Peptide with low content of d-CYS 38 epimer

Abstract

The present disclosure relates to a composition comprising a first compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 and a low amount of epimer impurities, a method of manufacturing thereof, a compound prepared by said method, an intermediate of said compound, and the use of such composition in medicine, such as in the treatment or prevention of an auto immune disease, diabetes, obesity, parodontitis and / or tissue transplant rejection.

Description

[0001] selectlON Therapeutics GmbH

[0002] S12906WO1

[0003] PEPTIDE WITH LOW CONTENT OF D-CYS38EPIMER

[0004] Technical field

[0005] The present disclosure relates to a composition comprising a first compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 and a low amount of epimer impurities, a method of manufacturing thereof, a compound prepared by said method, an intermediate of said compound, and the use of such composition in medicine, such as in the treatment or prevention of an auto immune disease, 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+ TE cells exhibit an approximately 4- to 5- fold increased expression of Kv1 .3, while human naive or TCM cells 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 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, capable of selectively binding to and inhibiting the activity of the potassium channel Kv1 .3.

[0011] However, the synthesis of peptides might turn out challenging.

[0012] In pharmaceutical industry, it is advantageous to use synthetic synthesis methods, such as solid-phase peptide synthesis (SPPS), for preparing peptides instead of recombinant approaches. However, SPPS often requires extensive optimization of resins, linkers, amino

[0013] DJB:MHO acid derivatives, coupling reagents, as well as protection-deprotection strategy, and cleavage reactions.

[0014] One of the most common and most difficult to control side reactions during SPPS is the epimerization of amino acids because the epimerization might affect the overall conformation of the molecule, eventually even altering the bioactivity of the peptide. Epimerized products have a high similarity of physical characteristics, thus making it difficult for them to be separated and purified. As regards amino acids in peptides, epimerization is very important in keeping the chirality of the assembled amino acids unchanged during the peptide synthesis and obtaining the desirable product without any problematic purification.

[0015] Summary of the invention

[0016] In view of the high therapeutic potential of compounds 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.

[0017] In that, the development and synthesis of Kv1 .3 channel inhibitors with high purity is highly desirable. The provision of such Kv1 .3 channel inhibitors with high purity does not only allow for a better understanding of structure-activity relationships but also enables moving forward into clinical trials. From a drug regulatory standpoint, it is essential to provide Kv1 .3 channel inhibitors that are intended for clinical trials or drug approval with high purity to receive regulatory approval.

[0018] The inventors of the present disclosure hence investigated into the therapeutic activity of epimers of Si-544 as disclosed in WO 2015 / 169901 A1 and derivatives thereof, as well as into conditions for preventing epimerization of peptide Kv1.3 channel inhibitors. Thus, the inventors of the present disclosure have found a way to provide Kv1 .3 channel inhibitor comprising or consisting of an amino acid sequence of SEQ ID No.: 1 or a pharmaceutically acceptable salt thereof with high purity and low content of epimers.

[0019] These and other objectives as they will become apparent from the ensuing description and claims are attained by the subject matter of the independent claims. Some of the preferred embodiments are defined by the dependent claims.

[0020] In a first aspect, the present invention relates to a composition comprising a) a compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 , 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 L-Cys38; or a pharmaceutically acceptable salt thereof; and b) a 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 D-Cys38; or a pharmaceutically acceptable salt thereof; characterized in that c) the composition comprises compound A in an amount of at least about 95 % (n / n) based on the total combined amount of substance of compound A and compound B.

[0021] According to a second aspect, the present invention relates to a composition of the first aspect of the present invention that was prepared by solid -phase peptide synthesis (SPPS).

[0022] According to a third aspect, the present invention relates to a solid-phase peptide synthesis method of preparing the composition according to the first aspect of the present invention, the method comprising the steps of a) providing a solid resin having Fmoc- L-Cys(PGI ) bound to said solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-L-Cys(PGI ) bound to said resin and washing to provide H-L-Cys(PGI ) bound to said resin; c) subsequent coupling, by stepwise solid-phase peptide synthesis, each 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; or building blocks consisting of said amino acids, wherein each of said amino acids optionally comprises a side-chain protection group, and wherein each of said subsequent couplings comprises adding a respective Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing to obtain a bound to said solid resin; d) cleaving a bond between said solid resin and Cys(PG1 ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain a composition comprising compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3 and compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4; and e) performing an oxidizing treatment to obtain the composition according to the first aspect of the present invention.

