D-CYS 38 epimer of KV1.3 inhibitor

Abstract

The present disclosure relates to compounds which are capable of selectively binding to and inhibiting the activity of the potassium channel Kv1.3. The invention also relates to a method of preparing said compounds, intermediates of said compounds, pharmaceutical compositions comprising such compounds and to the use of said compounds and said pharmaceutical compositions in medicine, including the treatment or prevention of autoimmune diseases, diabetes, obesity, parodontitis and / or tissue transplant rejection.

Description

[0001] selectlON Therapeutics GmbH

[0002] S12907WO

[0003] D-CYS38EPIMER OF KV1.3 INHIBITOR

[0004] Technical field

[0005] The present disclosure relates to compounds which are capable of selectively binding to and inhibiting the activity of the potassium channel Kv1 .3. The disclosure also relates to a method of preparing said compounds, intermediates of said compounds, pharmaceutical compositions comprising such compounds and to the use of said compounds and said pharmaceutical compositions in medicine, including the treatment or prevention of autoimmune diseases, diabetes, obesity, parodontitis and / or tissue transplant rejection.

[0006] Background

[0007] The voltage-gated Kv1 .3 K+channel is one of 76 potassium channels in the human genome and has been found to be present in human T lymphocytes. All human T cells express the Kv1.3 channel as well as the calcium-activated KCa3.1 , which together provide the counter-balancing potassium efflux for the calcium influx that is necessary for T cell activation and proliferation. The number of channels expressed by a given cell depends on its state of activation and differentiation. Antigen or mitogen stimulated CD4+ and CD8+ short-lived effector memory cells (TEM) exhibit an approximately 4- to 5- fold increased expression of Kv1 .3, while human naive or long-lived central memory cells (TCM) up-regulate the calcium-activated KCa3.1 channel to regulate membrane potential and Ca2+signaling in the activated state.

[0008] In view of this differential overexpression in TEM cells, the Kv1 .3 channel constitutes a promising new TEM-cell-specific therapeutic target for the treatment of autoimmune diseases, whose pathogenesis involves autoreactive TEM cells such as e.g. multiple sclerosis, rheumatoid arthritis, psoriasis and type-1 diabetes, but also for other chronic inflammatory diseases, such as e.g. parodontitits. The FDA-approved drug clofazimine inhibits Kv1.3 and is used for the treatment of psoriasis and graft-versus-host disease.

[0009] Furthermore, there is an indication that Kv1 .3 channels play a role in the regulation of body weight. The Kv1 .3 channel thus also constitutes a promising target for the treatment of obesity.

[0010] WO 2015 / 169901 A1 relates to peptides, such as Si-544 / cgtx-544, capable of selectively binding to and inhibiting the activity of the potassium channel Kv1 .3.

[0011] However, it may frequently turn out challenging to get approval for clinical trials of peptides due to the stringent requirements of regulatory agencies such as the FDA or EMEA for clinical trial approval. These include comprehensive documentation on the peptide’s safety, efficacy, and quality.

[0012] DJB:MHO One frequent problem is the lack of chemical stability of peptides, e.g. during manufacturing and thus inconsistent composition of production batches, e.g. due to epimerization.

[0013] Such epimerization is generally considered critical and usually disqualifies a drug candidate from clinical development, despite highly promising preclinical data. This is because different epimers of the same compound may have different effects on the body. In that, one epimer might be more effective at interacting with the biological target, while the other might be less effective or even inactive. Understanding which epimer is more active can guide dosage and administration strategies. The same holds true for off-target interactions: While one epimer might bind to the biological target in a highly selective manner, the other might show high binding to biological structures other than the biological target thus resulting in toxicity and / or the increased occurrence of side-effects. In other words, one epimer might be toxic or cause adverse effects, while the other might be safe.

[0014] Furthermore, the absorption, distribution, metabolism, and excretion (ADME) of epimers can differ significantly: One epimer might bind to an enzyme, such as a protease, with high affinity thus being metabolized more quickly (or more slowly) than the other, affecting how long it stays active in the body and its overall efficacy.

[0015] Also, understanding how two epimers interact with the biological target can provide insights into the mechanism of action of the peptide guiding further therapeutic strategies and drug design.

[0016] Independently, it is understood that regulatory agencies require detailed information about the properties of any compound before being tested in humans. Knowing the activity of each epimer helps in providing a comprehensive profile of the drug candidate, which is essential for regulatory approval.

[0017] Summarily, the biological activity of different epimers of a peptide drug candidate must be well-characterized to ensure the safety, efficacy, and consistency of the drug candidate before proceeding to clinical trials. This knowledge aids in optimizing therapeutic outcomes and minimizing risks to patients.

[0018] Summary of the invention

[0019] In view of the high therapeutic potential of inhibiting Kv1 .3 channels, there is an ongoing need for Kv1.3 channel specific therapeutic compounds that exhibit a strong and specific interaction with the Kv1 .3 channel and are capable of blocking or reducing its activity.

[0020] Before moving into clinical trials, further insights on the mode of action of Si-544 and its epimers are hence required. ln the course of their investigations, the inventors found that Si-544 might be susceptible to epimerization of Cys38during solid-phase peptide synthesis (SPPS). In that, the Cys38reacts from L-Cys to D-Cys.

[0021] Therefore, the inventors of the present disclosure have investigated into the understanding of the biological activity of the two different epimers of Si-544 and derivatives thereof.

[0022] In a first aspect, the present invention relates to compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 (such as D-Cys38-Si-544) or a pharmaceutically acceptable salt thereof.

[0023] In a second aspect, the present invention relates to a solid-phase peptide synthesis method of preparing the compound according to the first aspect of the present disclosure, the method comprising the steps of a) providing Fmoc-D-Cys(PGI ) bound to a solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc- D-Cys(PG1)-O-resin and washing to provide H-D-Cys(PG1 )-O-resin; c) subsequent coupling, by stepwise solid-phase peptide synthesis, each of amino acids Arg, Asn, Cys, Lys, Cys, Lys, Arg, Asn, Met, Cys, Lys, Gly, Tyr, Pro, Cys, Gly, Thr, Gin, Ala, Lys, Cys, Pro, Pro, Leu, Cys, Gin, Lys, Pro, Ser, Thr, Cys, Lys, Vai, Asn, lie, lie, and Thr, or peptide building blocks consisting of said amino acids, wherein each of said subsequent couplings comprises adding an Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing; d) cleaving a bond between said solid resin and D-Cys(PGI ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain a compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4; and e) performing an oxidizing treatment to obtain the compound according to the first aspect of the present disclosure.

[0024] In a third aspect, the present invention relates to a compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 (such as D-Cys38-, or a pharmaceutically acceptable salt thereof.

[0025] According to a fourth aspect, the present invention relates to a solid-phase peptide synthesis method of preparing the compound according to the third aspect of the present disclosure, the method comprising the steps of a) providing Fmoc-D-Cys(PGI ) bound to a solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-D-Cys(PG1)-O-resin and washing to provide H-D-Cys(PG1 )-O-resin; c) subsequent coupling, by stepwise solid-phase peptide synthesis, each of amino acids Arg, Asn, Cys, Lys, Cys, Lys, Arg, Asn, Met, Cys, Lys, Gly, Tyr, Pro, Cys, Gly, Thr, Gin, Ala, Lys, Cys, Pro, Pro, Leu, Cys, Gin, Lys, Pro, Ser, Thr, Cys, Lys, Vai, Asn, lie, lie, and Thr, or peptide building blocks consisting of said amino acids, wherein each of said subsequent couplings comprises adding an Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing; d) cleaving a bond between said solid resin and D-Cys(PGI ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain the compound according to the third aspect of the present disclosure.

[0026] According to a fifth aspect, the present disclosure relates to a compound prepared by the method according to the second aspect of the present disclosure or according to the fourth aspect of the present disclosure.

[0027] According to a sixths aspect, the present disclosure relates to a pharmaceutical composition comprising the compound according to the first, third, or fifth aspect of the present invention.

[0028] According to a seventh aspect, the present disclosure relates to a compound according to the present disclosure or a pharmaceutical composition according to the present disclosure for use in medicine.

[0029] According to an eighth aspect, the present invention relates to a compound according to the present disclosure or a pharmaceutical composition according to the present disclosure for use in a method of treatment or prevention of an auto immune disease, diabetes, obesity, parodontitis and / or tissue transplant rejection.

[0030] Brief description of the figures

[0031] Fig. 1: Representative current traces as determined for control, bath solution and 1 nM margatoxin in Example 2 on Kv1.3 channel.

[0032] Fig. 2: Representative current traces as determined for control, 1 nM, 3 nM, 10 nM, and 30 nM D-Cys38-Si-544 in Example 2 on Kv1.3 channel.

[0033] Fig. 3: Dose-response curve for D-Cys38-Si-544 on Kv1 .3 channel.

[0034] Fig. 4: Representative current traces as determined for control and 10 pM D-Cys38-Si-544 in Example 3 on hERG.

[0035] Fig. 5: Representative current traces as determined for control, bath solution and 1 pM E-4031 in Example 3 on hERG.

[0036] Detailed description of the invention

[0037] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. One and three letter amino acid abbreviations used herein correspond to IUPAC nomenclature (see e.g. European Journal of Biochemistry, 13829-37, 1984). Compound B

[0038] In a first aspect, the present disclosure relates to compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 (such as D-Cys38-Si-544) or a pharmaceutically acceptable salt thereof. In other words, the present disclosure in a first aspect relates to compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 (such as D-Cys38-Si-544), wherein Cys38is D-cysteine, and wherein compound B has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38; or a pharmaceutically acceptable salt thereof.

[0039] According to a particularly preferred embodiment of the present disclosure, compound B consists of an amino acid sequence of SEQ ID No.: 2 (that is D-Cys38-Si-544).

[0040] As shown in Example 2 and Example 3, the inventors of the present disclosure have surprisingly found that D-Cys38-Si-544 and Si-544 have essentially the same affinity to Kv1 .3 receptor and also does not bind to hERG.

[0041] In other words, the compound according to the first aspect of the present disclosure has surprisingly been found by the inventors of the present invention to be capable of selectively binding to the Kv1 .3 potassium channel over other potassium channels, such as e.g. hERG. Given the prevalence of Kv1 .3 in TEM cells, the compounds according to the present invention therefore constitute powerful therapeutic agents for TEM-COII mediated diseases, such as e.g. auto immune diseases. Furthermore, the disclosed compounds provide the advantage of reduced side effects, as they do not substantially modulate the activity of other potassium channels distributed to other types of cells or tissues.

