathways have provided an avenue to develop therapeutics with reduced KOR-related adverse effects (Machelska, Front Pharmacol 9, 2018, 1388). However, despite recent approval of a G- protein biased MOR agonist (oliceridine) for the treatment of moderate to severe pain, the role of p-arrestins in the development of side effects remains a hot-button issue in the opioid receptor field (Kliewer, Nat Commun 2019, 367). By contrast, the analgesic potential of KOR for therapeutic use has been particularly stressed by agonists that target KOR in the peripheral nervous system (Snyder, Neuron 2018, 1274-1288). This has been recently highlighted by clinical approval of peripherally restricted KOR peptide analgesic difelikefalin (CR845) which lacks centrally mediated KOR side effects (Davenport, Nat Rev Drug Discov 2020, 389-413). While targeting peripherally expressed KOR is a promising strategy to develop novel analgesics, the role of p-arrestins in the peripheral KOR signal transduction machinery remains poorly understood. It remains uncertain whether biased agonism offers therapeutic benefits (Che, Sci Signal 14, 2021, eaav0320). For example, 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, Difelikefalin 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 An? (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). Peptide stapling using cysteine crosslinking and thioether cyclization have emerged as attractive approaches to induce loss of conformational restraint of peptides by adopting preorganized, stable a-helical secondary structures and by locking peptides in an active conformation (Vinogradov, J Am Chem Soc 2019, 4167-4181, Lau, Chem Soc Rev 2015, 91-102, Li, Chem Rev 2020, 10079-10144). Peptide stapling using cysteine crosslinking and thioether cyclization have 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).

[0009] 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 crosslinking and thioether cyclization, especially with late-stage functionalization moieties, has only sparsely been employed for opioid receptor ligands. More particular, it has not yet been attempted for dynA.

[0010] DynAi-17 (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 crosslinking and thioether cyclization is also underutilized in the field of opioid receptors targeting and in the design of KOR ligands.

[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] There is still the need in the art to advance the status of peptide KOR ligands, in particular peptide KOR agonists, 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 agonists, with desired pharmacological properties (e.g. ((G-protein-)biased) KOR agonism, high selectivity, (merely) peripheral action and / or higher stability) to interrogate KOR pharmacology and to develop useful and improved therapeutic leads for KOR-related disorders.

[0015] 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 agonists. More specifically, the problem underlying the present invention is the provision of medically useful KOR agonists.

[0016] 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); and

[0022] (iii) each of L1and L2is independently selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2-CH2-, - CH(CH3)-CH2-, -CH2-CH(CH3)- and -CH(CH3)-CH(CH3)-, preferably -CH2. The above compound is also termed cyclic peptide of the invention or cyclized dynorphin A analog (dynA analog) of the invention.

[0023] In the context of the invention, constrained KOR-targeting dynAi-13 analogs were developed by using thioether cyclization and diverse peptide stapling approaches. More particular, peptide probes were designed based on dynAi-13 by using thioether cyclization and 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 peptide stapling strategy was utilized that could be implemented at a late stage, on unprotected peptide scaffolds (late-stage functionalization; cf. Figure 1). This stapling strategy is amenable to diversification. Further, C-terminal amidation was applied and the resulting effecs were tested.

[0024] The applied stapling strategy resulted in selective cysteine stapled (CSD) and thioether cyclized (TCD) dynAi-13 analogs (cyclized dynA analogs) with distinct pharmacological properties. Inter alia, the particular KOR-targeting dynAi-13 analog, "TCD(i,8)-OH", was obtained with surprisingly superior properties like G protein-biased KOR agonism and an improved selectivity for KOR over MOR and DOR. Further, the present invention provides for / offers the great potential of thioether cyclization to develop selective peptide probes to target / agonize KOR signaling, and to gain novel insight into KOR-mediated treatment of peripheral pain.

[0025] Evidence is provided in the context of the invention, and by the appended examples, that the cyclic peptide / cyclized dynA analogs as claimed and as provided / disclosed herein (particular constrained cyclized dynAi-13 analogs; also termed "thioether cyclized dynA analogs (TCD 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 agonists. More specifically, the present invention provides for even improved KOR agonists.

[0026] The pharmacological properties of the cyclized dynA analogs of the invention include high / full agonizing activity / potency, ((G-protein-)biased) agonizing activity, (extraordinary) high specificity to KOR (as compared to other opioid receptors, e.g. MOR and / or DOR), less adverse effects (e.g. no / reduced respiratory depression, constipation, tolerance or addiction), and / or peripheral activity / activity in the PNS. The cyclized dynA analogs of the invention are improved with respect to the agonizing activity / potency, specificity to KOR (e.g. MOR and / or DOR), less adverse effects (e.g. no / reduced respiratory depression, constipation, tolerance or addiction), and / or peripheral activity / activity in the PNS; in particular as compared to the known smallmolecule KOR agonists or other known peptide KOR agonists.

[0027] In particular, it is documented herein below and in the appended examples, that the constrained dynAi-13 analog termed "TCD(i,8)-OH" (see, for example, Figure 1 and Formula( IF) herein below) targets the KOR with a low nM affinity and a preference for modulating G protein over 0-arrestin signaling pathways ((G-protein-)biased agonizing activity). This is in agreement with Passoni et al. who demonstrated that thioether-cyclized carbetocin promotes Gqprotein signaling of the oxytocin receptor with no recruitment of p-arrestin-2 (J Neuroendocrinal 28, 2016, 1-10). Further, TCD(i,s)-OH targets the KOR with an up to and even more than 70-fold enhanced selectivity for KOR over MOR and / or DOR (as compared to dynAi-13 as such). Further, the selectivity of TCD(i,s)-OH for KOR is shown to be about 1 / 70 / 70 (KOR / MOR / DOR). Further, in a radioligand binding assay, TCD(i,s)-OH is shown to bind to hKOR with a Ki of ~60 nM, to hMOR with a Ki of ~1000 nM, and not to hDOR.

[0028] In addition, the ability of cyclized dynA analogs like TCD(i,s)-OH to retain inhibitory KOR activity in a dorsal root ganglion (DRG) neuronal primary cell model is shown. In particular, TCD(i,s)-OH does not elicit a significant calcium response in DRG neurons; in contrast to, for example, the non-biased KOR agonist U50,488 which induces calcium mobilization.

[0029] Moreover, in the context of the invention, a (dose-dependent) antinociceptive effect of TCD(i,8)-OH was shown in vivo (in a formalin test; as a model of (inflammatory) pain). In particular, the duration of pain behavior (time spent, for example, for licking / biting, lifting, and flinching of a formalin-injected paw) was significantly reduced by TCD(i,s)-OH in a dosedependent manner (cf. Figure 12A). The antinociceptive effect of TCD(i,s)-OH was shown to be based on KOR agonism (cf. Figure 12B). Further, the antinociceptive potency of TCD(i,s)-OH (in a formalin test) was shown to be higher (reduced ED50 value; reduced by about 10%) than the antinociceptive potency of U50,488 (cf. Table 3).

[0030] Further, without being bound by theory, the C-terminal amide of a subset of cyclized dynA analogs disclosed herein may interact with the acidic residues in the extracellular loop of KOR. Also without being bound by theory, herein disclosed cyclized dynA analogs thereby exhibit, for example, an increase! 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, the agonistic dynA analogs of the invention (like TCD(i,s)-OH) may, upon binding to KOR, stabilize the active state of KOR and promote a more active KOR- dynA analog complex. Also shown herein is the improved stability of C-terminally amidated cyclized dynA analogs disclosed herein, e.g. TCD(i,8)-NH2, towards proteolytic degradation in human serum.

[0031] In conclusion, by using the endogenous dynAi-13 as a molecular template and peptide thioether cyclization, the safe, potent, highly specific and( / or) peripherally-acting ((G-protein-)biased) KOR agonists of the invention were developed; and are provided herein.

[0032] The compound of the invention, i.e. the KOR agonist provided herein, comprises a peptide backbone (linear peptide precursor) which is derived from dynA, in particular from dynAi-13 (YGGFLRRIRPKLK; SEQ ID NO.7). The compound 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 agonist provided herein, 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 preferred 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.

[0044] 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.

[0045] 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).

[0046] 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.).

[0047] 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).

[0048] 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.

[0049] 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.

[0050] 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. 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.

[0051] 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).

[0052] 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).

[0053] 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.

[0054] 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.

[0055] In the cyclized dynA analog of the invention (see also Formula (I) and Formula (II)), each of L1and L2is independently selected from -CH2-, -CHfCHs)-, -C(CHs)2-, -CH2-CH2-, -CH(CH3)-CH2-, - CH2-CH(CH3)- and -CH(CH3)-CH(CH3)-. Preferably, at least one of L1and L2is -CH2-. Most preferably, both, L1and L2, are -CH2-.

[0056] 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).

[0057] The most preferred example of a cyclized dynA analog according to the invention (termed TCD(i,8)-OH) is a compound of Formula (I), wherein

[0058] (i) SQ1is YGGFLRR (SEQ ID NO.5);

[0059] (ii) SQ2is RPKLK (SEQ ID NO.3); and

[0060] (iii) both of L1and L2are -CH2-.

[0061] The valence bound formula of the most preferred example of a cyclized dynA analog according to the invention (termed TCD(i,s)-OH) is given below (Formula (II')):

[0062] Formula (II')

[0063] 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.

[0064] 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 or three), or (preferably) all of the desired and advantageous (biological) functions as described herein, in particular the (biological) functions of TCD(i,s)-OH. Most relevantly, the cyclized dynA analog according to the invention is a specific KOR agonist, preferably a specific a (G-protein-)biased KOR agonist. In particular, the cyclized dynA analog according to the invention is envisaged to bind to KOR (or to be capable of binding to KOR), and( / or) to be a specific (G-protein-)biased KOR agonist (in particular as compared to MOR and / or DOR). For example, the cyclized dynA analog according to the invention binds to KOR with a low nM affinity (see below and, for example, item (c), infra), has a preference for modulating G-protein over p-a rrestin signaling pathways (G-protein- biased agonizing activity; no / low recruitment of p-a rrestin, e.g. p-arrestin-1 and / or p-a rresti n- 2) (see below and, for example, item (d), infra), binds to KOR with an up to and even more than 70-fold enhanced selectivity for KOR over MOR and / or DOR (as compared to dynAi-i3-NH2 as such; for example, the selectivity of TCD(i,8)-OH for KOR is about 1 / 70 / 70 (KOR / MOR / DOR) (see below and, for example, item (b), infra), and( / or) does not elicit a (significant) calcium response in dorsal root ganglion (DRG) neurons (e.g. as compared to the non-biased KOR agonist U50,488; see below).