[0023] According to a fourth aspect, the present invention relates to a composition comprising a) a compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3, wherein Cys38is L-cysteine, and wherein compound C has no intramolecular disulfide bonds (such as Si-544-linear), or a pharmaceutically acceptable salt thereof; and b) a 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 (such as D-Cys38-Si-544-linear), or a pharmaceutically acceptable salt thereof; characterized in that c) the composition comprises compound C in an amount of at least about 95 % (n / n) based on the total combined amount of substance of compound C and compound D.

[0024] According to a fifth aspect, the present invention relates to a composition of the fourth aspect of the present invention that was prepared by solid-phase peptide synthesis (SPPS). According to a sixths aspect, the present invention relates to a solid-phase peptide synthesis method of preparing the composition according to the fourth aspect of the present invention, the method comprising the steps of a) providing a solid resin having Fmoc- L-Cys(PGI) bound to said solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-L-Cys(PGI ) bound to said resin and washing to provide H-L-Cys(PGI) bound to said resin; c) subsequent coupling, by stepwise solid-phase peptide synthesis, each 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; or building blocks consisting of said amino acids, wherein each of said amino acids optionally comprises a side-chain protection group, and wherein each of said subsequent couplings comprises adding a respective Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing to obtain a peptide bound to said solid resin; d) cleaving a bond between said solid resin and Cys(PG1 ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain a composition according to the fourth aspect of the present invention.

[0025] According to a seventh aspect, the present invention relates to a composition prepared by the method according to the third aspect of the present invention, or prepared by the method of the sixths aspect of the present invention.

[0026] According to an eighth aspect, the present invention relates to a pharmaceutical composition comprising a composition according to the present invention.

[0027] According to a ninth aspect, the present invention relates to a composition according to the invention or a pharmaceutical composition according to the invention for use in medicine.

[0028] According to a tenth aspect, the present invention relates to a composition according to the invention or a pharmaceutical composition according to the invention for use in a method of treatment or prevention of an auto immune disease, diabetes, obesity, parodontitis and / or tissue transplant rejection.

[0029] Brief description of the figures

[0030] Fig. 1 : Figure 1 shows a schematic overview of the solid-phase peptide synthesis according to Example 1 .

[0031] Detailed description of the invention

[0032] 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).

[0033] The composition of folded peptides

[0034] In a first aspect, the present disclosure relates to a composition comprising a) a compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 (such as Si-544), or a pharmaceutically acceptable salt thereof; and b) a 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; characterized in that c) the composition comprises compound A in an amount of at least about 95 % (n / n) based on the total combined amount of substance of compound A and compound B.

[0035] According to a further preferred embodiment of the present disclosure, the present disclosure relates to a composition comprising a) a compound A (also referred to as “Si-544”) consisting of an amino acid sequence of SEQ ID No.: 1 , or a pharmaceutically acceptable salt thereof; and b) a compound B (also referred to as “D-Cys38-Si-544”) consisting of an amino acid sequence of SEQ ID No.: 2, or a pharmaceutically acceptable salt thereof; characterized in that c) the composition comprises compound A in an amount of at least about 95 % (n / n) based on the total combined amount of substance of compound A and compound B.

[0036] According to another preferred embodiment of the present disclosure, compound A consists of an amino acid sequence of SEQ ID No.: 1 . According to another preferred embodiment of the present disclosure, compound B consists of an amino acid sequence of SEQ ID No.: 2.

[0037] In other words, the present disclosure relates to a composition comprising a) a compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 , 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 L-Cys38; or a pharmaceutically acceptable salt thereof; and b) a 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 D-Cys38; or a pharmaceutically acceptable salt thereof; characterized in that c) the composition comprises compound A in an amount of at least about 95 % (n / n) based on the total combined amount of substance of compound A and compound B. The inventors of the present disclosure have surprisingly found that Cys38of compound A according to the present disclosure may be prone to epimerization during preparation, that is the interconversion of one epimer to the other epimer. This is particularly true if compound A is to be prepared by solid-phase peptide synthesis: It is understood the Cys38is the “first” amino acid that is bound to a solid resin for the synthesis. Thus, Cys38not only experiences a high chemical stress during all subsequent coupling and de-protection steps, but must also be cleaved from the resin in the last step under harsh conditions.