[0042] Hence, the inventors of the present disclosure have surprisingly found that the compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 according to the first aspect of the present disclosure shows essentially the same pharmacodynamic properties as its epimer compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1.

[0043] This finding is highly surprising because Cys38plays an essential role for the folding of compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 due to the formation of a disulfide bond between Cys38and Cys23. In fact, the effect of epimerization on the pharmacodynamic properties of a peptide is expected to be even more significant when the respective D- or L-amino acid is cysteine that forms a disulfide bond in the folded peptide:

[0044] It is understood that disulfide bonds play a crucial role in stabilizing the three- dimensional structure of peptides which is essential for selective and high-affinity binding to of the peptide to its biological target. Thus, epimerization of cysteine from L- to its D-form may disrupt the precise spatial arrangement needed for disulfide bond formation, potentially leading to incorrect or unstable folding of the peptide. Disulfide bonds are typically formed between two L-cysteine residues. If one of these cysteines is epimerized to the D-form, it may not correctly pair with its intended partner, preventing the formation of the disulfide bond or leading to incorrect disulfide pairing. This can significantly alter the peptide's conformation and functionality.

[0045] Also, the biological activity of a peptide is highly dependent on the correct folding and the presence of functional disulfide bonds. Incorrect folding due to epimerization can lead to loss of activity or even the acquisition of new, unintended activities, which could be detrimental. The specific three-dimensional structure of a peptide is often critical for its interaction with biological targets such as receptors or enzymes. Epimerization that disrupts or changes the spatial arrangement of disulfide bonds can alter the binding affinity and specificity, affecting the peptide's efficacy and potency.

[0046] Even more, incorrectly folded peptides can be recognized as foreign by the immune system, potentially increasing the risk of immunogenic reactions.

[0047] Given these factors, the epimerization of cysteine in a peptide that relies on disulfide bonds for its structure and function is expected to have profound effects, making it particularly significant compared to the epimerization of other amino acids.

[0048] In conclusion, it is highly surprising and against all expectations that compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 according to the first aspect of the present disclosure shows essentially the same pharmacodynamics as its epimer compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 .

[0049] Method of preparation

[0050] In a second aspect, the present invention relates to a solid-phase peptide synthesis method of preparing the compound according to the first aspect of the present disclosure, the method comprising the steps of a) providing Fmoc-D-Cys(PGI ) bound to a solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc- D-Cys(PG1)-O-resin and washing to provide H-D-Cys(PG1 )-O-resin; c) subsequent coupling, by stepwise solid-phase peptide synthesis, each of amino acids Arg, Asn, Cys, Lys, Cys, Lys, Arg, Asn, Met, Cys, Lys, Gly, Tyr, Pro, Cys, Gly, Thr, Gin, Ala, Lys, Cys, Pro, Pro, Leu, Cys, Gin, Lys, Pro, Ser, Thr, Cys, Lys, Vai, Asn, lie, lie, and Thr, or peptide building blocks consisting of said amino acids, wherein each of said subsequent couplings comprises adding an Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing; d) cleaving a bond between said solid resin and D-Cys(PGI ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain a compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4; and e) performing an oxidizing treatment to obtain the compound according to the first aspect of the present disclosure. Solid-phase peptide synthesis (SPPS) is often preferred in the pharmaceutical industry over recombinant methods for several reasons: SPPS can rapidly produce peptides. Automated synthesizers can build peptides in a relatively short amount of time, making it suitable for high-throughput synthesis and iterative testing during drug development. On the other hand, recombinant methods involve cloning, expression, and purification steps that can be time-consuming and labor-intensive.

[0051] Furthermore, SPPS generally provides high control over the peptide sequence and purity. Each step of the synthesis can be monitored and optimized to ensure high-quality products. On the other hand, recombinant methods can result in heterogeneous products due to variations in post-translational modifications and the presence of host cell proteins, requiring additional purification steps to achieve the desired purity.

[0052] Additionally, SPPS is highly scalable for producing small to medium quantities of peptides, which is often sufficient for therapeutic peptides. The process can be scaled up without significant changes to the synthesis protocol.

[0053] Also, SPPS is particularly advantageous for synthesizing complex or cyclic peptides, which can be difficult to produce recombinantly due to challenges in folding and post- translational processing. Moreover, SPPS can be more cost-effective. It avoids the need for costly infrastructure and resources associated with maintaining cell cultures and fermentation systems; no cost-intensive setup is required.

[0054] Importantly, SPPS also allows for precise control over the synthesis process, which simplifies compliance with regulatory standards for pharmaceutical products, while recombinant methods require more complex regulatory pathways due to the use of genetically modified organisms and the need for stringent control over biological systems.

[0055] In summary, SPPS offers unparalleled flexibility, speed, and control, making it the method of choice for the development and production of therapeutic peptides in the pharmaceutical industry.

[0056] Therefore, the inventors of the present disclosure have extensively investigated into methods of preparing compound B as disclosed herein.

[0057] According to a preferred embodiment of the present disclosure, all steps a) to e) of the method according to the third aspect of the present disclosure are performed at a temperature of less than about 40 °C. According to another preferred embodiment of the present disclosure, all steps a) to e) of the method according to the third aspect of the present disclosure are performed at a temperature of about 35 °C or less. According to another preferred embodiment of the present disclosure, all steps a) to e) of the method according to the third aspect of the present disclosure are performed at a temperature of less than about 35 °C. According to a preferred embodiment of the present disclosure, all steps a) to e) of the method according to the third aspect of the present disclosure are performed at a temperature of more than about 10 °C. According to another preferred embodiment of the present disclosure, all steps a) to e) of the method according to the third aspect of the present disclosure are performed at a temperature of about 15 °C or more. According to another preferred embodiment of the present disclosure, all steps a) to e) of the method according to the third aspect of the present disclosure are performed at a temperature of about 20 °C or more.

[0058] Providing a solid resin having Fmoc-D-Cys(PGI) bound to said solid resin

[0059] In step a) of the method according to the second aspect of the present invention and according to the fourth aspect of the present invention, a solid resin having Fmoc- D-Cys(PGI) bound to said solid resin is provided, wherein PG1 is a side-chain protecting group. It is understood that for preparation of Fmoc-D-Cys(PG1 ) bound to a solid resin, an Fmoc-D-Cys(PG1)-OH with a free carboxyl group is reacted with the solid resin, such as under conditions of peptide coupling including DIC / Oxyma or TBTU / DIPEA mediated couplings.

[0060] In solid-phase peptide synthesis (SPPS), solid resins are used to anchor the growing peptide chain, allowing for sequential addition of amino acids. In that, a first amino acid is bound to said solid resin. Optionally, the first amino acid may be bound to said solid resin by a linker.

[0061] The solid resin as used in the method according to the third aspect of the present disclosure is not particularly limited. However, it is understood that the solid resin as used in the method according to the present disclosure is chemically resistant under the conditions used during coupling reactions, washing steps, and during Fmoc-deprotection. The solid resin may be in any form, such as beads.

[0062] According to a preferred embodiment of the present disclosure, the solid resin may be a commercially available resin. According to another preferred embodiment, the solid resin is selected from the group consisting of 2-chlorotrityl chloride resin, SASRIN resin, Wang resin, Rink amide resin, hydroxymethylphenoxy (HMP) resin, and TentaGel resin.

[0063] According to a particularly preferred embodiment of the present disclosure, the solid resin is a 2-chlorotrityl chloride resin (2-CT resin). As used herein, a 2-chlorotrityl chloride resin refers to a solid resin having a polystyrene backbone provided with a 2-chlorotrityl chloride linker.

[0064] According to another preferred embodiment of the present disclosure, the solid resin is a Wang resin. As used herein, a Wang resin refers to a solid resin having a polystyrene backbone provided with a p-benzyloxybenzyl alcohol linker. It is understood that a Wang resin is suitable for attaching cysteine via its carboxyl group and releasing peptides under acidic conditions from the resin, such as by addition of trifluoroacetic acid.

[0065] According to another preferred embodiment of the present disclosure, the solid resin is a Rink amide resin. As used herein, a Rink amide resin refers to a solid resin having a polystyrene backbone provided with a 4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)- phenoxy linker. It is understood that a Rink amide resin is suitable for attaching cysteine via its carboxyl group and releasing peptides from the resins under acidic conditions, such as by addition of trifluoroacetic acid (TFA).

[0066] According to another preferred embodiment of the present disclosure, the solid resin is a hydroxymethylphenoxy (HMP) resin. As used herein, a hydroxymethylphenoxy resin refers to a solid resin having a polystyrene backbone provided with a hydroxymethylphenoxy. It is understood that a HMP resin is suitable for attaching cysteine via its carboxyl group and releasing peptides from the resin under acidic conditions, such as by addition of trifluoroacetic acid (TFA).

[0067] According to another preferred embodiment of the present disclosure, the solid resin is a TentaGel resin. As used herein, a TentaGel resin refers to a solid resin having a composite backbone of polystyrene and polyethylene glycol (PEG) provided with a hydroxymethylphenoxy linker, a 4-hydroxymethylphenoxybutyric acid linker, a trityl linker, a 2-chlorotrityl linker, or the like. It is understood that a TentaGel resin is suitable for attaching cysteine via its carboxyl group and releasing peptides from the resin under acidic conditions, such as by addition of trifluoroacetic acid (TFA).

[0068] According to another preferred embodiment of the present disclosure, the solid resin is a SASRIN resin (super acid-sensitive resin). As used herein, a SASRIN resin refers to a solid resin having a polymer backbone provided with an extremely acid labile linker, such as 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetic acid. It is understood that a SASRIN is suitable for attaching cysteine via its carboxyl group and releasing peptides from the resin under acidic conditions, such as by addition of trifluoroacetic acid (TFA).

[0069] It is understood that any protective group of the linker is removed before coupling with the Fmoc-D-Cys(PG1 )-OH. Fmoc-D-Cys(PG1 )-OH is commercially available.

[0070] In the context of the present disclosure, PG1 is a side-chain protection group. In other words, PG1 is bound to the sulfur of cysteine. Said PG1 is bound to the sulfur of cysteine to prevent side reactions during subsequent coupling steps and only cleaved upon releasing the peptide from the resin.