[0065] 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 or three), or (preferably) all of the following biological functions: (a) the cyclized dynA analog is (is capable of acting as) an agonist of the KOR (partial agonist or, preferably, full agonist; in particular a (G-protein-)biased (partial or, preferably, full) agonist); e.g. in a cAMP inhibition assay (e.g. with a HEK293 mouse KOR cell line) (e.g. with (a potency / efficacy corresponding to) an Emaxof 80-200%, preferably 100-180%, more preferably 120-160%, more preferably (about) 137% (± ≤33%)), as compared to dynA1-13- NH2and / or an EC50value of 10-8to 10-6M, preferably 5x10-8M to 5x10-7M, more preferably 8x10-8M to 3x10-7M, more preferably (about) 1.3x10-7M (± ≤0.4x10-7M)); (b) the cyclized dynA analog specifically binds to KOR (but not to MOR and DOR; e.g. with a ≥15-fold, ≥30-fold, ≥40-fold, ≥50-fold, ≥50-fold, ≥60-fold or ≥ / about 70-fold selectivity for KOR over MOR and / or DOR; higher selectivity is preferred); (c) the cyclized dynA analog binds to KOR (is capable of binding to KOR) with an affinity corresponding to a Ki in the range of 10-8M to 10-6M, preferably in the range of 5x10-8M to 5x10-7M, more preferably in the range of 8x10-8M to 3x10-7M, preferably in the range of 1x10-7M to 2.5x10-7M (e.g. to a Ki value of about 1.5x10-7M (± ≤0.3 x10-7M)). In a radioligand binding assay (like a radioligand binding assay as described in the appended examples), the cyclized dynA analog may bind to KOR with an affinity corresponding to a Ki in the range of 10 to 1000 nM, preferably in the range of 20 to 500 nM, more preferably in the range of 30 to 200 nM, preferably in the range of 40 to 100 nM (e.g. to a Ki value of about 60 nM (± ≤10 nM)). In such an assay, TCD(1,8)-OH was shown to bind to hMOR with a Ki of ~1000 nM. Further, in such an assay, TCD(1,8)-OH was shown not to bind to hDOR; and / or (d) the cyclized dynA analog induces (is capable of inducing) only partial, or preferably no, β- arrestin recruitment, e.g. β-arrestin-2 (and / or β-arrestin-1) recruitment (e.g. ≤25%, ≤20%, ≤15% or ≤10% of the β-arrestin-2 (and / or -1) recruitment of dynA1-13-NH2); e.g. in a respective β-arrestin (-2 and / or -1) recruitment assay (e.g. with a HEK293 (mouse) KOR cell line; e.g. in a (kinetic) BRET assay (e.g. with EGFP / Nluc)) (e.g. partial or no β-arrestin (-2 and / or -1 recruitment up to a concentration of ≤15 µM, ≤10 µM, ≤5 µM, ≤1 µM, ≤0.5 µM, ≤0.1 µM, ≤0.05 µM, ≤0.01 µM; higher maximum concentration values preferred). The cyclized dynA analogs of the invention (in particular when being C-terminally amidated (C- terminal -NH2group instead of the C-terminal -OH group); cf. the appended examples) 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)); item (e). 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. For example, the cyclized dynA analog may be 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).

[0066] In one aspect (item (f)), a cyclized dynA analogof the invention may not be capable of passing the blood-brain barrier (BBB), and entering the CNS, respectively, (or only to a low or marginal extent). This applies, for example, in addition to one, more or all of the above functions (a) to (e). Such a cyclized dynA analog of the invention may peripherally act in accordance with the invention. Such a cyclized dynA analog may thus be particularly useful for peripheral applications, e.g. (chronic) peripheral pain treatment. Another peripheral action may be reducing or inhibiting peripheral immune cell invasion and / or (a) peripheral immune response(s) (like, for example infiltration of the CNS (e.g. the brain or spinal cord) by, for example, CD4+ and / or CD8+ T-cells).

[0067] In another aspect (item (g)), a cyclized dynA analog of the invention may be capable of modulating KOR function within the CNS; or even of passing the BBB, and entering the CNS (e.g. the brain and / or spinal cord) via the BBB, respectively. Further, a cyclized dynA analog may enter / pass the BBB and enter the CNS, respectively, under certain circumstances (for example when there is a leaky BBB; which may, for example, be associated with a disease, like a disease as defined herein (e.g. (a) certain inflammatory or autoimmune condition(s)). Thus, a cyclized dynA analog of the invention and as defined herein may be capable of penetrating the CNS (e.g. the brain and / or spinal cord) or of modulating KOR function in the CNS. This applies, for example, in addition to one, more or all of the above functions (a) to (e). A cyclized dynA analog which is capable of passing the BBB, and entering the CNS, respectively, or of modulation KOR function in the CNS, may be particularly useful for central applications, e.g. (chronic) central pain treatment or treatment of (CNS lesions in) multiple sclerosis (MS) or Alzheimer's disease (AD).

[0068] In one aspect of the invention, the cyclized dynA analog exhibits (or is capable of exhibiting) an antinociceptive effect; in particular an antinociceptive effect based on a KOR-agonizing activity. In this context, the cyclized dynA analog may lead in vivo to a short / shortened period of nociception and / or of pain behavior (e.g. as compared to a (saline) control without the cyclized dynA analog); e.g. a short / shortened period of <300 seconds, <200 seconds, <100 seconds, or about 50 seconds (e.g. in a mouse model of (inflamatory) pain; formalin test; e.g. about 15 minutes after formalin administration). An antinociceptive effect can, for example, be tested by a formalin test (a model of (inflamatory) pain), like a formalin test as described in Muratspahic (Nat. Commun 14:8064, 2023, 1-17), Dubuisson (Pain 4, 1977, 161-174), and in the appended examples. An antinociceptive effect can, for example, be determined by detecting the duration of a pain behavior of a test animal (like a mouse or a rat); after administering the test compound (cyclized dynA analog); and, for example, after pain has been induced (e.g. by formalin injection). For example, the time spent for licking / biting, lifting, and / or flinching of a (formalin) affected site of pain (e.g. formalin-injected paw) may be tested (dependent on the presence of (a certain amount / dose of) the test compound (cyclized dynA analog)). In the context of the testing of the antinociceptive effect, the test compound (cyclized dynA analog) may be administered prior to (or together with or after) a pain-inducing compound (e.g. formalin), and the antinociceptive effect may be determined afterwards. For example, the test compound may be administered 10-20 minutes prior to a pain-inducing compound (preferably about 15 minutes), and the antinociceptive effect may be determined 10-20 minutes after administration of the pain-inducing compound (preferably about 15 minutes thereafter). Suitable doses / concentrations of the test compound (cyclized dynA analog) can readily be determined (e.g. according to the guidance in Muratspahic (2023, loc. cit.)). In a mouse model, non-limiting examples of doses / concentrations may be in the range of 1-10 mg / kg, preferably 2-6 mg / kg, more preferably 3.0-5.5 mg / kg (e.g. about 5 mg / kg). In the context of this aspect of the invention, the antinociceptive potency of the cyclized dynA analog according to the invention may be higher (reduced ED50 value) than the antinociceptive potency of U50,488 (e.g. reduced by >5% or >10% as compared to U50,488). The antinociceptive potency of the cyclized dynA analog according to the invention may be in the range of an ED50 of 0.5 to 2.5 pmol / kg (95% CL) or, preferably, 1.0 to 2.0 pmol / kg (95% CL) For example, the antinociceptive potency of the cyclized dynA analog according to the invention may correspond to / be an ED50 of about 1.47 pmol / kg (95% CL). The antinociceptive potency and ED50 value, respectively, can be determined in, for example, a formalin test (a model of (inflamatory) pain), like a formalin test as described in Muratspahic (Nat. Commun 14:8064, 2023, 1-17), Dubuisson (Pain 4, 1977, 161-174), and herein elsewhere and in the appended examples. In the context of the invention, "pain-inducing compounds" not only encompasse componds which induce pain in a direct manner; the term also relates to compounds that induce other responses of the body which are associated with pain. For example, such a compound may induce an inflammatory response (including, for example, swelling and / or edema) which is associated with pain. Formalin is a typical example of such a compound. In one embodiment (preferred but not limiting) the cyclized dynA analog of the invention acts peripherally, i.e. targets the KOR in the peripheral nervous system (PNS). It is more preferred, that the cyclized dynA analog of the invention does not centrally act, i.e. does not target the KOR in the CNS. It is thus preferred, that the cyclized dynA analog of the invention does not cross the BBB and does not enter the CNS, respectively. However, due to the (G-Protein-)biased nature of the KOR agonistic activity of the cyclized dynA analog of the invention, a / some BBB mobility may be tolerated, even in peripheral applications.

[0069] 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 TCD(i,s)-OH. These functions include those which are described in items (a) to (g), supra. DynA (e.g. dynAi-13, dynAi-17, dynAi-i3-NH2 or dynAi-i?-NH2) and / or TCD(i,s)-OH (or any other TCD analog disclosed herein; cf. Figure 1) may be used as controls in this respect (for example, dynA as negative control and / or TCD(i,s)-OH 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 the passages below) and in the appended examples (see, for example, Example 1).

[0070] For example, whether a (cyclized) dynA analog is an agonist of the KOR / agonizes the KOR can be tested as described in Mu ratspa hie (J Med Chem 64, loc. cit.) or Duerrauer (Sci Rep 9, 2019, 19295), and as in appended Examples 1, 4 and 5 (cf. also Table 1 and Figures 3 and 4). For example, a cAMP assay (e.g. according to the Cisbio protocol) and / or Schild regression analysis may be applied in this respect. 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 6 (cf. also Figure 5A 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 5 (cf. also Table 1 and Figure 4). For example, whether a (cyclized) dynA analog is stable in (human) serum can be tested as described in Muratspahic (J Med Chem 64, loc. cit.) and in appended Examples 1 and 6 (cf. also Figure 10).

[0071] 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.

[0072] 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).

[0073] Further, multiple agonists of the KOR are known in the art (see, e.g. https: / / www.guidetopharmacology.org / GRAC / ObjectDisplayForward?objectld=318&familyld= 50&familyType=GPCR). Non limiting examples of known 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).

[0074] Agonists of the KOR as 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 agonist; cf. also one, more, or all of items (a) to (g), supra). For example, a KOR agonist known in the art may be used a control research tool / pharmacological probe when testing whether a given dynA analog (as, for example, structurally defined herein) acts just like the known KOR agonist (e.g. U50,488); or, if TCD(i,s)-OH is used as a further control research tool / pharmacological probe, like TCD(i,s)-OH itself.