[0038] However, it is of utmost importance to provide epimer-pure compounds for clinical trials for several reasons:

[0039] Different epimers of a compound can interact differently with biological targets. One epimer may often be more effective in binding to the desired target, such as Kv1 .3 channel, leading to better therapeutic outcomes. In other words, the pharmacological activity of different epimers can vary significantly. Using the most potent epimer can enhance the drug’s overall effectiveness.

[0040] The same holds true for binding to non-targets, such as potassium channels other than Kv1 .3: Non-target interactions of epimers can lead to unwanted side effects. By preparing and isolating the epimer that has the desired therapeutic effect and minimal off- target effects, the safety profile of a drug candidate or drug can be considerably improved.

[0041] Some epimers may even be toxic or produce harmful metabolites. Ensuring the compound used in clinical trials is free from toxic epimers further helps mitigate the risk of adverse effects in patients.

[0042] Epimers may also have different pharmacokinetic properties, such as absorption, distribution, metabolism, and excretion (ADME). One epimer might be absorbed better, distributed more effectively, or metabolized at a different rate than another. Ensuring epimer purity helps achieve predictable pharmacokinetic profiles.

[0043] Also, the bioavailability of the active compound in the body can be significantly influenced by the presence of different epimers. Pure epimers ensure consistent and reliable bioavailability.

[0044] Independently, regulatory agencies, such as the FDA or EMEA, generally require detailed characterization of the compound being tested, including its stereochemistry. Epimer purity simplifies this process and meets regulatory standards for drug approval.

[0045] Studying the pure epimers allows for a better understanding of the drug’s mechanism of action. This can lead to the development of better drugs and therapeutic strategies in the future.

[0046] In summary, providing epimer-pure compounds ensures the clinical trials are conducted with a compound that has the highest efficacy, best safety profile, and consistent pharmacokinetic properties, while also meeting regulatory requirements. Therefore, it is an object of the present invention to provide a composition comprising compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 , or a pharmaceutically acceptable salt thereof in a high epimer-purity and high epimeric excess over its D-Cys38epimer compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2, or a pharmaceutically acceptable salt thereof.

[0047] According to a preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound A or a pharmaceutically acceptable salt thereof in an amount of at least about 97 % (n / n) based on the total combined amount of substance of compound A and compound B. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound A or a pharmaceutically acceptable salt thereof in an amount of at least about 98 % (n / n) based on the total combined amount of substance of compound A and compound B. According to a further preferred embodiment of the present disclosure, the composition comprises compound A or a pharmaceutically acceptable salt thereof more preferably in an amount of more than about 98 % (n / n) based on the total combined amount of substance of compound A and compound B.

[0048] According to a preferred embodiment of the present disclosure, the composition comprises compound B or a pharmaceutically acceptable salt thereof in an amount of about 5 % (n / n) or less based on the total combined amount of substance of compound A and compound B. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of about 3 % (n / n) or less based on the total combined amount of substance of compound A and compound B. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of about 2 % (n / n) or less based on the total combined amount of substance of compound A and compound B. According to a further preferred embodiment of the present disclosure, the composition comprises compound B or a pharmaceutically acceptable salt thereof more preferably in an amount of less than about 2 % (n / n) based on the total combined amount of substance of compound A and compound B.

[0049] The inventors of the present disclosure have surprisingly found that compositions comprising compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 , or a pharmaceutically acceptable salt thereof, in a high purity, in particular in high excess of compound A over the epimer compound B (D-Cys38-Si-544) comprising or consisting of an amino acid sequence of SEQ ID No.: 2, or a pharmaceutically acceptable salt thereof, can be prepared. Said compositions overcome the need for epimer-pure compositions comprising compound A and, hence, enable studying compound A, that is a highly potent and selective inhibitor of Kv1 .3, in clinical trials.