[0071] According to a preferred embodiment of the present disclosure, PG1 is selected from the group consisting of triphenylmethyl (Trt), (4-methoxyphenyl)diphenylmethyl (Mmt), and tetrahydropyranyl (Thp). According to another preferred embodiment of the present disclosure, PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt) or triphenylmethyl (Trt). According to a further preferred embodiment of the present disclosure, PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt).

[0072] In other words, according to a preferred embodiment of the present disclosure, the first step of the method according to the second aspect of the present disclosure is providing a solid resin having Fmoc-D-Cys(Trt) bound to said solid resin, or having Fmoc-D-Cys(Mmt) bound to said solid resin, or having Fmoc-D-Cys(Thp) bound to said solid resin.

[0073] Each of said resins having Fmoc-D-Cys(PGI ) bound to said solid resin may be prepared from commercially available resins, such as resins disclosed herein, and commercially available Fmoc-D-Cys(PG1 )-OH, such as Fmoc-D-Cys(Thp)-OH, Fmoc- D-Cys(Mmt)-OH, or Fmoc-D-Cys(Trt)-OH.

[0074] Fmoc-D-Cys(Thp)-OH is also known under CAS number 167015-11-4. Fmoc- D-Cys(Mmt)-OH is also known under CAS number 1198791-73-9. Fmoc-D-Cys(Trt)-OH is also known under CAS number 167015-11-4.

[0075] Fmoc-deprotection

[0076] In step b) of the method according to the second aspect of the present invention and according to the fourth aspect of the present invention, a Fmoc-deprotection from the Fmoc- D-Cys(PGI) bound to said solid resin provided in the first step and washing is performed to provide H-D-Cys(PGI ) bound to said solid resin.

[0077] According to a preferred embodiment of the present disclosure, said Fmoc- deprotection is performed using a de-protection solution comprising piperidine (Pip) and DMF, wherein the concentration of piperidine is less than about 20 % (V / V) based on the total volume of the de-protection solution.

[0078] The inventors of the present disclosure have surprisingly found that Fmoc is efficiently deprotected and the amine terminus of D-Cys(PGI ) bound to said solid resins is efficiently provided if the amount of piperidine in the de-protections solution is less than about 20 % (V / V) based on the total volume of the de-protection solution.

[0079] According to another preferred embodiment of the present disclosure, said Fmoc- deprotection is performed using a de-protection solution comprising piperidine (Pip) and DMF, wherein the concentration of piperidine is in the range of from about 5 % (V / V) to about 15 % (V / V) based on the total volume of the de-protection solution. According to another preferred embodiment of the present disclosure, said Fmoc-deprotection is performed using a de-protection solution comprising piperidine (Pip) and DMF, wherein the concentration of piperidine is in the range of from about 7 % (V / V) to about 13 % (V / V) based on the total volume of the de-protection solution. According to another preferred embodiment of the present disclosure, said Fmoc-deprotection is performed using a de-protection solution comprising piperidine (Pip) and DMF, wherein the concentration of piperidine is in the range of from about 8 % (V / V) to about 12 % (V / V) based on the total volume of the de-protection solution. According to a further preferred embodiment of the present disclosure, said Fmoc- deprotection is performed using a de-protection solution comprising piperidine (Pip) and DMF, wherein the concentration of piperidine is in the range of from about 9 % (V / V) to about 11 % (V / V) based on the total volume of the de-protection solution. According to a yet further preferred embodiment of the present disclosure, said Fmoc-deprotection is performed using a de-protection solution comprising piperidine (Pip) and DMF, wherein the concentration of piperidine is about 10 % (V / V) based on the total volume of the de-protection solution.

[0080] Stepwise solid-phase peptide synthesis

[0081] In step c) of the method according to the second aspect of the present disclosure and according to the fourth aspect of the present disclosure, a stepwise solid-phase peptide synthesis is performed, in which the further amino acids forming compound B, and compound D are subsequently coupled to the respective preceding amino acid in the order of Arg, Asn, Cys, Lys, Cys, Lys, Arg, Asn, Met, Cys, Lys, Gly, Tyr, Pro, Cys, Gly, Thr, Gin, Ala, Lys, Cys, Pro, Pro, Leu, Cys, Gin, Lys, Pro, Ser, Thr, Cys, Lys, Vai, Asn, lie, lie, and Thr.

[0082] Each of said amino acids may be coupled to the each other in a separate stage or in the form of a building block consisting of the respective amino acids in the correct order for forming compound B, and / or compound D. Such building block may consist of two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, or ten amino acids selected from the above amino acids, wherein the amino acids forming the building block are in the correct order for forming compound B, and / or compound D.

[0083] In other words, the building blocks as disclosed herein may be any fragment of compound B, and / or compound D.

[0084] Additionally, it is understood that each of the coupling stages comprises adding a respective Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing.

[0085] During the step of adding the respective Fmoc-protected amino acid, a coupling reagent may be added. According to a preferred embodiment of the present disclosure, said coupling reagent comprises DIC / Oxyma or TBTU / DIPEA.

[0086] According to a preferred embodiment of the present disclosure, each of the Fmoc- deprotection stages in step c) of the method according to the second aspect of the present disclosure or the method according to the fourth aspect of the present disclosure is performed as described in step b) of the method of the second aspect of the present disclosure or the method according to the fourth aspect of the present disclosure.

[0087] As shown in example 1 , the inventors of the present disclosure have found that D-Cys38-Si-544 can thus be efficiently prepared at high yield. During the stepwise solid-phase peptide synthesis, Fmoc-protected amino acids are subsequently coupled to the respective preceding amino acid in the order of Arg, Asn, Cys, Lys, Cys, Lys, Arg, Asn, Met, Cys, Lys, Gly, Tyr, Pro, Cys, Gly, Thr, Gin, Ala, Lys, Cys, Pro, Pro, Leu, Cys, Gin, Lys, Pro, Ser, Thr, Cys, Lys, Vai, Asn, lie, lie, and Thr. According to a preferred embodiment of the present disclosure, each of said amino acids, in particular each of Arg, Asn, Cys, lys, Tyr, Thr, Gin, Ser may contain a side-chain protection group.

[0088] In other words, according to a preferred embodiment of the present disclosure, the method according to the second aspect of the present disclosure and / or the method according to the fourth aspect of the present disclosure comprises the following stages of the stepwise solid-phase peptide synthesis:

[0089] According to a preferred embodiment of the present disclosure, in a step c1 ), Fmoc- Arg(PG2)-OH is added to the H-D-Cys(PGI ) bound to said solid resin provided in step b) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a first intermediate.

[0090] According to a preferred embodiment of the present disclosure, PG2 is selected from the group of 2,2,4,6,7-pentamethyl-dihydrobenzofuranyl-5-sulfonyl (Pbf), 2, 2, 5,7,8- pentamethylchroman-6-sulfonyl (Pmc), and 4-Methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr).

[0091] According to another preferred embodiment of the present disclosure, Fmoc- Arg(PG2)-OH is Fmoc-Arg(Pbf)-OH. Fmoc-Arg(Pbf)-OH is also known under CAS number 154445-77-9.

[0092] According to another preferred embodiment of the present disclosure, Fmoc- Arg(PG2)-OH is Fmoc-Arg(Pmc)-OH. Fmoc-Arg(Pmc)-OH is also known under CAS number 119831-72-0.

[0093] According to another preferred embodiment of the present disclosure, Fmoc- Arg(PG2)-OH is Fmoc-Arg(Mtr)-OH. Fmoc-Arg(Mtr)-OH is also known under CAS number 98930-01-9.

[0094] According to a preferred embodiment of the present disclosure, in a step c2), Fmoc- Asn(PG3)-OH or Fmoc-Asn-OH is added to the first intermediate provided in step c1 ) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a second intermediate.

[0095] According to a preferred embodiment of the present disclosure, PG3 is selected from the group consisting of trityl (Trt), tert-butylcarbonyl (Boc), and acetamidomethyl (Acm).

[0096] According to another preferred embodiment of the present disclosure, Fmoc- Asn(PG3)-OH is Fmoc-Asn(Boc)-OH.

[0097] According to another preferred embodiment of the present disclosure, Fmoc- Asn(PG3)-OH is Fmoc-Asn(Acm)-OH. According to a further preferred embodiment of the present disclosure, Fmoc- Asn(PG3)-OH is Fmoc-Asn(Trt)-OH. Fmoc-Asn(Trt)-OH is also known under CAS number 132388-59-1.

[0098] According to a preferred embodiment of the present disclosure, in a step c3), Fmoc-L-Cys(PG1)-OH is added to the second intermediate provided in step c2) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a third intermediate.

[0099] According to a preferred embodiment of the present disclosure, PG1 is selected from the group consisting of triphenylmethyl (Trt), (4-methoxyphenyl)diphenylmethyl (Mmt), and tetrahydropyranyl (Thp).

[0100] According to another preferred embodiment of the present disclosure Fmoc- Cys(PG1)-OH is Fmoc-L-Cys(Thp)-OH. Fmoc-L-Cys(Thp)-OH is also known under CAS number 1673576-83-4.

[0101] According to another preferred embodiment of the present disclosure Fmoc- L-Cys(PG1)-OH is Fmoc-L-Cys(Mmt)-OH. Fmoc-L-Cys(Mmt)-OH is also known under CAS number 177582-21-7.

[0102] According to another preferred embodiment of the present disclosure Fmoc- Cys(PG1)-OH is Fmoc-Cys(Trt)-OH. Fmoc-L-Cys(Trt)-OH is also known under CAS number 103213-32-7.

[0103] According to a preferred embodiment of the present disclosure, in a step c4), Fmoc- Lys(PG4)-OH is added to the third intermediate provided in step c3) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a fourth intermediate.

[0104] According to a preferred embodiment of the present disclosure, PG4 is selected from the group consisting of tert-butyloxycarbonyl (Boc), carbobenzyloxy (Cbz), and 4-Methyltrityl (Mtt).

[0105] According to another preferred embodiment of the present disclosure Fmoc- Lys(PG4)-OH is Fmoc-Lys(Boc)-OH. Fmoc-Lys(Boc)-OH is also known under CAS number 71989-26-9.