[0075] As mentioned, the cyclized dynA analog of the invention is envisaged to act as a (h)KOR agonist / agonist of (h)KOR. The meaning of "(h)KOR agonist" and "agonist 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).

[0076] In the context of the invention, being an "agonist" of the KOR and exhibiting "agonistic" function on the KOR, respectively, means that the KOR activity, i.e. the relevant biological function(s) of the KOR, is(are) increased or induced. More particular, being an "agonist" of the KOR and exhibiting "agonistic" function on the KOR, respectively, means in the context of the invention that a KOR-mediated opioid stimulus, e.g. intracellular cAMP reduction, is increased or induced. This, for example, leads to an activation (increase or induction) of the RAF / MEK1 / 2 / ERK1 / 2 and / 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). As mentioned, a preferred "agonist" of the KOR modulates the G protein over the 0-arrestin signaling pathway.

[0077] For example, a KOR ligand is agonizing (activating), if it agonizes KOR with the same, or a similar, potency and / or efficacy as a known KOR agonist (e.g. U50,488 or dynAi-13). A similar potency and / or efficacy as compared to a known KOR agonist means, for example, ± 1-40%, preferably ± 2-20%). Further, a ligand is agonizing, for example, if it agonizes KOR with the same or a similar Emax as a known KOR agonist (e.g. U50,488 or dynAi-13); e.g. in a cAMP inhibition assay (see also Examples 1, 4 and 5; cf. also Table 1, Figures 3 and 4)). For example, a KOR ligand is agonizing, if it agonizes KOR with an Emax of 60-180%, 80-140%, or (about) <100% (± 1-5 %), for example (about) <140% (± 1-5 %), as compared to, for example, dynAi-13. In addition, or alternatively, a KOR ligand is agonizing, if it agonizes KOR with an EC50 of 1010to 10-6M, preferably IO-9M to 10-7M, more preferably 5xl0-8M to 5xl0-7M, more preferably 8xl0-8M to 3xl0-7M, more preferably (about) 1.3xl0-7M (± <0.4xl0-7M); e.g. in a cAMP inhibition assay (see above).

[0078] If not mentioned differently herein, the above also applies to the meaning of "full" agonism / "fully" agonizing.

[0079] A KOR ligand is partially agonizing, if it agonizes KOR only with a reduced potency and / or efficacy as compared to a known KOR agonist (e.g. U50488 or dynAi-13). A KOR ligand is partially agonizing, for example, if it agonizes KOR with (an) increased value(s) for Emax and / or EC50 as compared to a known KOR agonist (e.g. U50,488 or dynAi-13); e.g. in a cAMP inhibition assay (see herein elsewhere). For example, a KOR ligand is partially agonizing, if it agonizes KOR with an Emax of <80 %, 20-80 %, 40-80 % or 50-80 % as compared to, for example, dynAi-13. A partially agonizing KOR ligand may, for example, agonize KOR with an EC50 of 10-5-10-9M, 10-5-10-8M, 10-5-10-7M, 10-6-5xl0-7M e.g. in a cAMP inhibition assay (see herein elsewhere).

[0080] Whether a (cyclized) dynA analog is capable of acting as a (G-protein-)biased (agonizing) KOR ligand, or of inducing no or only partial 0-arrestin recruitment (e.g. p-arrestin-2 (and / or -1) recruitment), respectively (e.g. item (d), supra), can, for example, be determined / tested by a respective 0-arrestin recruitment assay (e.g. for p-arrestin-2 and / or -1). Such an assay is, for example, disclosed herein (e.g. Examples 1, 4 and 7) or in WO 2021 / 186035 or Duerrauer (Sci Rep 9, 2019, 19295). In principle, as regards “biased” agonizing KOR ligands, the definitions given herein elsewhere apply here, mutatis mutandis. Being a biased agonizing KOR ligand in accordance with the invention means that it does not recruit, or recruits only partially, β-arrestin (e.g. β-arrestin-2 and / or -1) and / or one Gα-subunit over other Gα-subunits; G-protein bias may include preferential coupling of Gαi1, Gαi2, Gαi3, GαoA, GαoB,Gαz, Gαgastducin, Gα15, Gαq, or Gαsover other aforementioned ones. No recruitment, or only partial recruitment, of β-arrestin-2 (and / or -1) is of particular relevance for the present invention. A KOR ligand is biased, for example, if it only partially recruits β-arrestin (-2 and / or -1), for example if it recruits β-arrestin (-2 and / or -1) with a reduced potency and / or efficacy as compared to a known (unbiased) KOR agonist (e.g. U50,488 or dynA1-13), and / or with the same or a similar (e.g. ± 1-10%), potency and / or efficacy as a known biased agonist (e.g. collybolide, noribogaine, B-64, nalfurafine). A KOR ligand is biased, for example, if it induces β-arrestin-2 (and / or -1) recruitment with a reduced Emaxas compared to a known (unbiased) KOR agonist (e.g. U50,488 or dynA1-13); e.g. in an appropriate recruitment assay, like an appropriate β-arrestin (-2 and / or -1) recruitment assay (cf. Examples 1, 4 and 7). For example, a KOR ligand is biased, if it induces β-arrestin (-2 and / or -1) recruitment with an Emax of < 80%, < 60%, or < 50% as compared to, for example, dynA1-13. In addition, or alternatively, a KOR ligand is biased, for example, if it induces β-arrestin (-2 and / or -1) recruitment with an EC50 of > 80 nM, > 100 nM, or > 120 nM; e.g. in a β-arrestin (-2 and / or -1) recruitment assay (cf. Examples 1, 4 and 7). In general, the lower the β-arrestin (-2 and / or -1) recruitment, the lower is the biase fator; and the lower is the profile of adverse effects. Usually ≤50% β-arrestin (-2 and / or -1) recruitment is ideal. Whether a (cyclized) dynA analog crosses the BBB and enters the CNS, respectively, or modulates KOR function in the CNS, can, for example, be tested as described in Erli (J Med Chem 60, 2017, 7579-90). For example, motor coordination in, for example, the rotarod 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 IVIS 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 (Muratspahić, loc. cit.; Thell, Proc Natl Acad Sci USA 113(15), 2016, 3960-5). 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, agonizing 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.

[0081] 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, agonizing 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.

[0082] 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.

[0083] The cyclized dynA analog of the invention, or the pharmaceutical composition comprising it, may be for use in the treatment, amelioration or prophylaxis of a KOR-related disease, in particular of a disease resulting from or coming along with a decreased KOR activity and / or which is treatable by (biased) KOR agonism / an increased KOR activity. 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 below / elsewhere).

[0084] In case the cyclized dynA analog according to the invention is incapable of entering the BBB, or incapable of modulating KOR function in the CNS, it may particularly be useful in treating, ameliorating or preventing diseases or disorders which manifest / occur in the periphery / PNS (outside the CNS (spinal cord and brian)). Respective examples are (chronic) peripheral pain, and the like, or (peripheral) inflammation, and the like.

[0085] In case the cyclized dynA analog according to the invention is capable of entering the BBB, or able to modulate KOR functionin the CNS, it may particularly be useful in treating, ameliorating or preventing diseases or disorders which manifest / occur centrally / in the CNS (inside the spinal cord and brian). Respective examples are (CNS lesions in) multiple sclerosis (MS) or Alzheimer's disease (AD), and the like.

[0086] The cyclized dynA analog of the invention, or the pharmaceutical composition comprising it, may be for use in the treatment of pain, including acute and chronic pain, nociceptive pain, inflammatory pain, visceral pain, cancer-related pain, postoperative pain, pain resulting from injury, musculoskeletal and neuropathic pain, but is not limited to these. The cyclized dynA analog of the invention, or the pharmaceutical composition comprising it, may also be for use in the treatment of pruritus, e.g. uremic pruritus (nalfurafine and difelikephalin have, for example, been clinically used to treat uremic pruritus); and neurological disorders, such as epilepsy (preclinical data of efficacy of common KOR drugs are available in the literature). In particular, in view of the data provided herein, it is highly credible that the cyclized dynA analog of the invention, and the pharmaceutical composition comprising it, would be active against such disorders as well.

[0087] Furthermore, the cyclized dynA analog of the invention, and the pharmaceutical composition comprising it, may, due their KOR agonist activity, have an anti-itching effect. The cyclized dynA analog of the invention, and the pharmaceutical composition comprising it, may thus be used for the treatment of itching and disorders associated with itching / itching disorders.

[0088] The present invention further relates to a cyclized dynA analog of the invention, and to a pharmaceutical composition comprising it, and optionally a pharmaceutical carrier, for use in (i) treating, ameliorating or preventing (chronic) pain, in particular (chronic) abdominal pain, (chronic) visceral pain or (chronic) central pain, neuropathic pain (e.g. central or, preferred, peripheral (neuropathic) pain) and / or pain resulting from / coming along with MS; (chronic) peripheral pain is preferred;

[0089] (ii) treating, ameliorating or preventing inflammation and / or an inflammatory disease (e.g. inflammatory bowel disease etc.);

[0090] (iii) neuroprotection, more specifically remyelination (e.g. for therapy of CNS lesions in MS or AD);

[0091] (iv) treating, ameliorating or preventing MS (e.g. treatment by therapy or prophylactic / preventive treatment);

[0092] (v) remyelination (i.e. its induction or increase), in particular of oligodendrocytes, and / or improving (e.g. reducing or curing / healing) CNS lesions (e.g. brain lesions); and / or

[0093] (vi) preventing or reducing demyelination (in particular of oligodendrocytes) and / or preventing the formation of CNS lesions (e.g. brain lesions) and / or reducing existing CNS lesions (e.g. brain lesions).

[0094] The treatment, in particular the neuroprotection treatment and / or the treatment of MS and of related diseases, defects and / or symptoms, may also comprise or result in promoting OPC differentiation, remyelination and / or (subsequent) functional recovery of neurons.

[0095] The meanings of demyelinating diseases, neurological disorders and / or nerve-related diseases, in particular of MS and related diseases, defects and symptoms, are well known to the skilled person. They are, for example, described in Ciccarelli (Lancet Neurol. 13(8), 2014, 807-22) and Sospedra (Annu Rev Immunol. 23, 2005, 683-747).

[0096] Examples of demyelinating diseases, neurological disorders and / or nerve-related diseases to be treated in accordance with the invention are selected from the group consisting of MS, AD, optic neuritis, Devic's disease, inflammatory demyelinating diseases, central nervous system neuropathies, myelopathies (like Tabes dorsalis), leukoencephalopathies and leukodystrophies or are selected from the group consisting of Guillain-Barre syndrome and its chronic counterpart, chronic inflammatory demyelinating polyneuropathy, anti-MAG (myelin- associated glycoprotein) peripheral neuropathy, Charcot Marie Tooth (CMT) disease, copper deficiency and progressive inflammatory neuropathy.