[0050] According to a preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound A or a pharmaceutically acceptable salt thereof in an amount of from about 95 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound A and compound B. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound A or a pharmaceutically acceptable salt thereof in an amount of from about 97 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound A and compound B. According to a further preferred embodiment of the present disclosure, the composition comprises compound A or a pharmaceutically acceptable salt thereof in an amount of from about 98 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound A and compound B.

[0051] According to a preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 % (n / n) to about 5 % (n / n) based in the total combined amount of substance of compound A and compound B. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 % (n / n) to about 3 % (n / n) based in the total combined amount of substance of compound A and compound B. According to a further preferred, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 % (n / n) to about 2 % (n / n) based in the total combined amount of substance of compound A and compound B.

[0052] The above disclosed ratio of the amounts of compound A and compound B comprised in the composition according to the first aspect of the present invention may also be expressed as a weight ratio. In this, it is understood that the anion of a pharmaceutical salt of compound A is the same as the anion of a pharmaceutical salt of compound B.

[0053] In a further embodiment of the first aspect, the present invention relates to a composition comprising a) a compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 , or a pharmaceutically acceptable salt thereof; and b) a compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2, or a pharmaceutically acceptable salt thereof; characterized in that c) the composition comprises compound A in an amount of at least about 95 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to a preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound A or a pharmaceutically acceptable salt thereof in an amount of at least about 97 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound A or a pharmaceutically acceptable salt thereof in an amount of at least about 98 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to a further preferred embodiment of the present disclosure, the composition comprises compound A or a pharmaceutically acceptable salt thereof more preferably in an amount of more than about 98 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof.

[0054] According to a preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of about 5 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of about 3 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of about 2 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to a further preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof more preferably in an amount of less than about 2 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof.

[0055] According to a preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound A or a pharmaceutically acceptable salt thereof in an amount of from about 95 wt.-% to about 99.5 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound A or a pharmaceutically acceptable salt thereof in an amount of from about 97 wt.-% to about 99.5 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to a further preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound A or a pharmaceutically acceptable salt thereof in an amount of from about 98 wt.-% to about 99.5 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof.

[0056] According to a preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 wt.-% to about 5 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 wt.-% to about 3 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof. According to a further preferred embodiment, the composition according to the first aspect of the present disclosure comprises compound B or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 wt.-% to about 2 wt.-% based on the total combined weight of compound A or the pharmaceutically acceptable salt thereof and compound B or the pharmaceutically acceptable salt thereof.

[0057] The inventors of the present disclosure have surprisingly found that compositions comprising compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 , or a pharmaceutically acceptable salt thereof, in a high purity, in particular in high excess of compound A over the epimer compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2, or a pharmaceutically acceptable salt thereof, can be prepared. Said compositions overcome the need for epimer-pure compositions of compound A and, hence, enable studying compound A (e.g., Si-544), that is a highly potent and selective inhibitor of Kv1 .3, in clinical trials.

[0058] In that, the composition according to the first aspect of the present disclosure may be prepared in a convenient manner by solid-phase peptide synthesis (SPPS) which is often preferred in the pharmaceutical industry over recombinant methods for several reasons as explained herein.

[0059] Therefore, the composition of the first aspect of the present disclosure solves the object of providing a composition comprising the highly potent and highly selective Kv1.3 inhibitor compound A (e.g., Si-544) with high purity including high enantiomeric purity and high epimeric excess by convenient and scalable SPPS. Thus, compound A (e.g., Si-544 can now be studied in clinical trials and — pending the outcome of such trials — advanced to regulatory approval.

[0060] Composition prepared by solid-phase peptide synthesis

[0061] According to a second aspect, the present invention relates to a composition of the first aspect of the present invention that was prepared by solid -phase peptide synthesis (SPPS).

[0062] It is understood that the composition according to the second aspect of the present invention is characterized by the same features as the composition of the first aspect of the present invention. In particular, the composition according to the second aspect of the invention comprises, preferably consists of, the same components in the same amounts as the composition according to the first aspect of the present disclosure.