[0106] According to another preferred embodiment of the present disclosure Fmoc- Lys(PG4)-OH is Fmoc-Lys(Cbz)-OH. Fmoc-Lys(Cbz)-OH is also known under CAS number 86060-82-4.

[0107] According to another preferred embodiment of the present disclosure Fmoc- Lys(PG4)-OH is Fmoc-Lys(Mtt)-OH. Fmoc-Lys(Mtt)-OH is also known under CAS number 167393-62-6.

[0108] According to a preferred embodiment of the present disclosure, in a step c5), Fmoc- Cys(PG1)-OH as disclosed herein is added to the fourth intermediate provided in step c4) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a fifth intermediate.

[0109] According to a preferred embodiment of the present disclosure, in a step c6), Fmoc- Lys(PG4)-OH as disclosed herein is added to the fifth intermediate provided in step c5) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a sixths intermediate.

[0110] According to a preferred embodiment of the present disclosure, in a step c7), Fmoc- Arg(PG2)-OH as disclosed herein is added to the sixths intermediate obtained from step c6) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a seventh intermediate.

[0111] According to a preferred embodiment of the present disclosure, in a step c8), Fmoc- Asn(PG3)-OH as disclosed herein or Fmoc-Asn-OH is added to the seventh intermediate provided in step c7) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide an eighth intermediate.

[0112] According to a preferred embodiment of the present disclosure, in a step c9), Fmoc- Met-OH added to the eighth intermediate provided in step c8) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a ninth intermediate. Fmoc-Met-OH is known under CAS number 71989-28-1 .

[0113] According to a preferred embodiment of the present disclosure, in a step c10), Fmoc- Cys(PG1)-OH as disclosed herein is added to the ninth intermediate provided in step c9) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a tenth intermediate.

[0114] According to a preferred embodiment of the present disclosure, in a step c11 ), Fmoc- Lys(PG4)-OH as disclosed herein is added to the tenth intermediate provided in step c10) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide an 11thintermediate.

[0115] According to a preferred embodiment of the present disclosure, in a step c12), Fmoc- Gly-OH added to the 11thintermediate provided in step c11 ) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 12thintermediate. Fmoc-Gly-OH is known under CAS number 29022-11-5.

[0116] According to a preferred embodiment of the present disclosure, in a step c13), Fmoc- Tyr(PG5)-OH is added to the 12thintermediate provided in step d 2) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 13thintermediate.

[0117] According to a preferred embodiment of the present disclosure, PG5 is tert-butyl (tBu) or trityl (Trt) or 2-chlorotrityl (2-CITrt). According to another preferred embodiment of the present disclosure Fmoc- Tyr(PG5)-OH is Fmoc-Tyr(tBu)-OH. Fmoc-Tyr(tBu)-OH is also known under CAS number 71989-38-3.

[0118] According to another preferred embodiment of the present disclosure Fmoc- Tyr(PG5)-OH is Fmoc-Tyr(Trt)-OH. Fmoc-Tyr(Trt)-OH is also known under CAS number 133180-02-6.

[0119] According to another preferred embodiment of the present disclosure Fmoc- Tyr(PG5)-OH is Fmoc-Tyr(2-CITrt)-OH. Fmoc-Tyr(2-CITrt)-OH is also known under CAS number 350241-80-4.

[0120] According to a preferred embodiment of the present disclosure, in a step c14), Fmoc- Pro-OH added to the 13thintermediate provided in step c13) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 14thintermediate. Fmoc-Pro-OH is known under CAS number 71989-31 -6.

[0121] According to a preferred embodiment of the present disclosure, in a step c15), Fmoc- Cys(PG1 )-OH as disclosed herein is added to the 14thintermediate provided in step c14) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a 15thintermediate.

[0122] According to a preferred embodiment of the present disclosure, in a step c16), Fmoc- Gly-OH added to the 15thintermediate provided in step d 5) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 16thintermediate. Fmoc-Gly-OH is known under CAS number 29022-11 -5.

[0123] According to a preferred embodiment of the present disclosure, in a step c17), Fmoc- Thr(tBu)-OH added to the 16thintermediate provided in step c16) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 17thintermediate. Fmoc-Thr(tBu)-OH is known under CAS number 71989-35-0.

[0124] According to a preferred embodiment of the present disclosure, in a step c18), Fmoc- Gln(Trt)-OH added to the 17thintermediate provided in step c17) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 18thintermediate. Fmoc-Gln(Trt)-OH is also known under CAS number 132327-80-1.

[0125] According to a preferred embodiment of the present disclosure, in a step c19), Fmoc- Ala-OH added to the 18thintermediate provided in step d 8) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 19thintermediate. Fmoc-Ala-OH is also known under CAS number 35661 -39-3.

[0126] According to a preferred embodiment of the present disclosure, in a step c20), Fmoc- Lys(PG4)-OH as disclosed herein is added to the 19thintermediate provided in step c19) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a 20thintermediate. According to a preferred embodiment of the present disclosure, in a step c21 ), Fmoc- Cys(PG1 )-OH as disclosed herein is added to the 20thintermediate provided in step c20) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a 21stintermediate.

[0127] According to a preferred embodiment of the present disclosure, in a step c22), Fmoc- Pro-OH added to the 21stintermediate provided in step c21 ) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 22ndintermediate. Fmoc-Pro-OH is known under CAS number 71989-31 -6.

[0128] According to a preferred embodiment of the present disclosure, in a step c23), Fmoc- Pro-OH added to the 22ndintermediate provided in step c22) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 23rdintermediate. Fmoc-Pro-OH is known under CAS number 71989-31 -6.

[0129] According to a preferred embodiment of the present disclosure, in a step c24), Fmoc- Leu-OH added to the 23rdintermediate provided in step c23) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 24thintermediate. Fmoc-Leu-OH is known under CAS number 35661 -60-0.

[0130] According to a preferred embodiment of the present disclosure, in a step c25), Fmoc- Cys(PG1 )-OH as disclosed herein is added to the 24thintermediate provided in step c24) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a 25thintermediate.

[0131] According to a preferred embodiment of the present disclosure, in a step c26), Fmoc- Gln(Trt)-OH added to the 25thintermediate provided in step c25) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 26thintermediate. Fmoc-Gln(Trt)-OH is also known under CAS number 132327-80-1 .

[0132] According to a preferred embodiment of the present disclosure, in a step c27), Fmoc- Lys(PG4)-OH as disclosed herein is added to the 26thintermediate provided in step c26) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a 27thintermediate.

[0133] According to a preferred embodiment of the present disclosure, in a step c28), Fmoc- Pro-OH added to the 27thintermediate provided in step c27) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 28thintermediate. Fmoc-Pro-OH is known under CAS number 71989-31 -6.

[0134] According to a preferred embodiment of the present disclosure, in a step c29), Fmoc- Ser(PG7)-OH is added to the 28thintermediate provided in step c28) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 29thintermediate. According to a preferred embodiment of the present disclosure, PG7 is tert-butyl (tBu) or trityl (Trt).

[0135] According to another preferred embodiment of the present disclosure Fmoc- Ser(PG7)-OH is Fmoc-Ser(tBu)-OH. Fmoc-Ser(tBu)-OH is also known under CAS number 71989-33-8.

[0136] According to another preferred embodiment of the present disclosure Fmoc- Ser(PG7)-OH is Fmoc-Ser(Trt)-OH. Fmoc-Ser(Trt)-OH is also known under CAS number 111061-56-4.

[0137] According to a preferred embodiment of the present disclosure, in a step c30), Fmoc- Thr(tBu)-OH added to the 29thintermediate provided in step c29) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 30thintermediate. Fmoc-Thr(tBu)-OH is known under CAS number 71989-35-0.

[0138] According to a preferred embodiment of the present disclosure, in a step c31 ), Fmoc- Cys(PG1 )-OH as disclosed herein is added to the 30thintermediate provided in step c30) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a 31stintermediate.

[0139] According to a preferred embodiment of the present disclosure, in a step c32), Fmoc- Lys(PG4)-OH as disclosed herein is added to the 31stintermediate provided in step c31 ) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc- deprotection, and washing to provide a 32ndintermediate.

[0140] According to a preferred embodiment of the present disclosure, in a step c33), Fmoc- Val-OH is added to the 32ndintermediate provided in step c32) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 33rdintermediate. Fmoc-Val-OH is also known under CAS number 68858-20-8.

[0141] According to a preferred embodiment of the present disclosure, in a step c34), Fmoc- Asn(PG3)-OH as disclosed herein or Fmoc-Asn-OH is added to the 33rdintermediate provided in step c33) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 34thintermediate.

[0142] According to a preferred embodiment of the present disclosure, in a step c35), Fmoc- lle-OH is added to the 34thintermediate provided in step c34) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 35thintermediate. Fmoc-lle-OH is also known under CAS number 71989-23-6.

[0143] According to a preferred embodiment of the present disclosure, in a step c36), Fmoc- lle-OH is added to the 35thintermediate provided in step c35) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 36thintermediate. Fmoc-lle-OH is also known under CAS number 71989-23-6. According to a preferred embodiment of the present disclosure, in a step c37), Fmoc- Thr(tBu)-OH added to the 36thintermediate provided in step c36) under addition of a coupling agent as disclosed herein, subjected to washing, Fmoc-deprotection, and washing to provide a 37thintermediate. Fmoc-Thr(tBu)-OH is known under CAS number 71989-35-0.

[0144] Optionally, further amino acids may be coupled to the 37thintermediate provided in step c37).

[0145] According to another preferred embodiment of the present disclosure, step c) may be performed with suitable building blocks consisting of from 2 to 10 of the amino acids as disclosed herein.

[0146] Releasing the peptide from the resin and deprotection

[0147] In step d) of the method according to the second aspect of the present invention and according to the fourth aspect of the present invention, the peptide is released from the solid resin by cleaving a bond between said solid resin and D-Cys(PGI ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4. In other words, step d) of the method according to the second aspect of the present disclosure and according to the fourth aspect of the present disclosure yields a compound according to the third aspect of the present disclosure.

[0148] According to a preferred embodiment of the present disclosure, step d) comprises the addition of trifluoroacetic acid (TFA). It is understood that the addition of TFA is sufficient to cleave both the bond between D-Cys38and the solid resin. In other words, the solid resin is adapted in that the bond between D-Cys38and the solid resin is hydrolyzed upon addition of TFA.