[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 pain or inflammation in an acute manner. 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 ((G-protein-)biased) KOR-agonizing 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 ≤1h, ≤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. Further, the cyclized dynA analog according to the invention, or the pharmaceutical composition comprising it, may be used when a short / shortened period of nociception and / or of pain behavior 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 a short / shortened period of nociception and / or of pain behavior of ≤1h, ≤45 min, ≤30 min, ≤15 min, ≤10 min, or even ≤5 min, for example after pain onset and / or upon administration (e.g. s.c.), is desired, e.g. in the context of the herein defined treatment, amelioration or prevention / prophylaxis. Immediate efficacy is also within the scope of the invention (e.g. efficacy of ≤1 min after pain onset and / or upon administration). Furthermore, the cyclized dynA analog according to the invention may, in principle, be short- acting, medium-acting or long-acting. Accordingly, the cyclized dynA analog according to the invention may be a short-acting, medium-acting or long-acting agonist 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, 2.0h, 2.5h, 3.0h, 3.5h). As used herein “medium-acting” may refer to a time (e.g. half-life) of >4 to 36 hours (e.g. about 10h, 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 and medium-acting efficacy may, for example, be particularly useful for the treatment of acute-, nociceptive-, post-operative-, inflammatory-, visceral-, neuropathic, and / or musculoskeletalpain, like pain resulting from injury, surgery, trauma, inflammation, intestinal disease, hypersensitivity (e.g. due to prolonged pain medication), cancer etc. Treatment options based on long-acting efficacy may, for example, be particularly useful for the treatment of chronic-, neuropathic-, and / or inflammatory pain, like cancer-related pain, chronic intestinal / visceral pain, pain derived from autoimmune disorders etc. 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 (time- independent) medical use of the cyclized dynA analog of the invention is also within the scope of the invention. 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 KOR-dependent adverse effects (predominantly centrally-mediated (or (also) peripherally-mediated)), more particular adverse effects resulting from β-arrestin recruitment (e.g. β-arrestin-2 and / or β- arrestin-1 recruitment) and / or from recruitment of one G protein alpha anlog over other G- proteins, may be reduced / ameliorated or avoided in accordance with the invention. Such adverse effects are, for example, (adverse effects related to) opioid crisis / tolerance and / or dysphoria, sedation, diuresis and / or hallucinations. Thus, 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. 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.

[0098] 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.

[0099] 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.

[0100] In accordance with the invention, the active agent (cyclic peptide / 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).

[0101] 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).

[0102] 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.

[0103] The cyclized dynA analog may, for example, be administered subcutaneously, intravenously, 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.v., i.p. or oral administration and / or comprises a pharmaceutically acceptable carrier for s.c., i.v., 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.

[0104] 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.

[0105] 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)).

[0106] In one aspect, the cyclized dynA analog, or the pharmaceutical composition comprising it, is (to be) administered systemically.

[0107] A preferred, however non-limiting, mode of administration scheme is s.c. administration.

[0108] 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.

[0109] 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).

[0110] 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).

[0111] Non-limiting examples of one or more additional active agent(s) may be selected from the group consisting of KOR agonists (e.g. https: / / www.guidetopharmacology.org / GRAC / ObjectDisplayForward?objectld=318&familyld= 50&familyType=GPCR) and / or (other) pain or anti-inflamation therapeutics.

[0112] 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.

[0113] 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.

[0114] In the following, several non-limiting administration schemes, and the use of corresponding suitable pharmaceutically acceptable carriers, are described.

[0115] 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.

[0116] 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.

[0117] 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. 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.

[0118] 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).

[0119] 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.

[0120] 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) / container(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 pain and / or inflammation. 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.

[0121] 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.

[0122] The present invention further relates to a method of preparing the cyclized dynA analog of the invention.

[0123] 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").

[0124] 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. 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 thioether cyclized peptides like the particular thioether cyclized peptides as exemplified herein, may, for example, be generated / synthesized as described in Example 1, "Peptide synthesis" and "Peptide purification", below).

[0125] 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.

[0126] In particular, the method for preparing the cyclized dynA analog of the invention may comprise at least one, preferably all three, of the following steps (most preferred in this order):

[0127] (i) preparing a linear peptide (peptide backbone / linear peptide precursor) having the structure Br-L2-CH2C(=O)-SQ1-Z-SQ2-OH, wherein L2, SQ1and SQ2are defined as herein elsewhere and Z is -NH-C(H)(-L1-SRs)-C(=O)-, wherein L1is defined as herein elsewhere, preferably Z is a cysteine residue or a homocysteine residue, more preferably a cysteine residue, wherein the cysteine residue or homocysteine residue is optionally protected at the sulfur and Rsis hydrogen or a protecting group;

[0128] (ii) if the cysteine residue or homocysteine residue is protected at the sulfur or Rsis a protecting group: removing the protecting group of the cysteine residue or homocysteine residue, or the protecting group of Rs(such that Rsis H), respectively;

[0129] (iii) allowing the product of step (ii) to form a cyclic peptide; 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.

[0130] In the context of the method of preparing the cyclized dynA analog of the invention, step (i) of said method may comprise solid phase peptide synthesis (SPPS), such as Fmoc SPPS, e.g. by using a Wang resin or a 2-chlorotrityl chloride resin, or Boc SPPS, e.g. by using a PAM resin.

[0131] In the context of the method of preparing the cyclized dynA analog of the invention, Rsmay be a methyltrityl (Mtt) protecting group (e.g. Z is a cysteine residue protected at the sulfur with Mtt or a homocysteine residue protected at the sulfur with Mtt) and / or step (ii) may involve the use of trifluoroacetic acid, preferably trifluoroacetic acid in dichloromethane, preferably at a temperature in the range of from 10 to 40°C.

[0132] In the context of the method of preparing the cyclized dynA analog of the invention, step (iii) may comprise adding a base, preferably an amine, more preferably triethylamine, such as a solution of triethylamine in an aprotic solvent, preferably triethylamine in dimethyl formamide, preferably at a temperature in the range of from 10 to 40°C.

[0133] The skilled person is readily able to choose (an) appropriate protecting group(s) to be used in the context of the method of preparing the cyclized dynA analog of the invention.

[0134] Respective guidance and protecting groups is / are known in the art (see, for example, Greene's Protective Groups in Organic Synthesis, Peter et. AL, 2007 John Wiley & Sons, Inc., 2007, 001:10.1002 / 0470053488; https: / / onlinelibrary.wiley.com / doi / book / 10.1002 / 0470053488).

[0135] Non-limiting examples of thiol protecting groups which may be used are, in particular when more elaborate chemical synthesis strategies like, for example Boc-SPPS, are employed: Meb (p-methylbenzyl), Mob (p-methoxybenzyl), tBu (tert-butyl), Bn (benzyl), Fm (9- fluorenylmethyl), Dnpe (2-(2,4-dinitrophenyl)ethyl) and Fmoc (9-fluorenylmethoxycarbonyl).

[0136] 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.

[0137] Examples of variations of the side chain protecting groups that may be used are:

[0138] 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).

[0139] 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).

[0140] 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 (propargyloxycarbonyl), Nvoc (4-nitroveratryloxycarbonyl), Azoc (azidomethoxycarbonyl).

[0141] Cys: See the thiol protecting groups above.

[0142] 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, Example 1).

[0143] 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 (IK), the peptide backbone (linear peptide precursor), SQ1, SQ2, L1and / or L2, as well as with respect to the thioether cyclization, applies also here, mutatis mutandis.

[0144] 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 (in particular TCD(i,s)-OH and TCD(i,io)- OH). The present invention also relates to a C-terminally amidated form of a cyclized dynA analog as disclosed herein, e.g. to a C-terminally amidated form of a particular cyclized dynA analog as depicted in Figure 1 (in particular of TCD(i,s)-OH and TCD(i,io)-OH; e.g. TCD(i,8)-NH2 and TCD(i,io)-NH2). 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 -OH group is replaced by an -NRH group (wherein R is selected from H, methyl and ethyl, preferably selected from H and methyl, more preferably R is H). 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 (IK), the peptide backbone (linear peptide precursor), SQ1, SQ2, L1and / or L2, amidation, as well as with respect to the the thioether cyclization, applies also here, mutatis mutandis.

[0145] The present invention further relates to the following items:

[0146] 1. A cyclic peptide / cyclized dynorphin A analog (dynA analog) of Formula (I), below: Formula (I), wherein

[0147] (i) SQ1consists of 5 to 8 amino acid residues and comprises

[0148] (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

[0149] (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);

[0150] (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); and

[0151] (iii) each of L1and L2is independently selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2-CH2- , -CH(CH3)-CH2-, -CH2-CH(CH3)- and -CH(CH3)-CH(CH3)-, preferably -CH2. 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). 6. The cyclic peptide / cyclized dynA analog according to any one of items 1 to 5, wherein said cyclic peptide / cyclized dynA analog (a) is an agonist (in particular a (G-protein-)biased agonist) of the kappa opioid receptor (KOR); (b) specifically binds to KOR (but not to MOR and DOR); (c) binds to KOR with an affinity corresponding to a Ki in the range of 10-8M to 10-6M, preferably the range of 3x10-7M to 10-7M (e.g. to a Ki value of about 1.5x10-7M)); and / or (d) induces only partial, or preferably no, β-arrestin recruitment, e.g. β-arrestin-2 and / or β-arrestin-1 recruitment (e.g. ≤25%, ≤20%, ≤15% or ≤10% of the β-arrestin recruitment of dynA1-13-NH2; e.g. up to a concentration of ≤10 µM of the cyclic peptide / cyclized dynA analog). 7. The cyclic peptide / cyclized dynA analog according to any one of items 1 to 6, wherein (i) SQ1is YGGFLRR (SEQ ID NO.5); (ii) SQ2is RPKLK (SEQ ID NO.3); and (iii) each of L1and L2is -CH2-. 8. A pharmaceutical composition comprising the cyclic peptide / cyclized dynA analog according to any one of items 1 to 7, and optionally a pharmaceutical carrier. 9. 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) treating pain, in particular (chronic) peripheral pain; and or (ii) treating inflammation (e.g. inflammatory bowel disease etc.). 10. 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. 11. 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)). 12. A method of preparing the cyclic peptide / cyclized dynA analog of any one of items 1 to 7, said method comprising the steps of (i) preparing a linear peptide having the structure Br-L2-CH2C(=O)-SQ1-Z-SQ2-OH, wherein L2, SQ1and SQ2are as defined in any one of items 1 to 3 and Z is -NH- C(H)(-L1-SRs)-C(=O)-, wherein L1is defined as in item 1, preferably Z is a cysteine residue or a homocysteine residue, more preferably a cysteine residue, wherein the cysteine residue or homocysteine residue is optionally protected at the sulfur and Rsis hydrogen or a protecting group;

[0152] (ii) if the cysteine residue or homocysteine residue is protected at the sulfur or Rsis a protecting group: removing the protecting group of the cysteine residue or homocysteine residue, or the protecting group of Rs(such that Rsis H), respectively;

[0153] (iii) allowing the product of step (ii) to form a cyclic peptide; 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.