[0063] Method of preparation of linear and folded peptides

[0064] According to a third aspect, the present invention relates to a solid-phase peptide synthesis method of preparing the composition according to the first aspect of the present invention, the method comprising the steps of a) providing a solid resin having Fmoc- L-Cys(PGI) bound to said solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-L-Cys(PGI ) bound to said resin and washing to provide H-L-Cys(PGI) bound to said resin; c) subsequent coupling, by stepwise solid-phase peptide synthesis, each 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; or building blocks consisting of said amino acids, wherein each of said amino acids optionally comprises a side-chain protection group, and wherein each of said subsequent couplings comprises adding a respective Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing to obtain a peptide bound to said solid resin; d) cleaving a bond between said solid resin and Cys(PG1 ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain a composition comprising compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3 and compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4; and e) performing an oxidizing treatment to obtain the composition according to the first aspect of the present invention.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] Therefore, the inventors of the present disclosure have extensively investigated into methods of preparing compound A as disclosed herein in high purity, in particular with a high diastereomeric excess or epimeric excess, by SPPS.

[0072] 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.

[0073] 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.

[0074] The inventors of the present disclosure have surprisingly found that the temperature has a decisive influence on the epimerization of Gly38during preparation of a peptide. As shown in example 3, the amount of compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 (such as D-Cys38-Si-544-linear) is considerably increased when the steps of SPPS are carried out under suboptimal conditions of temperature of 35 °C.

[0075] Providing a solid resin having Fmoc-L-Cys(PGI) bound to said solid resin

[0076] In step a) of the method according to the third aspect of the present invention and / or according to the sixths aspect of the present invention, a solid resin having Fmoc- L-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-L-Cys(PG1 ) bound to a solid resin, an Fmoc-L-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.

[0077] 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.

[0078] The solid resin as used in the method according to the third aspect of the present disclosure and / or according to the sixths 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.

[0079] 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.

[0080] 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. As is shown in Examples 1 to 3, the inventors of the present disclosure have surprisingly found that compound A and compound C can be advantageously synthesized on and cleaved from a 2-chlorotrityl chloride resin with low formation of the respective D-Cys38epimer, i.e. compound B and compound D.

[0081] 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.

[0082] 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).

[0083] 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).

[0084] 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).

[0085] 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).

[0086] It is understood that any protective group of the linker is removed before coupling with the Fmoc-L-Cys(PG1 )-OH. Fmoc-L-Cys(PG1 )-OH is commercially available.

[0087] 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.

[0088] 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 tetrahydropyranyl (Thp). According to a further preferred embodiment of the present disclosure, PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt).

[0089] As shown in examples 1 and 2, the inventors of the present disclosure have surprisingly found that the compound A as disclosed herein can be prepared with high purity when using any one of the PG1 as disclosed herein. This is particularly true when using 4-methoxyphenyl)diphenylmethyl (Mmt) or (4-methoxyphenyl)diphenylmethyl (Mmt), and tetrahydropyranyl (Thp) as PG1 .

[0090] In other words, in a preferred embodiment of the present disclosure, the first step of the method according to the third aspect of the present disclosure and / or according to the sixths aspect of the present disclosure is providing a solid resin having Fmoc-L-Cys(Trt) bound to said solid resin, or having Fmoc-L-Cys(Mmt) bound to said solid resin, or having Fmoc-L-Cys(Thp) bound to said solid resin.

[0091] Each of said resins having Fmoc-L-Cys(PGI ) bound to said solid resin may be prepared from commercially available resins, such as resins disclosed herein, and commercially available Fmoc-L-Cys(PG1 )-OH, such as Fmoc-L-Cys(Thp)-OH, Fmoc- L-Cys(Mmt)-OH, or Fmoc-L-Cys(Trt)-OH.

[0092] As used herein, the solid resin having Fmoc-L-Cys(PGI ) bound to said solid resin comprises less than 0.5 % (n / n), preferably less than 0.3 % (n / n) of solid resin having Fmoc- D-Cys(PGI) bound to said solid resin, based on the total amount of solid resin having Fmoc- Cys(PG1) bound to said solid resin.

[0093] Fmoc-L-Cys(Thp)-OH is also known under CAS number 1673576-83-4. Fmoc- L-Cys(Mmt)-OH is also known under CAS number 177582-21-7. Fmoc-L-Cys(Trt)-OH is also known under CAS number 103213-32-7.