[0149] Furthermore, it is a preferred embodiment of the present disclosure that all side chain protection groups PG1 , PG2, PG3, PG4, PG5, PG6, and PG7 are cleaved off the respective amino acids upon addition of trifluoroacetic acid (TFA).

[0150] According to another preferred embodiment of the present disclosure, step d) further comprises a step of purifying compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4.

[0151] According to another preferred embodiment of the present disclosure, the step of purifying comprises a purification by preparative reversed -phase high performance liquid chromatography (HPLC) to yield a purified compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4. According to another preferred embodiment of the present disclosure, the step of purifying further comprises a lyophilizing step to yield a lyophilized purified compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4. OxidizinQ treatment

[0152] In step e) of the method according to the second aspect of the present invention, the compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 obtained from step d) of the method according to the second aspect of the present disclosure is subjected to an oxidizing treatment to obtain the compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 according to the first aspect of the present disclosure. In other words, step e) of the method according to the second aspect of the present invention comprises performing an oxidizing treatment to yield a compound according to the first aspect of the present disclosure.

[0153] It is understood that the oxidizing treatment results in the formation of disulfide bonds between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38.

[0154] In other words, compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 is reacted to compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2.

[0155] According to a preferred embodiment of the present disclosure, the oxidizing treatment in step e) of the method according to the second aspect of the present disclosure is performed using cystine or a mixture of cysteine and cystine.

[0156] Adding an oxidizing agent after solid-phase peptide synthesis (SPPS) serves the oxidative folding and formation of disulfide bonds in peptides and proteins containing cysteine residues. Thereby, the mixture of cysteine and cystine forms a redox buffer system. Cysteine can donate electrons to reduce disulfide bonds, while cystine can accept electrons to form new disulfide bonds. This dynamic equilibrium helps in the correct pairing of cysteine residues to form disulfide bonds.

[0157] The concentrations and ratio of cysteine to cystine are not particularly limited.

[0158] According to another preferred embodiment of the present disclosure, step e) further comprises a step of purifying the compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2.

[0159] According to another preferred embodiment of the present disclosure, the step of purifying the compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 comprises a purification by preparative reversed-phase high performance liquid chromatography (HPLC) to yield a purified compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2. According to another preferred embodiment of the present disclosure, the step of purifying the compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 further comprises a lyophilizing step to yield a the purified, lyophilized compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2.

[0160] According to another preferred embodiment of the present disclosure, the step of purifying the compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 further comprises a step of ion exchange to yield a pharmaceutically acceptably salt of the compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2. Preferably, the step of ion exchange is performed prior to the lyophilizing step.

[0161] Further steps

[0162] According to another preferred embodiment of the present disclosure, step d) further comprises a step of purifying compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4.

[0163] According to another preferred embodiment of the present disclosure, the step of purifying comprises a purification by preparative reversed -phase high performance liquid chromatography (HPLC) to yield a purified compound D.

[0164] According to another preferred embodiment of the present disclosure, the step of purifying further comprises a lyophilizing step to yield a lyophilized purified compound D.

[0165] According to another preferred embodiment of the present disclosure, step e) further comprises a step of purifying compound B.

[0166] According to another preferred embodiment of the present disclosure, the step of purifying comprises a purification by preparative reversed -phase high performance liquid chromatography (HPLC) to yield a purified compound B.

[0167] According to another preferred embodiment of the present disclosure, the step of purifying further comprises a lyophilizing step to yield a lyophilized purified compound B.

[0168] According to another preferred embodiment of the present disclosure, the step e) further comprises a step of ion exchange to yield a pharmaceutically acceptable salt of compound B.

[0169] Linear peptide

[0170] In a third aspect, the present disclosure relates to a compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 (such as D-Cys38-Si-544-linear) or a pharmaceutically acceptable salt thereof. In other words, the present disclosure in a third aspect relates to a compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 (such as D-Cys38-Si-544-linear), wherein Cys38is D-cysteine, and wherein compound D has no intramolecular disulfide bonds; or a pharmaceutically acceptable salt thereof. According to a particularly preferred embodiment, the present disclosure relates to a compound D consisting of an amino acid sequence of SEQ ID No.: 4 (D-Cys38-Si-544-linear) or a pharmaceutically acceptable salt thereof.

[0171] It is understood that the compound according to the third aspect of the present invention is an important intermediate in the preparation of the compound according to the first aspect of the present disclosure. However, the compound of the third aspect of the present disclosure is different from the compound of the first aspect of the present disclosure in that compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 according to the third aspect of the present disclosure does not have intramolecular disulfide bonds.

[0172] Method of preparation of linear peptides

[0173] According to a fourth aspect, the present invention relates to a solid-phase peptide synthesis method of preparing the compound according to the third aspect of the present disclosure, the method comprising the steps of a) providing Fmoc-D-Cys(PGI ) bound to a solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-D-Cys(PG1)-O-resin and washing to provide H-D-Cys(PG1 )-O-resin; c) subsequent coupling, by stepwise solid-phase peptide synthesis, each of amino acids Arg, Asn, Cys, Lys, Cys, Lys, Arg, Asn, Met, Cys, Lys, Gly, Tyr, Pro, Cys, Gly, Thr, Gin, Ala, Lys, Cys, Pro, Pro, Leu, Cys, Gin, Lys, Pro, Ser, Thr, Cys, Lys, Vai, Asn, lie, lie, and Thr, or peptide building blocks consisting of said amino acids, wherein each of said subsequent couplings comprises adding an Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing; d) cleaving a bond between said solid resin and D-Cys(PGI ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain the compound according to the third aspect of the present disclosure.

[0174] It is understood that the method according to the fourth aspect of the present invention is a method for preparing the important intermediate according to the third aspect of the present disclosure. However, the method of the fourth aspect of the present disclosure is different from the method of the second aspect of the present disclosure in that the method of the fourth aspect of the present disclosure does not comprise a method step of performing an oxidizing treatment.

[0175] Therefore, it is understood that all features discussed under steps a), b), c), and d) of the method according to the second aspect of the present disclosure are the same for the method according to the fourth aspect of the present disclosure. In other words, steps a), b), c), and d) of the method according to the fourth aspect of the present disclosure are characterized by the same features and preferred embodiments as disclosed herein in respect of steps a), b), c), and d) of the method according to the second aspect of the present disclosure.

[0176] Compound prepared by method

[0177] According to a fifth aspect, the present disclosure relates to a compound prepared by the method according to the second aspect of the present disclosure or according to the fourth aspect of the present disclosure.

[0178] It is understood that the compounds according to the fifth aspect of the present invention are characterized by the same features as the compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 according to the first aspect of the present disclosure, or by the same features as the compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 according to the third aspect of the present disclosure, respectively.

[0179] Pharmaceutical composition

[0180] According to a sixths aspect, the present disclosure relates to a pharmaceutical composition comprising the compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 according to the first aspect of the present disclosure, the compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 according to the third aspect of the present disclosure, and / or the compound according to the fifth aspect of the present disclosure.

[0181] It is understood that the pharmaceutical compositions according to the sixths aspect of the present disclosure in particular solves the object of providing a pharmaceutical composition comprising the highly potent and highly selective Kv1 .3 inhibitor compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2.

[0182] According to a preferred embodiment, the pharmaceutical composition according to the sixths aspect of the present disclosure is suitable for oral, rectal, nasal, sublingual, percutaneous, topic, intravenous, or intramuscular administration.

[0183] According to a preferred embodiment, the pharmaceutical composition according to the sixths aspect of the present disclosure is suitable for oral administration, for example in the form of inhalable powder pills, tablets, lacquered tablets, sugar-coated tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions.

[0184] According to a preferred embodiment, the pharmaceutical composition according to the sixths aspect of the present disclosure is suitable for rectal administration, for example in the form of a suppository.

[0185] According to a preferred embodiment, the pharmaceutical composition according to the sixths aspect of the present disclosure is suitable for topic administration, for example for topic administration on the skin. Thus, the pharmaceutical composition according to the sixths aspect of the present disclosure is preferably in the form of a cream, an ointment, a gel, a lotion, a paste, a foam, a spray, a powder, a dermal patch, a transdermal patch, a solution, or a suspension.

[0186] According to a preferred embodiment, the pharmaceutical composition according to the sixths aspect of the present disclosure is suitable for intranasal application. Thus, the pharmaceutical composition according to the sixths aspect of the present disclosure is preferably in the form of a nasal spray, nasal drop, nasal gel, nasal powder, nasal inhaler, nasal ointment, or nasal film.

[0187] According to a preferred embodiment, the pharmaceutical composition according to the sixths aspect of the present disclosure is suitable for sublingual application. Thus, the pharmaceutical composition according to the sixths aspect of the present disclosure is preferably in the form of a sublingual tablet, sublingual film, sublingual strip, sublingual spray, sublingual drops, or a sublingual troche.

[0188] According to a preferred embodiment, the pharmaceutical composition according to the sixths aspect of the present disclosure is suitable for percutaneous application. Thus, the pharmaceutical composition according to the sixths aspect of the present disclosure is preferably in the form of a transdermal patch, cream, ointment, a gel, a lotion, a paste, a foam, a spray, a powder, a dermal patch, a solution, or a suspension.

[0189] According to a preferred embodiment, the pharmaceutical composition according to the sixths aspect of the present disclosure is suitable for intravenous application. Thus, the pharmaceutical composition according to the sixths aspect of the present disclosure is preferably in the form of a solution, an emulsion, or a suspension.

[0190] According to a preferred embodiment, the pharmaceutical composition according to the sixths aspect of the present disclosure is suitable for intramuscular application. Thus, the pharmaceutical composition according to the sixths aspect of the present disclosure is preferably in the form of a solution, an emulsion, a suspension, or a depot-injection.

[0191] For the production of pills, tablets, sugar-coated tablets and hard gelatin capsules it is possible to use, for example, lactose, starch, for example maize starch, or starch derivatives, talc, stearic acid or its salts, etc. Carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc. Suitable carriers for the preparation of solutions, for example of solutions for injection, or of emulsions or syrups are, for example, water, physiological sodium chloride solution, alcohols such as ethanol, glycerol, polyols, sucrose, invert sugar, glucose, mannitol, vegetable oils, etc. The pharmaceutical compositions can also contain additives, for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.