[0154] 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 Wang resin or a 2-ch lorotrityl chloride resin, or Boc solid phase peptide synthesis (SPPS), e.g. by using a PAM resin.

[0155] 14. The method of item 12 or 13, wherein Rsis a methyltrityl (Mtt) protecting group (e.g. Z is a cysteine residue protected at the sulfur with Mtt or a homocysteine residue protected at the sulfur with Mtt) and / or step (ii) involves the use of trifluoroacetic acid, preferably trifluoroacetic acid in dichloromethane, preferably at a temperature in the range of from 10 to 40°C.

[0156] 15. The method of any of items 12 to 14, wherein step (iii) comprises adding a base, preferably an amine, more preferably triethylamine, such as a solution of triethylamine in an aprotic solvent, preferably triethylamine in dimethyl formamide, preferably at a temperature in the range of from 10 to 40°C.

[0157] 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.

[0158] 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.

[0159] 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).

[0160] 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.

[0161] The present invention is further described by reference to the following non-limiting figures and examples.

[0162] The Figures show:

[0163] Figure 1. Structures of cysteine stapled and thioether cyclized dynAi.13 analogs. Peptides have been synthesized as described in the section Materials and methods (Example 1). Late-stage functionalization moieties incorporated into dynAi-13 sequence between tyrosine at position 1 and isoleucine at position 8 (left) or proline at the position 10 (right) include the dichloroacetone (DCA; (sometimes also termed "ace")), m-dibromooxylene (mXYL), dichlorotetrazine (tet) and methylene group (CH2). DynAi-13 with disulfide bonds (ox) were synthesized for comparison. Cysteine stapled dynA1-13 analogs were named CSD, whereas thioether cyclized dynA1-13 analogs are abbreviated as TCD. Figure 2. Capability of dynA1-13-OH ligands to recruit β-arrestin-2. Ligands at a concentration of 10 µM were added in the BRET assay following establishment of the baseline for 5 min with furimazine (Nluc substrate). CSD-ox(1,8)-OH (2ndbar), CSD-ox(1,10)-OH (3rdbar), CSD-CH2(1,8)-OH (4thbar), CSD-CH2(1,10)-OH (5thbar), CSD-mXYL(1,8)-OH (6thbar), CSD-mXYL(1,10)-OH (7thbar), CSD- tet(1,8)-OH (8thbar), CSD-tet(1,10)-OH (9thbar), CSD-dca(1,8)-OH (10thbar), CSD-dca(1,10)-OH (11thbar), TCD(1,8)-OH (12thbar) and TCD(1,10)-OH (13thbar) were subsequently measured for 30 min. DynA1-13-NH2(10 µM) was used as positive control (1stbar). Data are from three independent experiments and are shown as mean ± SD. Figure 3. Functional cAMP and radioligand binding assays of CSD-CH2(1,8)-OH, CSD-CH2(1,10)- OH, CSD-dca(1,8)-OH and TCD(1,8)-OH at the KOR. A) KOR activation upon treatment with CSD- CH2(1,8)-OH (n=3, solid line, open squares), CSD-CH2(1,10)-OH (n=3, dashed line, triangles), CSD- dca(1,8)-OH (n=2, dashed line, inverted triangles) and TCD(1,8)-OH (n=3, solid line, diamonds) was concentration-dependently measured by quantifying cAMP in HEK293 cells stably expressing mouse κ-opioid receptor (KOR). Dynorphin A 1-13-NH2 (dyn, black line and circles) was used as positive control (n=3). Data represent mean ± SD and are normalized to the percentage of maximum activation of dynA1-13-NH2 detected at the highest concentration. B) Radioligand binding assay was carried out using 1 nM of [3H]-diprenorphine (DPN) in HEK293 cell membrane preparations stably expressing mouse KOR. Cells were treated with indicated concentrations of CSD-CH2(1,8)-OH (n=3, solid line, open squares), CSD-CH2(1,10)-OH (n=3, dashed line, triangles), CSD-dca(1,8)-OH (n=2, dashed line, inverted triangles), TCD(1,8)-OH (n=3, solid line, diamonds) and dynA1-13-NH2 (n=3, solid black line, black circles). The specific binding was calculated by subtracting total from the nonspecific binding. Data are mean ± SD and are normalized to the percentage of maximum binding of the dynorphin A 1-13-NH2. Figure 4. Pharmacology of C-terminally amidated peptide ligands at the KOR. A) Bioluminescence resonance energy transfer (BRET) assay was employed to measure the transient interaction of κ-opioid receptor (KOR) with β-arrestin-2 in HEK293 cells over time following cell treatment with 10 µM of CSD-CH2(1,8)-NH2(n=3, open 2ndbar), CSD-CH2(1,10)-NH2(n=3, 3rdbar), CSD-dca(1,8)-NH2(n=3, 4thbar) and TCD(1,8)-NH2(n=3, 5thbar). DynA1-13-NH2was used a positive control (n=3, 1st, black bar) and furimazine as a substrate for luciferase which was incubated for 5 min prior to the establishment of baseline and ligand treatment. B) Gαi- mediated cAMP inhibition of mouse KOR was measured in HEK293 cells stably expressing mouse κ-opioid receptor (KOR) after the cell treatment with CSD-CH2(1,8)-NH2 (n=3, solid line, open squares), CSD-CH2(1,10)-NH2 (dashed line, triangles), CSD-dca(1,8)-NH2 (dashed line, inverted triangles) and TCD(1,8)-NH2 (n=3, solid line, diamonds). DynA1-13-NH2 was positive control (n=3, solid black line, black circles). Data are shown as mean ± SD. Normalization of data was performed according to the percentage of the maximum activation of dynA1-13-NH2. C) The affinity of CSD-CH2(1,8)-NH2(n=3, solid line, open squares), CSD-CH2(1,10)-NH2(n=3, dashed line, triangles), CSD-dca(1,8)-NH2(n=3, dashed line, inverted triangles) and TCD(1,8)-NH2(n=2, solid line, diamonds) 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 dynA1-13-NH2is included for comparison. Data are 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. Figure 5. Selectivity profile of CSD and TCD peptide ligands. CSD and TCD peptide ligands were measured for displacing 1 nM of tritiated diprenorphine ([3H]-DPN, 1stblack bar) in radioligand binding studies in HEK293 cells stably expressing A) mouse µ-opioid receptor (MOR) and B) mouse δ-opioid receptor (DOR) (n=2). DynA1-13-NH2 served as positive control (10 µM, n=2; 2ndbar). To determine specific binding, total from non-specific binding was subtracted. CSD-CH2(1,8) peptides are shown in 3rdand 7th, empty bars, CSD-CH2(1,10) ligands in 4thand 8thfilled bars and CSD-dca(1,8) as well as TCD(1,8) peptides are displayed in 5thand 9thfilled and 6thand 10thfilled bars, respectively. Figure 6. KOR biotinylation assay. A) Representative Western blot image of κ-opioid receptor (KOR) internalization following treatment with 10 µM TCD(1,8)-OH (TCD) and U50,488 (U50) for 30 min at 37°C. HEK293 cells were transiently transfected with the plasmid encoding KOR tagged with enhanced green fluorescence protein (EGFP). Cells stripped with dithiothreitol to remove any non-internalized biotin are denoted strip whereas nt cells are non-treated cells. Total represents the total amount of the receptor at the cell surface. B) Densitometric analysis of the bands at 70 kDa (n=2). Figure 7. KOR ligands TCD(1,8)-OH and CSD(1,8)-NH2do not stimulate calcium mobilization. A) Representative traces of calcium signals in dorsal root ganglion (DRG) cultures stimulated by control (CTRL, black traces, n=648), U50,488 (U5010 µM, blue traces, n=1467), U50 (10 µM) in combination with norbinaltorphimine (nor-BNI, 0.5 µM, red traces, n=537), TCD(1-8)-OH (10 µM, light green traces, n=638), TCD(1-8)-OH (100 µM, dark green traces, n=704), U50 (10 µM) in combination with CSD-CH2(1,8)-NH2 (1 µM, violet traces, n=1126) and U50 (10 µM) in combination with CSD-CH2(1,8)-NH2 (10 µM, cyan traces, n=720). The antagonists nor-BNI and CSD-CH2(1,8)-NH2 were pre-applied followed by addition of the agonist. Carvacrol (100 µM) and capsaicin (1 µM) served as controls for TRPA1 and TRPV1, respectively. CTRL was an external solution. A potassium chloride (KCl) solution (60 mM) added at the end of each recording was used as positive control for detecting viable cells and generated similar response amplitudes. B) Quantification by area under curve (AUC) of control (CTRL, 1st, black circle), U50,488 (U5010 µM, 2ndcircle), (nor-BNI, 0.5 µM, 3rdcircle), TCD(1-8)-OH (10 µM, 4thcircle), TCD(1-8)-OH (100 µM, 5thcircle), U50 (10 µM) in combination with CSD-CH2(1,8)-NH2(1 µM, 6thcircle) and U50 (10 µM) in combination with CSD-CH2(1,8)-NH2(10 µM, 7thcircle) for the interval 120-180 s (the duration of the KOR agonist application). Data are shown as mean ± 95% C.I. from two independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. Two asterisks indicate significance between U50 (10 µM) and U50 (10 µM) + nor-BNI (0.5 µM) as well as U50 (10 µM) and TCD(1,8)-OH (10 µM) (**p < 0.05) while three asterisks represent significance between U50 (10 µM) and U50 (10 µM) + CSD-CH2(1,8)-NH2 (1 µM) as well as U50 (10 µM) and U50 + CSD- CH2(1,8)-NH2(10 µM) (***p < 0.001). Four asterisks indicate significance between U50 (10 µM) and CTRL (****p < 0.0001) Comparison between U50 (10 µM) and TCD(1,8)-OH (100 µM) (ns) as well as CTRL and U50 (10 µM) + nor-BNI (0.5 µM), CTRL and TCD(1,8)-OH (10 µM), CTRL and TCD(1,8)-OH (100 µM), CTRL and U50 (10 µM) + CSD-CH2(1,8)-NH2 (1 µM) as well as CTRL and U50 + CSD-CH2(1,8)-NH2 (10 µM) was not significant (ns). Figure 8. BRET assay of CSD-OH and TCD-OH ligands at the KOR. Kinetic bioluminescence resonance energy transfer (BRET) reported by nanoLuc. Substances at a concentration of 10 µM were applied after 300 s to measure β-arrestin recruitment at the KOR of A) CSD-ox(1,8)-OH (n=3, empty squares), B) CSD-ox(1,10)-OH (n=3, filled squares), C) CSD-CH2(1,8)-OH (n=3, empty triangles), D) CSD-CH2(1,10)-OH (n=3, filled triangles), E) CSD-mXYL(1,8)-OH (n=3, empty inverted triangles), F) CSD-mXYL(1,10)-OH (n=3, filled inverted triangles), G) CSD-tet(1,8)-OH (n=3, empty diamonds), H) CSD-tet(1,10)-OH (n=3, filled diamonds), I) CSD-dca(1,8)-OH (n=3, empty hexagons), J) CSD-dca(1,10)-OH (n=3, filled hexagons), K) TCD(1,8)-OH (n=3, empty circles), L) TCD(1,10)-OH (n=3, filled circles). DynA1-13-NH2served as control (n=3, filled black circles in all panels). Figure 9. BRET assay of C-terminally amidated peptide ligands at the KOR. A-D) Bioluminescence resonance energy transfer (BRET) assay was used to measure the ability of C- terminally amidated peptide ligands to induce β-arrestin-2 recruitment in HEK293 cells transiently expressing mouse κ-opioid receptor (KOR). A saturating concentration of 10 µM of CSD-CH2(1,8)-NH2(n=3, open squares), CSD-CH2(1,10)-NH2(n=3, squares), CSD-dca(1,8)-NH2(n=3, squares) and TCD(1,8)-NH2 (n=3, squares) was tested. Substances were applied after 300 s, dynA1- 13-NH2 was used a positive control (n=3, filled black circles in all panels). Figure 10. Stability assay of C-terminally amidated CSD and TCD peptides. Stability of dynA1-13-NH2(n=3, black solid line, black circles), CSD-CH2(1,8)-NH2(n=3, solid line, empty squares), CSD- CH2(1,10)-NH2(n=3, dashed line, triangles), CSD-dca(1,8)-NH2(dashed line, inverted triangles) and TCD(1,8)-NH2was measured in human plasma. Figure 11.1H NMR spectrum of TCD(1,8)-OH at 600 MHz in H2O / D2O (9:1, v / v). NMR was performed as described in Example 1. Figure 12. Antinociceptive effect of TCD(1,8)-OH in the formalin test, as a model of inflammatory pain, after s.c. administration to male mice. Saline or TCD(1,8)-OH were s.c. injected 15 min before formalin and the duration of pain behavior (time spent licking / biting, lifting, and flinching of the formalin-injected paw) was counted for 15 min, starting 15 min after formalin injection. (A) Dose-dependent antinociceptive effect of TCD(1,8)-OH. Mice received saline (control, n=8) or different doses of TCD(1,8)-OH (1 mg / kg, 0.60 µmol / kg, n=6; 2.5 mg / kg, 1.49 µmol / kg, n=6; 5 mg / kg, 2.98 µmol / kg, n=6). (B) Antagonism by nor-BNI of the antinociceptive effect of TCD(1,8)-OH. Mice received s.c. saline (control, n=8), TCD(1,8)-OH (5 mg / kg, 2.98 µmol / kg, n=6) or were s.c. pretreated with nor-BNI (10 mg / kg) for 24 h before TCD(1,8)-OH (n=6). Values represent means ± SEM. ***P < 0.001 vs. saline group;###P < 0.001 vs. TCD(1,8)-OH-treated group; one-way ANOVA with Tukey’s post hoc test. 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. 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. Further, reference is made herein to the following amino acid sequences (and also to the respectiove coding nucleotide sequences): SEQ ID NO.1 (N-terminal “message” motif of dynA1-13and dynA1-17) YGGF SEQ ID NO.2 (N-terminal part of C-terminal “address” sequence of dynA1-13and dynA1-17) LRRI SEQ ID NO.3 (C-terminal part of C-terminal “address” sequence of dynA1-13; most preferred example of SQ2) RPKLK SEQ ID NO.4 (C-terminal part of C-terminal “address” sequence of dynA1-13; example of SQ2) IRPKLK SEQ ID NO.5 (N-terminal part of dynA1-13 and dynA1-17; most preferred example of SQ1) YGGFLRR SEQ ID NO.6 (N-terminal part of dynA1-13 and dynA1-17; example of SQ1) YGGFLRRI SEQ ID NO.7 (Dynorphin A1-13(dynA1-13)) YGGFLRRIRPKLK SEQ ID NO.8 (C-terminal “address” sequence of dynA1-13) LRRIRPKLK SEQ ID NO.9 (Dynorphin A1-17(dynA1-17)) YGGFLRRIRPKLKWDNQ SEQ ID NO.10 (C-terminal “address” sequence of dynA1-17) LRRIRPKLKWDNQ SEQ ID NO.11 (most preferred example of the peptide backbone of the cyclized dynA analog of the invention) YGGFLRRCRPKLK SEQ ID NO.12 (example of the peptide backbone of the cyclized dynA analog of the invention) YGGFLRRICPKLK SEQ ID NO.13 (example of the peptide backbone of the cyclized dynA analog of the invention) YGGFLRCIRPKLK Tripeptide (no SEQ ID NO. assigned; N-terminal part of C-terminal “address” sequence of dynA1-13and dynA1-17) LRR 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. Example 1: Materials and Methods Materials Dynorphin (dyn) A1-13amide 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 Gikit 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). Peptide Synthesis 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 10 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. TCD peptides were manually bromoacetylated at their N-termini by bromoacetic anhydride. The bromoacetic anhydride was produced via reaction of bromoacetic acid (10 eq.) with DCC (10 eq.) in DMF.

[0164] Following bromoacetylation for 3-4 h at room temperature, peptides were treated with 1% TFA in dichloromethane for 60 min at room temperature to remove Mtt protecting group from the cysteine residue. Subsequent cyclization was initiated by adding 1% triethylamine in 3-6 mL of DMF and incubating the mixture overnight at the room temperature. Each peptide was cleaved from the resin using TFA / TIPS / H2O (5:2.5:2.5) for 2 h and the crude peptide were isolated via precipitated with cold Et20. The isolates were redissolved in H2O / ACN (1:1) and lyophilized to yield a white powder. Each peptide was purified by reversed-phase high performance liquid chromatography (RP-HPLC) prior to oxidation and stapling.

[0165] Peptide Purification

[0166] 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.

[0167] NMR

[0168] 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.TH, TOCSY and NOESY experiments were run for each sample. An exemplaryTH NMR spectrum is provided herein as Figure 11.

[0169] Stability Assay 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.

[0170] Cell culture and Transfection

[0171] 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.).

[0172] Radioligand Displacement Binding Assays

[0173] 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

[0174] 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.

[0175] Bioluminescence Resonance Energy Transfer (BRET) Assay

[0176] 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. Animals and Drug Administration

[0177] Breeding, euthanasia and all procedures of animal handling are performed according to regulations of animal care and welfare. Experiments are carried out in accordance with the European Communities Council Directive of 24 November 1986 (86 / 609 / EEC). Behavioral experiments are performed in male CD1 mice (8-10 weeks old) purchased from Janvier Labs (Le Genest-Saint-lsle, France). All animal care and experimental procedures are in accordance with the ethical guidelines for the animal welfare standards of the European Communities Council Directive (2010 / 63 / EU), and are approved by the Committee of Animal Care of the Austrian Federal Ministry of Science and Research. Mice are 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)-NH2are prepared in sterile physiological 0.9% saline solution. Test compounds or vehicle (saline) are s.c. administered in a volume of 10 pL / g body weight.

[0178] Isolation of Dorsal Root Ganglion (DRG) Neurons

[0179] 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.

[0180] Single-cell Calcium Microfluorimetry

[0181] 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.

[0182] Reversible biotinylation internalization assay

[0183] HEK293 cells (2.4 x 106), seeded into one 10 cm dish and incubated overnight at 37°C, were transfected with plasmid DNA encoding KOR-EGFP at a cell confluency of ~80% using. Three hours post transfection, all cells were split equally over 10 wells (4 + 6) of two 6-well dishes (~ 500.000 cells per well). After 18 h, transfection efficiency was assesed by epifluorescence microscopy and reversible biotinylation was conducted. Cells were washed with PBS supplemented with 1 mM MgCl2 and 0.1 mM CaCl2 (PBS2+) and further incubated two times with PBS2+containing 1,6 mg mL1Pierce Premium Grade Sulfo-NHS-SS-Biotin (PG82077; Thermo Scientific) (750 pL per well) for 30 min at 4°C and under gentle agitation. Following, reaction was quenched by washing two times with 100 mM glycine in PBS2+(cells were incubated in 2ndwash step for 10 min on ice). Three internalization solutions containing either 10 pM TCD(i,8)-OH or 10 pM U50,488 or vehicle were prepared in PBS2+(2 mL each) additionally containing 0.2% (w / v) IgG and protease free bovine serum albumin (A3059, Sigma-Aldrich) as well as 0.18% (w / v) glucose monohydrate. Internalization solutions were kept at 37°C and after complete quenching, cells were washed once in PBS2+. Whereas the 6-well dish with only 4 cellcontaining wells remained on ice, the other (6 cell-containing wells) was transferred to room temperature, whereby PBS2+was removed. Subsequently, internalization solutions (1 mL per well) were added to each well in duplicates and cells were incubated at 37°C 5% CO2 for 35 min to initiate internalization of proteins. "Internalization" samples were then incubated on ice and all cell-containing wells were washed once with chilled Tris-buffered saline (TBS; 20 mM Tris- HCI, pH = 7.5; 150 mM NaCI). To strip the cellular surface from the biotinylation (except for the "total"-controls) TBS containing 150 mM Tris(2-carboxyethyl)phosphine (pH = 8.1) was added at 4°C for 15 min and under gentle agitation. After repeating the stripping process for the 2ndtime, cells were washed three times with chilled TBS and lysed in RIPA lysis-buffer buffer (20 mM Tris-HCI, pH = 7.6; 150 mM NaCI; 1 mM EDTA; 1% Triton X-100; 0.1% SDS; 0.5% sodium deoxycholate; protease inhibitor cocktail; phosSTOP) (500 pL per well) directly on the dish for 30 min at 4°C and under gentle agitation. Lysates were re-suspended thoroughly and centrifuged at 16 000 g for 45 min at 4°C. Streptavidin agarose (20357, Thermo Scientific) (~ 25 pL packed resin per sample) was equilibrated in RIPA-buffer and lysates were incubated with streptavidin agarose overnight at 4°C under end-over-end rotation. The following day, agarose was spun down and 50 pL supernatants were separated. Agarose was washed three times with 1 mL of RIPA-buffer without sodium deoxycholate and protein / phosphatase inhibitors followed by elution of immobilized biotinylated proteins with 100 pL of 2x Laemmli SDS-PAGE sample buffer at 90°C for 10 minutes. In the same step, the 50 pL supernatant-samples were denatured with 50 pL of 2x sample buffer and analyzed by western blotting. Immunoblots were analyzed with ImageJ and internalization rates (IR) were calculated using the following equation: IR = internal - strip / total.