[0192] In some embodiments of the present disclosure, the pharmaceutical composition may be a sustained release formulation.

[0193] In some embodiments of the present disclosure, the pharmaceutical composition according to the invention may further comprise at least one immunosuppressive agent which is suitable for the treatment of autoimmune diseases. Examples of such immunosuppressive agents include e.g. cortisol, hydrocortisol, dexamethasone, cyclophosphamide, nitrosoureas, methotrexate, mercaptopurine, mitomycin C, bleomycin, mithramycin, cyclosporine, rapamycin, azathioprine, prednisone and deoxyspergualin and interferons.

[0194] Medical use

[0195] According to a seventh aspect, the present disclosure relates to a compound according to the present disclosure, in particular compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2, or a pharmaceutical composition according to the first aspect of the present disclosure, in particular a pharmaceutical composition comprising compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2, for use in medicine.

[0196] The inventors of the present disclosure have surprisingly found (cf. Example 2 and Example 3 of the present disclosure) that compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 according to the first aspect of the present disclosure has a high affinity and selectivity towards Kv1 .3 channels. Thus, in particular compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 according to the first aspect of the present disclosure may be advantageously used in medicine.

[0197] According to an eighths aspect, the present invention relates to a compound according to the present disclosure or a pharmaceutical composition according to the present disclosure for use in a method of treatment or prevention of an auto immune disease, diabetes, obesity, parodontitis and / or tissue transplant rejection.

[0198] For the purposes of the present disclosure the term “treatment” refers to the curative alleviation of a disease while the term “prevention” refers to preventive prophylaxis. It is to be understood that both terms do not imply a complete remission or prevention of the respective disease but rather that there is an improvement compared to a situation where no pharmaceutically active agent is administered for either curative or preventive purposes.

[0199] In a particularly preferred embodiment the present invention relates to compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2 according to the first aspect of the present disclosure or pharmaceutical composition according to the disclsoure for use in the treatment or prevention of an autoimmune disease.

[0200] In the context of the present invention the term “auto immune disease” or “auto immune diseases” refers to a disease state caused by an inappropriate immune response that is directed to a self-encoded entity, i.e. an autoantigen. Encompassed within the definition are any of a number of disorders caused by an immune system defect that allows the body to attack its own tissues. In a preferred embodiment, the auto immune disease is a T cell mediated autoimmune disorder.

[0201] Examples of auto immune diseases that may be treated or prevented by the compounds and pharmaceutical compositions of the present invention include e. g. multiple sclerosis, rheumatoid arthritis, psoriasis, type-1 diabetes, vasculitis, Hashimoto's disease, asthma, atopic dermatitis, autoimmune eye diseases, Sjogren's syndrome, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anaemia, autoimmune hepatitis, autoimmune oophoritis, Coeliac disease, Crohn's disease, gestational pemphigoid, Goodpasture's syndrome, Grave's disease, Guillian-Barre syndrome, idiopathic thrombocytopenic purpura, Kawasaki's disease, lupus erythematosus, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, polyarthritis (in dogs), primary biliary cirrhosis, Reiter's syndrome, Takayasu's arteritis, temporal arteritis, warm autoimmune haemolytic anaemia, Wegener's granulomatosis, ANCA-associated systemic vasculitis, Churg-Strauss syndrome, microscopic polyangiitis, colitis, inflammatory bowel diseases, uveitis and psoriatic arthritis.

[0202] Some of these auto-immune diseases have been linked to TEM-cells for example ANCA-associated systemic vasculitis (Abdulahad, Nephrology, 2009 vol. 14 pp 26), Churg- Strauss syndrome, Wegener's granulomatosis , microscopic polyangiitis (Berden, Arthritis & Rheumatism, 2009, vol.60, pp 1578) , Ankylosing spondylitis, Behcet’s disease, colitis, Crohn’s disease, Inflammatory Bowel Diseases, multiple sclerosis, psoriasis, rheumatoid Arthritis, Sjogren’s Syndrome, Type 1 Diabetes Mellitus (Ulivieri, Expert Rev. Vaccines, 2013 vol. 12 pp 297), System lupus erythematosus (Devarajan, Immunol. Res, 2013 vol. 57 pp 12), uveitis (Amadi — Obi, Nephrology, 2009, vol 14 pp 26), and psoriatic arthritis (De Vlam, Acta Derm. Venereol, 2014, vol. 94, pp 627).

[0203] The same applies to obesity (Xu, Human Molecular Genetics, 2003, vol. 12, pp 551).

[0204] In a preferred embodiment the auto immune disease to be treated or prevented by the compounds and pharmaceutical compositions of the present invention is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, psoriasis, type-1 diabetes and vasculitis. ln another aspect the present invention relates to a method of treating or preventing an auto immune disease, obesity, parodontitis and / or tissue transplant rejection in a mammal by administering a compound according to the invention or a pharmaceutical composition according to the invention to a mammal in need thereof.

[0205] Preferably the auto immune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, psoriasis, type-1 diabetes and vasculitis.

[0206] Definitions and general embodiments

[0207] As used herein, the term “compound A” refers to a compound or peptide comprising or consisting of an amino acid sequence SEQ ID No.: 1 , that is represented by the following formula:

[0208] H-Thr1-lle2-lle3-Asn4-Val5-Lys6-Cys7-Thr8-Ser9-Pro10-Lys11-Gln12-Cys13-Leu14-Pro15- Pro16-Cys17-Lys18-Ala19-Gln20-Thr21-Gly22-Cys23-Pro24-Tyr25-Gly26-Lys27-Cys28-Met29-Asn30- Arg31-Lys32-Cys33-Lys34-Cys35-An s36-Arg37-Cys38-O H wherein Cys38is L-cysteine, and wherein compound A has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38.

[0209] As used herein, the terms “compound A consisting of an amino acid sequence SEQ ID No.: 1”, “Si-544”, and “cgtx-544” are used interchangeably.

[0210] As used herein, the term “compound B" refers to a compound or peptide comprising or consisting of an amino acid sequence SEQ ID No.: 2, that is represented by the following formula:

[0211] H-Thr1-lle2-lle3-Asn4-Val5-Lys6-Cys7-Thr8-Ser9-Pro10-Lys11-Gln12-Cys13-Leu14-Pro15- Pro16-Cys17-Lys18-Ala19-Gln20-Thr21-Gly22-Cys23-Pro24-Tyr25-Gly26-Lys27-Cys28-Met29-Asn30- Arg31-Lys32-Cys33-Lys34-Cys35-An s36-Arg37-Cys38-O H wherein Cys38is D-cysteine, and wherein compound B has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38.

[0212] As used herein, the terms “compound B consisting of an amino acid sequence SEQ ID No.: 2”, “D-Cys38-Si-544”, and “D-Cys38-cgtx-544” are used interchangeably.

[0213] Hence, it is understood that Si-544 and D-Cys38Si-544 as disclosed herein differ in the configuration of one amino, that is Cys38. Thus, Si-544 and D-Cys38-Si-544 are epimers. Both Si-544 and D-Cys38-Si-544 as disclosed herein have the same molecular formula C173H297N55O49S9 (net) and the same molecular mass of 4212.1 g / mol (net).

[0214] As used herein, the term “compound C” refers to a compound or peptide comprising or consisting of an amino acid sequence SEQ ID No.: 3, that is represented by the following formula H-Thr1-lle2-lle3-Asn4-Val5-Lys6-Cys7-Thr8-Ser9-Pro10-Lys11-Gln12-Cys13-Leu14-Pro15- Pro16-Cys17-Lys18-Ala19-Gln20-Thr21-Gly22-Cys23-Pro24-Tyr25-Gly26-Lys27-Cys28-Met29-Asn30- Arg31-Lys32-Cys33-Lys34-Cys35-An s36-Arg37-Cys38-O H wherein Cys38is L-cysteine, and wherein compound C has no intramolecular disulfide bonds.

[0215] As used herein, the terms “compound C consisting of an amino acid sequence SEQ ID No.: 3”, “Si-544-linear”, and “cgtx-544-linear” are used interchangeably.

[0216] As used herein, the term “compound D” refers to a peptide comprising or consisting of an amino acid sequence SEQ ID No.: 4, that is represented by the following formula:

[0217] H-Thr1-lle2-lle3-Asn4-Val5-Lys6-Cys7-Thr8-Ser9-Pro10-Lys11-Gln12-Cys13-Leu14-Pro15- Pro18-Cys17-Lys18-Ala19-Gln20-Thr21-Gly22-Cys23-Pro24-Tyr25-Gly26-Lys27-Cys28-Met29-Asn30- Arg31-Lys32-Cys33-Lys34-Cys35-An s36-Arg37-Cys38-O H wherein Cys38is D-cysteine, and wherein compound D has no intramolecular disulfide bonds.

[0218] As used herein, the terms “compound D consisting of an amino acid sequence SEQ ID No.: 4, “D-Cys38-Si-544-linear”, and “D-Cys38-cgtx-544-linear” are used interchangeably.

[0219] Hence, it is understood that Si-544-linear and D-Cys38-Si-544-linear as disclosed herein differ in the configuration of one amino, that is Cys38. Thus, Si-544-linear and D-Cys38-Si-544-linear are epimers. Both Si-544-linear and D-Cys38-Si-544-linear as disclosed herein have the same molecular formula C173H297N55O49S9 (net) and the same molecular mass of 4220.1 g / mol (net).

[0220] As used herein, the term “pharmaceutically acceptable salt" refers to a salt of a compound as disclosed herein that retains the desired biological activity of the aboveidentified compound and include pharmaceutically acceptable acid addition salts. Suitable pharmaceutically acceptable acid addition salts of the compound comprising or consisting of an amino acid sequence of SEQ ID No.: 1 may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, trifluoroacetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic, and arylsulfonic acid.

[0221] As used herein, any reference to an amino acid or derivative, or protected form thereof relates to the L-form of said respective amino acid unless indicated otherwise.

[0222] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present disclosure, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising”. If a group is defined to comprise at least a certain number of embodiments herein, this is also to be understood to disclose a group, which preferably consists only of these embodiments. Furthermore, if a composition is defined using the term “comprising”, it may additionally comprise other elements not explicitly listed, however, not further amounts of an element listed. As such, if, e.g., a de-protection solution comprises piperidine in a concentration of 10 % (V / V) based on the total volume of the de-protection solution, said de-protection solution may comprise elements other than piperidine and DMF, however, not additional amounts of piperidine thereby exceeding the amount of 10 % (VAX).