[0184] Formalin test

[0185] The formalin test (see also Dubuisson, Pain 4, 1977, 161-174) was performed as in Mu ratspa hie, Nat. Commun 14:8064, 2023, 1-17 (if not described otherwise herein). In particular, the formalin test was performed in male GDI mice (8-10 weeks old, 30-35 g body weight). Following a habituation period of 15 min to individual transparent observation chambers, male mice were s.c. administered saline (control) or TCD(i,s)-OH (1 mg / kg, 0.60 pmol / kg, n=6; 2.5 mg / kg, 1.49 pmol / kg, n=6; 5 mg / kg, 2.98 pmol / kg, n=6) in this very assay, 15 min prior to injection of 20 pL of 5% formalin aqueous solution to the plantar surface of the right hindpaw. The time (in sec) each animal spent licking, biting, lifting, and flinching of the formalin-injected paw (pain behavior) was recorded in 5 min intervals between 15 and 30 min after the injection of formalin (Phase II reaction). For the antagonism study, norBNI (10 mg / kg) was s.c. administered 24 h before TCD(i,s)-OH (5 mg / kg, 2.98 pmol / kg, n=6) and pain behavior was assessed as described above. Antinociceptive activity in the formalin test, as percentage decrease in duration of pain behavior compared to the saline (control) group, was calculated according to the following formula: 100* [C-T / C], where C is the mean time in control (saline) group and T is the time in the drug treated group (Puls, Pharmaceuticals 15(6), 2022, 1-21). Dose-response relationships of percentage inhibition of pain behavior were constructed, and the dose necessary to produce a 50% effect (EDso) and 95% confidence limits (95% CL) was calculated according to the method of Litchfield and Wilcoxon (Litchfield, J. Pharmacol. Exp. Ther. 96, 1949, 99-113). U50,488 may be used as a control / comparison compound (for example U50,488 at doses of 1.1, 2.1 and 5.4 pmol kg-1; as described in Muratspahic, Nat. Commun 14:8064, 2023, 1-17). For details pertaining to the particular formalin test as exemplarily performed in the context of the invention, see also Fig.12 and its caption.

[0186] Data and Statistical Analysis 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).

[0187] 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 (Ki) 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 are 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.

[0188] Example 2: Design and Synthesis of dynAi-i3-OH analogs

[0189] 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.). However, late-stage functionalization of cysteines and thioether cyclization have not been investigated yet to design dynAi-13 analogs as KOR ligands.

[0190] First, applied herein was a stapling strategy to constrain dynAi-13 by manually coupling 3- (tritylthio)propionic acid to the N-terminus of the tyrosine at the position 1 of the message sequence to yield N-terminal thiol and replacing either isoleucine at the position 8 or proline at the position 10 of the address sequence by cysteine residues to give second thiol for oxidation and stapling. Isoleucine and proline residues were chosen since previous studies demonstrated that these residues are not critical for the pharmacological activity of dynAi-13 (Naqvi, Peptides 1998, 1277-1292). These peptides were named Cysteine Stapled Dyn (CSD) analogs.

[0191] Second, thioether cyclization using bromo-acetylation technique has been incorporated between the N-terminal tyrosine and either the cysteine at the position 8 or cysteine at the position 10. Accordingly, these peptides were named Thioether Cyclized Dyn (TCD) analogs. In addition, since the native structure of dynAi-13 has been reported with carboxyl group at the C- terminus dynAi-13 analogs with C-terminal carboxylic acid were first designed (Goldstein, Proc Natl Acad Sci 1979, 6666-6670).

[0192] Overall, these strategies yielded ten cysteine stapled dynAi-13 analogs with diverse crosslinkers and two thioether-bridged dynAi-13 peptides (Figure 1). The crosslinkers ranged from the methylene (CH2) group over dichloroacetone (dca or ace), m-dibromoxylene (mXYL) to dichlorotetrazine (tet) moieties. In addition, dynAi-13 with disulfide bonds (ox) without stapling groups has been synthesized as a control compound.

[0193] Example 3: DynAi n-OH analogs elicit reduced p-arrestin-2 recruitment

[0194] To identify KOR peptide ligands which lack p-arrestin recruitment, twelve initially designed peptides were screened in a kinetic BRET assay using HEK293 cells transiently expressing mouse KOR coupled to EGFP and p-arrestin-2 fused to nanoluc (NIuc). The data revealed that dynAi-13 analogs recruit p-arrestin-2 yet with distinct levels of BRET efficiency when using a saturating concentration of 10 pM (Figure 2; Figure 8). For instance, as compared to the native dynAi-13- NH2, CSD-CH2(i,8)-OH, CSD-CH2(I,IO)-OH, CSD-dca(i,s)-OH exhibited minor P-arrestin-2 recruitment with efficiencies ranging from 14% and 25% to 18%, respectively, while TCD(i,s)-OH displayed no detectable p-arrestin-2 recruitment up to concentrations of 10 pM (Figure 2; Figure 8). By contrast, CSD-ox(i,s)-OH and CSD-dcai,io)-OH recruited p-arrestin-2 to an extent similar to the native dynAi-13 (Figure 2; Figure 8).

[0195] Example 4: TCD(i,s)-OH exhibits preference for G protein over p-arrestin-2 at the KOR

[0196] The four most promising ligands were CSD-CH2(i,8)-OH, CSD-CH2(i,io)-OH, CSD-dca(i,8)-OH and TCD(i,8)-OH induced only minimal or no p-arrestin-2 recruitment, as evident from their low levels of BRET efficiency (see above and Figures 2 and 8). The ability of these compounds for KOR-mediated, adenylyl cyclase-dependent cAMP inhibition was next tested. In the functional cAMP assay, CSD-CH2(i,8)-OH and CSD-CH2(i,io)-OH were inactive up to concentrations of 10 pM, suggesting potential antagonism of these ligands at the KOR, whereas CSD-dca(i,8)-OH partially activated KOR with an EC50 of 270 nM and an Emax of 57% (Figure 3A, Table 1). By contrast, TCD(1,8)-OH is a full agonist at the KOR with an EC50 and an Emax of 127 nM and 136%, respectively. This indicates that TCD(1,8)-OH preferentially modulates G protein over β-arrestin- 2 pathways as it does not show β-arrestin-2 recruitment up to concentrations of 10 µM (Figure 3A, Table 1). In addition to functional cAMP assay, radioligand binding studies were conducted to examine the affinity of ligands towards the KOR. Herein, all four ligands displayed KOR affinity in the nanomolar range (Figure 3B, Table 1). Whereas CSD-CH2(1,8)-OH and CSD-CH2(1,10)-OH exhibited Kivalues of 77 and 33 nM, respectively, CSD-dca(1,8)-OH and TCD(1,8)-OH bound to the KOR with Kivalues of 91 and 152 nM, respectively (Figure 3B, Table 1). Example 5: Pharmacological Properties of C-terminally Amidated CSD Ligands at the KOR A C-terminal amidation was introduced to the most promising peptides, i.e. CSD-CH2(1,8)-OH, CSD-CH2(1,10)-OH, CSD-ace(1,8)-OH and TCD(1,8)-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 (cf. Figure 1). Following synthesis, peptides were subjected to pharmacological characterization. Peptides were first screened in the kinetic β-arrestin-2 recruitment assay and observed results similar to previously characterized analogs were observed in that all four peptides exhibited diminished β-arrestin-2 recruitment up to concentrations of 10 µM (Figure 4A). A cAMP assay was then conducted to explore the potency and efficacy of newly synthesized KOR ligands to modulate G protein signaling. Intriguingly, compared to counterparts with COOH at the C-terminus, peptides comprising C-terminal amidation displayed disparate potencies and efficacies (Figure 4B). CSD-CH2(1,8)-NH2 was inactive at the KOR in the agonist-detected mode compared to CSD- CH2(1,10)-NH2 which acted as a partial agonist following C-terminal amidation with an EC50 and Emax of 540 nM and 49%, respectively (Figure 4B, Table 1). On the other hand, C-terminal amidation of CSD-dca(1,8)-NH2 and TCD(1,8)-NH2 decreased their potencies to EC50 = 740 nM and EC50= 1400 nM, respectively (Figure 4B, Table 1). Similarly, this applies to TCD(1,8)-NH2(Figure 4B, Table 1). To assess the affinity of C-terminally amidated peptides, radioligand binding studies were carried out. Herein, the affinity of CSD-CH2(1,8)-NH2(Ki= 7 nM), CSD-CH2(1,10)-NH2(6 nM) and CSD-dca(1,8)-NH2(32 nM) improved by approximately 12-, 6- and 3-fold, respectively (Figure 4C, Table 1). By contrast, the affinity of TCD(1,8)-NH2slightly improved (Ki= 130 nM) (Figure 4C, Table 1). Example 6: Increased Selectivity and Stability of KOR CSD and TCD Ligands Next, the selectivity profiles of four peptides with the C-terminal COOH and four peptides comprising an NH2at the C-terminus were examined by measuring their binding affinity in HEK293 cell membrane preparations stably expressing mouse MOR and DOR. A saturating concentration of 10 µM for each peptide ligand was tested and it was demonstrated that all eight peptides are KOR selective as they did not significantly bind to MOR and DOR when using a concentration of 10 µM (Figure 5A and B). Further, the stability of four peptides with the C- terminal COOH in human serum was first measured by reversed-phase high performance liquid chromatography (RP-HPLC). Interestingly, these peptides degraded within 15 min, similar to the native dynA1-13-NH2(data not shown). Since C-terminal amidation improved binding affinity of CSD-CH2(1,8)-NH2, CSD-CH2(1,10)-NH2, CSD-dca(1,8)-NH2and TCD(1,8)-NH2, stability of these peptides was then determined in human serum by RP-HPLC. In fact, C-terminal amidation of CSD and TCD peptides improved their stability as compared to the linear dynA1-13-NH2with TCD(1,8)-NH2being the most stable peptide ligand (Figure 10). Example 7: G protein-biased TCD(1,8)-OH exhibits impaired KOR internalization It was explored whether the TCD(1,8)-OH agonist which exhibits selectivity for G protein and lacks β-arrestin-2 recruitment (Figure 2B, Figure 3A, Figure supplement 1) is associated with reduced KOR internalization. To measure KOR internalization, a reversible biotinylation assay was conducted by using sulfo-N-hydroxysuccinimide (NHS)-SS-biotin that reacts with primary amines to label cell surface KOR (Hislop, Methods Mol Biol 2011, 425-440). As a positive control, a selective KOR agonist was used, U50,488. U50,488 was reported to promote robust KOR internalization (McLaughlin, J Biol Chem 2003, 34631-34640, Chen, Biochem J 2016, 497-508). By applying this strategy, it was demonstrated that TCD(1,8)-OH (10 µM) induces KOR endocytosis following an exposure for 30 min at 37°C yet to a lesser extent than U50,488 (10 µM) (Figure 6). These data support the notion that TCD(1,8)-OH manifests attenuated β-arrestin- 2 recruitment and KOR internalization. Example 8: TCD(1,8)-OH does not induce calcium mobilization while CSD(1,8)-NH2 blocks U50,488-induced calcium response in DRGs Over the past years, studies unveiled that β-arrestins and MOR interact with transient receptor potential (TRP) channels to regulate intracellular events in dorsal root ganglion (DRG) neurons (Scherer, Proc Natl Acad Sci 2017, 13561-13566, Dembla, Elife 2017, e26280, Quallo, Elife 2017, e26138). However, β-arrestin-dependent KOR signaling in DRGs is less explored. To disentangle this phenomenon, it was tested whether the G protein-biased KOR agonist TCD(1,8)-OH (Figures 2, 3 and 4) and the KOR antagonist CSD-CH2(1,8)-NH2(Figures 2, 3 and 4) are capable of modulating calcium mobilization in DRGs known to express KOR (Snyder, Neuron 2018, 1274- 1288, Scherer, Proc Natl Acad Sci 2017, 13561-13566, Dembla, Elife 2017, e26280). Using single- cell calcium microfluorimetry and control agonists carvacrol and capsaicin for TRPA1 and TRPV1, respectively, TCD(1,8)-OH was applied alone and CSD-CH2(1,8)-NH2 in combination with U50,488 (Figure 7A and B). As compared to balanced (non-biased) KOR ligand U50,488, TCD(1,8)- OH elicited slight yet non-significant calcium signaling when applying a concentration of 100 µM (Figure 7A and B). To confirm antagonistic activity of CSD-CH2(1,8)-NH2, it was co-applied with U50,488 and it was observed that CSD-CH2(1,8)-NH2significantly blocks the action of U50,488 (Figure 7A and B). This effect was further supported by KOR selective antagonist norbinaltorphimine (nor-BNI) which inhibited the activity of U50,488 (Figure 7A and B). Taken together, these findings suggest that TCD(1,8)-OH is incapable of inducing calcium mobilization whereas CSD-CH2(1,8)-NH2inhibits the action of U50,488. Example 9: TCD(1,8)-OH exhibits a KOR-dependent antinociceptive effect in vivo; with an antinociceptive potency which is higher than the antinociceptive potency of U50,488. The antinociceptive effect of TCD(1,8)-OH and its antinociceptive potency, was tested in a formalin test (an inflammatory pain model) as described above in Example 1 and in the legend to Figure 1. It was shown in the context of this test, that the duration of pain behavior (time spent for licking / biting, lifting, and flinching; of a formalin-injected paw) was significantly reduced by TCD(1,8)-OH in a dose-dependent manner (Figure 12A). Further, the antinociceptive effect of TCD(1,8)-OH was shown to be based on KOR agonism (Figure 12B). Moreover, the antinociceptive potency of TCD(1,8)-OH was shown to be higher than the antinociceptive potency of U50,488 (reduced ED50 value; reduced by about 10%; Table 3). Reference is further made herein to the following table(s): Table 1: Pharmacological properties of CSD and TCD peptide leads at the KOR Data are from two to three independent experiments; dyn, dynorphin; n.a., not activeiFunctional affinity of 32 nM was derived from Schild regression analysis Table 2: Characterization and purity analysis of TCD and CSD peptide ligands determined by