[0223] The term “about" and “approximately” in conjunction with a numerical value refers to normal deviations of said numerical value. It is to be understood that the terms “about” and “approximately” can each mean a deviation of ± 10 %, preferably ± 5 %, more preferably ± 2.5 % of said numeric value as indicated.

[0224] As used herein, the term “room temperature” means 25 °C.

[0225] As used in this specification and in the intended claims, the singular forms of “a” and “an” also include the respective plurals unless the context clearly dictates otherwise.

[0226] Examples

[0227] In the following section, particular examples illustrating various embodiments and aspects of the invention are presented. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description.

[0228] All materials were obtained from commercial suppliers, unless specified otherwise.

[0229] Abbreviations

[0230] The following abbreviations may appear in the examples:

[0231] ATP: adenosine triphosphate

[0232] CHO Chinese Hamster Ovary

[0233] C-type: Rapid inactivation process responsible for hERG rectification

[0234] DMSO: Dimethylsulfoxide

[0235] EGTA: Ethylene glycol-bis(IS-aminoethyl ether)- -tetraacetic acid HEPES: N-2-hydroxyethylpiperazine- -2-ethanesulfonic acid hERG: human-ether-a-go-go related gene IKr: rapid delayed rectifier potassium current SEM: standard error of the mean

[0236] Example 1 - Preparation of compound B consisting of an amino acid sequence of SEQ ID No.: 2 (D-Cys38-Si-544)

[0237] Compound B consisting of an amino acid sequence of SEQ ID No.:2 was prepared. For that, a Fmoc-D-Cys(Trt)-OH was coupled to a 2-chlorotirtyl chloride resin in the presence of DIC / Oxyma or TBTU / DIPEA. Subsequently, the Fmoc-protection group was removed by addition of a solution of 10 % (VAX) piperidine in DMF and shaking the suspension for an appropriate time.

[0238] Subsequently, stepwise solid-phase peptide synthesis was performed using an Fmoc- strategy as disclosed herein. Each of the respective Fmoc-protected amino acids was subsequently coupled to the N-terminal amine of a respective peptide coupled to the solid resin. After washing, the Fmoc protection group was by adding a solution of 10 % (VA / ) piperidine in DMF and shaking the suspension for an appropriate time thereby revealing a new N-terminal amine to which a further amino acid may be attached, followed by washing. After each coupling reaction, a capping step was performed using acetic anhydride in DMF to block free / V-terminus of any unreacted amino acid.

[0239] After subsequently coupling all 38 amino acids forming the peptide of SEQ ID No.: 2, the peptide was cleaved from the solid resin and all side-chain protection groups were removed by adding a mixture of TFA / H2O / EDT / TIS (85:3:10:2, V / VA / A / ) and stirring for 2.5 h.

[0240] The crude product was purified by preparative reversed phase HPLC on a C18 column (Waters BEH C18 column). The eluent system was acetonitrile + 0.05 % TFA (solvent A) and water + 0.05 % TFA (solvent B) with a gradient: 0 min: 5 % solvent B 30 min: 35 % solvent B. The flow rate was 0.4 mL / min.

[0241] The linear peptide compound D consisting of an amino acid SEQ ID No.: 4 (D-Cys38-Si-544-linear) was obtained with a purity of > 90 % determined by RP-HPLC.

[0242] The linear peptide compound D consisting of an amino acid SEQ ID No.: 4 was subjected to an oxidation treatment by adding a mixture of cysteine and cysteine for forming disulfide bonds and folding.

[0243] After oxidation treatment, the crude peptide was purified by preparative RP-HPLC as described above The resulting fractions were subjected to lyophilization to compound B consisting of an amino acid SEQ ID No.: 2 (D-Cys38-Si-544) with a purity of > 95 % determined by RP-HPLC.

[0244] Example 2 - Effect on Kv1 .3 currents recorded from stably transfected CHO cells at controlled room temperature

[0245] The whole-cell patch-clamp technique was used to investigate the effects of four concentrations of D-Cys38-Si-544 on Kv1.3 stably expressed in CHO cells.

[0246] Materials and methods

[0247] Test system: CHO cells stably expressing the Kv1 .3 channel were obtained from

[0248] B’SYS (B'SYS GmbH, 4108 Witterswil, Switzerland). All experiments were performed with manual Patch-Clamping (Amplifier: EPC-10, HEKA Electronics; Headstage: Preamplifier EPC-10, HEKA Electronics; Software: PatchMaster, HEKA Electronics, Version v2x73.2).

[0249] Bath solution: 137 mM sodium chloride, 4 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 10 mM HEPES, 10 mM D-Glucose; pH (NaOH) 7.4.

[0250] Pipette solution: 130 mM potassium chloride, 1 mM magnesium chloride, 5 mM Mg- ATP, 10 mM HEPES, 5 mM EGTA; pH (KOH) 7.2.

[0251] To achieve an appropriate level of statistical significance each concentration of the test item was analyzed in sufficient numbers (n = 3). The four test item concentrations (1 nM, 3 nM, 10 nM and 30 nM of D-Cys38-Si-544 trifluoroacetate as prepared in Example 1 in bath solution) were applied on every cell cumulatively.

[0252] Negative control: As negative control, three additional experiments (n=3) were treated with bath solution only for the same duration as the longest experiment treated with test compound.

[0253] Positive control: As positive control, and if possible, the same cells were treated with a specific Kv1.3 current blocker (Margatoxin, supplied by Sigma) at a concentration of 1 nM (in bath solution).

[0254] Experimental procedure: The 35 mm culture dishes upon which cells are seeded at a density allowing single cells to be recorded was placed on the dish holder of the microscope and continuously perfused (at approximately 1 mL / min) with the bath solution. All solutions applied to cells including the pipette solution were maintained at room temperature (19 °C to 30 °C). After formation of a Gigaohm seal between the patch electrodes and individual Kv1.3 stably transfected OHO cells (pipette resistance range: 2.0 MQ to 7.0 MQ, seal resistance range > 1 GQ) the cell membrane across the pipette tip was ruptured to assure electrical access to the cell interior (whole-cell patch-configuration). If the quality of the seal was poor (seal resistance < 1 GQ), the process of seal formation was repeated with a different cell and a new pipette.

[0255] As soon as a stable seal could be established the following voltage protocol was applied: Holding potential: -80 mV, depolarization to +40 mV for 1500 ms at a frequency of 0.2 Hz. Kv1.3 outward peak current was measured as maximal current amplitude during the voltage pulse from -80 mV to +40 mV. This voltage protocol was run at least 10 times at intervals of 5 s. If current amplitude is judged to be too low for measurement, another cell was recorded.

[0256] Once control recordings have been accomplished, cells were continuously perfused with test solutions (1 nM, 3 nM, 10 nM and 30 nM of D-Cys38-Si-544 trifluoroacetate as prepared in Example 1 in bath solution), bath solution only, or 1 nM Margatoxin, respectively. Representative current traces as recorded for control, bath solution, 1 nM margatoxin, and tests solutions are shown in Figs. 1 and 2, respectively. During wash-in of the test item the voltage protocol indicated above was run continuously again at 5 s intervals until the steadystate level of current block was reached.

[0257] IC50 Determination: Since Kv1 .3 outward peak currents were inhibited by the test item by more than 50%, a concentration-response curve was determined and the IC50 (given in weight and molar concentrations) was calculated using SigmaPlot 11 .0. The dose-response curve was fitted to a 2-parameter logistic function (amax = 100 %): wherein X is the drug concentration, ICso is the concentration of drug at half maximal inhibition and H is the Hill coefficient.

[0258] Multi-sample as performed to test statistical significance of all test item concentrations assayed (GraphPad Prism 5).

[0259] Data compilation and statistical analysis (raw data): The values (in A) of the peak amplitudes of outward currents and the bath temperature of each voltage step were printed for compilation and analysis at the end of each experiment. Using SigmaPlot 11 .0 the recorded current amplitudes at the steady state level of current inhibition were compared to those from control conditions measured in the pre-treatment phase of the same cell. The amount of current block was calculated as percentage of control. To determine whether the observed current inhibition was due to a test item interaction with the Kv1 .3 channel or due to current rundown, these residual currents were compared to those measured in bath solution treated cells. Data from three individual experiments per test item concentration were collected and the corresponding mean values and standard errors calculated. Multi-sample analysis (ANOVA followed by Dunnett’s test) was performed to test statistical significance of all test item concentrations assayed (GraphPad Prism 5).

[0260] Results

[0261] A total of 7 cells were used for compilation of data and analysis as summarized in

[0262] Table 1.

[0263] Table 1 Summary of experiments

[0264] Experimental results:

[0265] A summary of Kv1 .3 current inhibition is provided in Table 2, representative current traces are provided in FIG Table 2 Summary of Kv1.3 current inhibition

[0266] One wav analysis of Variance and Dunnett

[0267] Analysis of Variance (ANOVA), multi sample comparison (Dunnett) was conducted with GraphPad Prism 5. According to One Way Analysis of Variance the means of each group (test concentrations) were significantly different. Post testing for comparisons versus Bath solution treated group according to Dunnett’s test revealed significant differences for all tested concentrations equal or higher than 1 .0 nM. Table 3 Dunnett’s multiple comparison test

[0268] IC50 determination:

[0269] Since D-Cys38-Si-544 reduced the Kv1 .3 peak current amplitude by more than 50 %, the dose response curve was constructed with a sigmoidal two parameter equation (using SigmaPlot 11.0): wherein X is the drug concentration, ICso isthe concentration of drug at half maximal inhibition and H is the Hill coefficient. Dose-response curve for D-Cys38-Si-544 is shown in Fig.3.

[0270] IC50 value was estimated to be 4.42 nM, with a Hill coefficient of 0.90.

[0271] Conclusion

[0272] In this study the test item D-Cys38-Si-544 trifluoroacetate salt was tested at the concentrations of 1 nM, 3 nM, 10 nM and 30 nM for its effects on the Kv1 .3 peak currents recorded in CHO cells stably transfected with cDNA encoding this potassium channel.

[0273] Since Kv1 .3 peak currents were inhibited by the test item by more than 50 %, a concentration-response curve was determined and the IC50 was calculated to be 4.42 nM with a Hill coefficient of 0.90.