[0197] VIALDI-TOF mass spectrometry and RP-HPLC

[0198] 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.

[0199] Table 3: Antinociceptive potencies of TCD(i,s)-OH and U50,488 (in the formalin test) aData from Muratspahic, Nat. Commun. 2023 loc. cit.

[0200] If not indicated differently, the following abbreviations are used herein:

[0201] BRET, bioluminescence resonance energy transfer; cAMP, cyclic adenosine monophosphate; CSD, cysteine stappled dynorphin; TCD, thioether cyclized dynorphin; DOR, 6-opioid receptor; DRG, dorsal root ganglion; dynA, dynorphin A; EGFP, enhanced green fluorescence protein; ESIMS, 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); and(iii) each of L1and L2is independently selected from -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2-CH2- , -CH(CH3)-CH2-, -CH2-CH(CH3)- and -CH(CH3)-CH(CH3)-.

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 5, wherein said cyclic peptide / cyclized dynA analog(a) is an agonist of the kappa opioid receptor (KOR);(b) specifically binds to KOR;(c) binds to KOR with an affinity corresponding to a Ki in the range of IO-8M to IO-6M or in the range of 3xl0-7M to IO-7M; or binds to KOR in a radioligand binding assay with an affinity corresponding to a Ki in the range of 10 to 1000 nM, in the range of 20 to 500 nM, in the range of 30 to 200 nM or in the range of 40 to 100 nM;(d) induces only partial or no p-a rrestin recruitment; and / or(e) exhibits an antinociceptive effect.

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 a G-protein-biased agonist of the KOR;(b) binds to the p-opioid receptor (MOR) in a radioligand binding assay with a low affinity corresponding to a Ki > about 1000 nM; and / or does not bind to the 6-opioid receptor (DOR);(c) binds to KOR with an affinity corresponding to a Ki value of about 1.5xl0-7M; or binds to KOR in a radioligand binding assay with an affinity corresponding to a Ki value of about 60 nM;(d) induces only partial or no p-arrestin-2 and / or p-arrestin-1 recruitment; and / or(e) exhibits an antinociceptive effect with an antinociceptive potency which is higher than the antinociceptive potency of U50,488.

9. The cyclic peptide / cyclized dynA analog according to any one of claims 1 to 8, wherein said cyclic peptide / cyclized dynA analog (a) is a short-acting, medium-acting or long-acting agonist of the KOR; (b) binds to KOR with a ≥15-fold, ≥30-fold, ≥40-fold, ≥50-fold, ≥50-fold, ≥60-fold or ≥ / about 70-fold selectivity over MOR and / or DOR; (c) induces only ≤25%, ≤20%, ≤15% or ≤10% of the β-arrestin recruitment of dynA1-13- NH2; and / or (d) exhibits an antinociceptive effect in an in vivo formalin test.

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); and (iii) each of L1and L2is -CH2-.

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 (i) treating, inhibiting or preventing pain and / or inducing an antinociceptive effect in a patient; (ii) treating, inhibiting or preventing inflammation / an inflammatory disease; or (iii) treating, inhibiting or preventing pruritus and / or itching / itching disorders.

13. The pharmaceutical composition for use according to claim 12, wherein said pain is acute pain or chronic pain.

14. The pharmaceutical composition for use according to claim 12 or 13, wherein said pain is peripheral pain or central pain.

15. The pharmaceutical composition for use according to any one of claims 12 to 14, wherein (i) said pain is selected from the group consisting of nociceptive pain, inflammatory pain, visceral pain, cancer-related pain, postoperative pain, pain resulting from injury, musculoskeletal pain and neuropathic pain; or(ii) said inflammation / inflammatopry disease is inflammatory bowel disease.

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

17. The pharmaceutical composition according to claim 11 or 16 or the pharmaceutical composition for use according to any one of claims 12 to 16, 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.

18. The pharmaceutical composition according to any one of claims 11, 16 and 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 systemically.

19. The pharmaceutical composition according to any one of claims 11 and 16 to 18 or the pharmaceutical composition for use according to any one of claims 12 to 18, wherein said cyclic peptide / cyclized dynA analog or said pharmaceutical composition is administered parenterally, perorally, rectally or bronchially.

20. 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 Br–L2–CH2C(=O)–SQ1–Z–SQ2–OH, wherein L2, SQ1and SQ2are as defined in any one of claims 1 to 3 and Z is –NH– C(H)(–L1–SRS)–C(=O)–, wherein L1is defined as in claim 1, preferably Z is a cysteine residue or a homocysteine residue, more preferably a cysteine residue, wherein the cysteine residue or homocysteine residue is optionally protected at the sulfur and RSis hydrogen or a protecting group; (ii) if the cysteine residue or homocysteine residue is protected at the sulfur or RSis a protecting group: removing the protecting group of the cysteine residue or homocysteine residue, or the protecting group of RS, respectively; (iii) allowing the product of step (ii) to form a cyclic peptide; 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.

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

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

23. The method of claim 21 or 22, wherein said SPPS is Fmoc SPPS by using a Wang resin or a 2-chlorotrityl chloride resin, or wherein said SPPS is Boc SPPS by using a PAM resin.

24. The method of any one of claims 20 to 23, wherein RSis a methyltrityl (Mtt) protecting group and / or step (ii) involves the use of trifluoroacetic acid.

25. The method of claim 24, wherein Z is a cysteine residue protected at the sulfur with Mtt or a homocysteine residue protected at the sulfur with Mtt.

26. The method of claim 24 or 25, wherein step (ii) involves the use of trifluoroacetic acid in dichloromethane.

27. The method of any one of claims 24 to 26, wherein step (ii) involves the use of trifluoroacetic acid at a temperature in the range of from 10 to 40°C.

28. The method of any of claims 20 to 27, wherein step (iii) comprises adding a base.

29. The method of claim 28, wherein said base is an amine.

30. The method of claim 29, wherein said amine is triethylamine.

31. The method of any one of claims 28 to 30, wherein step (iii) comprises addinga solution of triethylamine in an aprotic solvent.

32. The method of any one of claims 28 to 31, wherein step (iii) comprises adding triethylamine in dimethyl formamide.

33. The method of any one of claims 28 to 32, wherein said adding a base is at a temperature in the range of from 10 to 40°C.

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