[0274] The test system was validated using the reference item Margatoxin at a concentration of 1 nM, which effectively blocked the Kv1.3 peak current in this study (23.20 ± 5.17 % relative current, mean ± SEM of n=3 cells).

[0275] Thus, D-Cys-Si-544 has a similar or even higher affinity to Kv1.3 channel compared to Si-544 (IC50 of 6.9 nM as determined in WO 2018 / 169901 A1 ). Example 3 - Effect on hERG inactivating tail currents recorded from stably transfected CHQ cells at controlled room temperature

[0276] The whole-cell patch-clamp technique was used to investigate the effects of 10 pM D-Cys38-Si-544 on the hERG (human-ether-a-go-go related gene) potassium channel stably expressed in OHO cells.

[0277] Materials and methods

[0278] Test system: CHO cells stably expressing the hERG channel (Source: B'SYS GmbH, 4108 Witterswil, Switzerland).

[0279] All experiments were performed with manual Patch-Clamping (Amplifier: EPC-10, HEKA Electronics; Headstage: Preamplifier EPC-10, HEKA Electronics; Software: PatchMaster, HEKA Electronics, Version v2x73.2).

[0280] Bath solution: 137 mM sodium chloride, 4 mM potassium chloride, 1 .8 mM calcium chloride, 1 mM magnesium chloride, 10 mM HEPES, 10 mM D-Glucose; pH (NaOH) 7.4.

[0281] Pipette solution: 120 mM potassium gluconate, 10 mM potassium chloride, 1.5 mM Mg-ATP, 10 mM HEPES, 5 mM EGTA; pH (KOH) 7.3.

[0282] To achieve an appropriate level of statistical significance, the test item (10 pM D-Cys38-Si-544 trifluoroacetate in bath solution) was analyzed in sufficient numbers (n = 3).

[0283] Negative control: As negative control, three additional experiments (n = 3) were treated with bath solution only for the same duration as the longest experiment treated with test item.

[0284] Positive control: As positive control, the same cells as for the negative control were treated with E-4031 (supplied from Alomone labs, Israel) at a concentration of 1 pM (in bath solution).

[0285] Experimental procedure: The 35 mm culture dishes upon which cells are seeded at a density allowing single cells to be recorded was placed on the dish holder of the microscope and continuously perfused (at approximately 1 mL / min) with the bath solution. All solutions applied to cells including the pipette solution were maintained at room temperature (19 °C to 30 °C). After formation of a Gigaohm seal between the patch electrodes and individual hERG stably transfected CHO cells (pipette resistance range: 2.0 MQ to 7.0 MQ, seal resistance range > 1 GO) the cell membrane across the pipette tip was ruptured to assure electrical access to the cell interior (whole-cell patch-configuration). If the quality of the seal was poor (seal resistance < 1 GQ), the process of seal formation was repeated with a different cell and a new pipette.

[0286] As soon as a stable seal could be established the following voltage protocol was applied: Holding potential: -80 mV, depolarization to +40 mV for 500 ms, followed by a voltage ramp to -80 mV (100 ms duration). Stimulation frequency: 0.2 Hz (every 5 s). 200 ms before the depolarization to +40 mV, a 100 ms pulse to -90 mV was applied. This voltage step was used to calculate the seal resistance for each applied voltage pulse. Data was digitized at 5 kHz. hERG current was measured as maximal current amplitude during the voltage ramp from +40 mV to -80 mV minus the current amplitude at -80 mV before depolarization to +40 mV. If current amplitude is judged to be too low for measurement (<500 pA), another cell was recorded.

[0287] Once control recordings have been accomplished (less than 10 % change of current amplitude within 125 s), cells were continuously perfused with test solution, bath solution only, or E-4031 . During wash-in of the test item the voltage protocol indicated above was run continuously again at 5 s intervals until the steady-state level of current block was reached.

[0288] IC50 Determination: Since hERG tail currents were inhibited by the test item by less than 50%, no concentration-response curve was determined and the IC50 was not calculated.

[0289] Data compilation and statistical analysis (raw data): The values (in A) of the peak amplitudes of outward currents and the bath temperature of each voltage step were printed for compilation and analysis at the end of each experiment. Using SigmaPlot 11 .0 the recorded current amplitudes at the steady state level of current inhibition were compared to those from control conditions measured in the pre-treatment phase of the same cell. The amount of current block was calculated as percentage of control. T o determine whether the observed current inhibition was due to a test item interaction with the hERG channel or due to current rundown, these residual currents were compared to those measured in bath solution treated cells. Data from three individual experiments per test item concentration were collected and the corresponding mean values and standard errors calculated. Since only one test item concentration was tested, the statistical significance was tested using Student's T-Test.

[0290] Results

[0291] A total of 6 cells were used for compilation of data and analysis as summarized in Table 4 Summary of experiments:

[0292] Experimental results:

[0293] A summary of hERG current inhibition is provided in Table 5, representative current traces are provided in Figs. 4 and 5. Table 5 Summary of summary of hERG current inhibition

[0294] Student’s T-Test

[0295] Student’s T-Tests were performed to test statistical significance of 10 pM D-Cys38-Si-544 versus bath solution (>215 s perfused) using GraphPad Prism 5.

[0296] Table 6 Student’s T-Test Conclusion

[0297] In this study the test item D-Cys38-Si-544 trifluoroacetate salt was tested at the concentration of 10 pM for its effects on the hERG outward currents recorded in CHO cells stably transfected with cDNA encoding this cardiac potassium channel.

[0298] Since hERG tail currents were not inhibited by the test item by more than 10 %, only one concentration was measured, no concentration-response curve was determined and the IC50 was not calculated. The test item did not block the hERG current significantly at a concentration of 10 pM.

[0299] The test system was validated using the reference item E-4031 at a concentration of 1 pM, which effectively blocked the hERG current in this study (6.17 ± 0.55% relative current, mean ± SEM of n = 3 cells).

[0300] Thus, it is understood that D-Cys38-Si-544 binds to Kv1.3 with high selectivity.

Claims

Claims1 . Compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2, wherein Cys38is D-cysteine, and wherein compound B has four intramolecular disulfide bonds, wherein the four intramolecular disulfide bonds are between Cys7and Cys28, between Cys13and Cys33, between Cys17and Cys35, and between Cys23and Cys38, or a pharmaceutically acceptable salt thereof.

2. A solid-phase peptide synthesis method of preparing the compound according to claim 1 , the method comprising the steps of a) Providing Fmoc-D-Cys(PGI ) bound to a solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-D-Cys(PG1 )-O-resin and washing to provide H-D-Cys(PG1 )-O-resin; c) Subsequent coupling, by stepwise solid-phase peptide synthesis, each of amino acids Arg, Asn, Cys, Lys, Cys, Lys, Arg, Asn, Met, Cys, Lys, Gly, Tyr, Pro, Cys, Gly, Thr, Gin, Ala, Lys, Cys, Pro, Pro, Leu, Cys, Gin, Lys, Pro, Ser, Thr, Cys, Lys, Vai, Asn, lie, lie, and Thr, or peptide building blocks consisting of said amino acids, wherein each of said subsequent couplings comprises adding an Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing; d) Cleaving a bond between said solid resin and D-Cys(PGI ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain a compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4; and e) Performing an oxidizing treatment to obtain the compound according to claim 1 .

3. Compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4, wherein Cys38is D-cysteine, and wherein compound D has no intramolecular disulfide bonds, or a pharmaceutically acceptable salt thereof.

4. A solid-phase peptide synthesis method of preparing the compound according to claim 3, the method comprising the steps of a) Providing Fmoc-D-Cys(PGI ) bound to a solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-D-Cys(PG1 )-O-resin and washing to provide H-D-Cys(PG1 )-O-resin; c) Subsequent coupling, by stepwise solid-phase peptide synthesis, each of amino acids Arg, Asn, Cys, Lys, Cys, Lys, Arg, Asn, Met, Cys, Lys, Gly, Tyr, Pro, Cys, Gly, Thr, Gin, Ala, Lys, Cys, Pro, Pro, Leu, Cys, Gin, Lys, Pro, Ser, Thr, Cys, Lys, Vai, Asn, lie, lie, and Thr, or peptide building blocks consisting of said amino acids, wherein each of said subsequent couplings comprises adding an Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing;d) Cleaving a bond between said solid resin and D-Cys(PGI ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain the compound according to claim 3.

5. The method according to claim 2, wherein the oxidizing treatment is performed using a mixture of cysteine / cystin.

6. The method according to any one of claims 2, 4, or 5, wherein PG1 is selected from the group consisting of triphenylmethyl (Trt), (4-methoxyphenyl)diphenylmethyl (Mmt), and tetrahydropyranyl (Thp); preferably wherein PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt) or triphenylmethyl (Trt); more preferably wherein PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt).

7. The method according to any one of claims 2, or 4 to 6, wherein all Fmoc deprotections are performed using a de-protection solution comprising piperidine and DMF, wherein the concentration of piperidine is less than about 20 % (V / V) based on the total volume of the de-protection solution, preferably wherein the concentration of piperidine is in the range of from about 5 % (V / V) to about 15 % (V / V), more preferably in the range of from about 7 % (V / V) to about 13 % (V / V), even more preferably in the range of from about8 % (V / V) to about 12 % (V / V), yet more preferably in the range of from about 9 % (V / V) to about 11 % (V / V), most preferably wherein the concentration of piperidine is about 10 % (V / V) based on the total volume of the de-protection solution.

8. The method according to any one of claims 2, or 4 to 7, wherein step d) comprises the addition of trifluoroacetic acid (TFA).

9. The method according to any one of claims 2, or 4, to 8, wherein all steps a) to e) are performed at a temperature of less than 40 °C, preferably at a temperature of 35 °C or less.

10. The method according to any one of claims 2, or 4 to 9, the method further comprising an ion-exchange step, and / or a lyophilization step.

11. A compound prepared by the method according to any one of claims 2, or 4 to 10.

12. A pharmaceutical composition comprising the compound according to any one of claims 1 , 3, or 11 .

13. A compound according to any one of claims 1 , 3, or 11 for use in medicine.

14. A compound according to any one of claims 1 , 3, or 11 for use in a method of treatment or prevention of an auto immune disease, diabetes, obesity, parodontitis and / or tissue transplant rejection.

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