[0094] Fmoc-deprotection

[0095] In step b) of the method according to the third aspect of the present invention and / or according to the sixths aspect of the present invention, a Fmoc-deprotection from the Fmoc- L-Cys(PGI) bound to said solid resin provided in the first step and washing is performed to provide H-L-Cys(PGI ) bound to said solid resin.

[0096] 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.

[0097] As shown in examples 1 to 3, the inventors of the present disclosure have surprisingly found that the reaction conditions during Fmoc-deprotection contribute to the epimerization of Cys38thereby forming a D-Cys. However, if the amount of piperidine in the de-protections solution is less than about 20 % (VAX) based on the total volume of the de-protection solution, epimerization can be substantially reduced.

[0098] In particular, if PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt) or tetrahydropyranyl (Thp), the presence of high amounts of piperidine in the de-protection solution does not increase epimerization as shown in example 1 .

[0099] According to another preferred embodiment of the present disclosure, all Fmoc- deprotection steps are performed using a de-protection solution comprising piperidine (Pip) and DMF, wherein the concentration of piperidine is in the range of from about 5 % (VAX) to about 20 % (V / V) based on the total volume of the de-protection solution. According to another preferred embodiment of the present disclosure, all Fmoc-deprotection steps are 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 15 % (V / V) based on the total volume of the de-protection solution. According to another preferred embodiment of the present disclosure, all Fmoc-deprotection steps are 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, all Fmoc-deprotection steps are 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 10 % (V / V) based on the total volume of the de-protection solution. According to a yet further preferred embodiment of the present disclosure, all Fmoc-deprotection steps are 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.

[0100] According to another preferred embodiment of the present disclosure, if PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt) or tetrahydropyranyl (Thp), the 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 30 % (V / V) based on the total volume of the de-protection solution. According to another preferred embodiment of the present disclosure, if PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt) or tetrahydropyranyl (Thp), 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 25 % (V / V) based on the total volume of the de-protection solution. According to another preferred embodiment of the present disclosure, if PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt) or tetrahydropyranyl (Thp), 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 % (VAX) to about 20 % (V / V) based on the total volume of the de-protection solution. According to a further preferred embodiment of the present disclosure, if PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt) or tetrahydropyranyl (Thp), 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 15 % (V / V) based on the total volume of the de-protection solution. According to a yet further preferred embodiment of the present disclosure, if PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt) or tetrahydropyranyl (Thp), 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 deprotection solution.

[0101] According to another preferred embodiment of the present disclosure, if PG1 is triphenylmethyl (Trt), 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, if PG1 is triphenylmethyl (Trt), 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, if PG1 is triphenylmethyl (Trt), 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, if PG1 is triphenylmethyl (Trt), 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, if PG1 is triphenylmethyl (Trt), 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. The inventors of the present disclosure have surprisingly found that when PG1 is Trt, epimerization can be substantially reduced by reducing the amount of piperidine in the deprotection solution as shown in example 1.

[0102] As shown in example 1 , the epimerization of cysteine from L-Cys to D-Cys can be substantially reduced when adjusting the concentration of piperidine in the de-protection solution in accordance with the above ranges.

[0103] Stepwise solid-phase peptide synthesis

[0104] In step c) of the method according to the third aspect of the present invention and / or according to the sixths aspect of the present invention, a stepwise solid-phase peptide synthesis is performed, in which the further amino acids forming compound A, compound B, compound C, 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.

[0105] 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 A and / or compound C. 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 A, compound B, compound C, and / or compound D.

[0106] In other words, the building blocks as disclosed herein may be any fragment of compound A, and / or compound C.

[0107] Additionally, it is understood that each of the coupling stages comprises adding a respective Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing.

[0108] 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.

[0109] According to a preferred embodiment of the present disclosure, each of the Fmoc- deprotection stages in step c) of the method according to the third aspect of the present disclosure and / or the method according to the sixths aspect of the present disclosure is performed as described in step b) of the method of the third aspect of the present disclosure and / or the method according to the sixths aspect of the present disclosure.

[0110] As shown in example 1 , the inventors of the present disclosure have surprisingly found that the reaction conditions during Fmoc-deprotection contribute to the epimerization of Cys38thereby forming a D-Cys. However, 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, epimerization can be substantially reduced.

[0111] 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.

[0112] As shown in example 1 and 2, the epimerization of cysteine from L-Cys to D-Cys can be substantially reduced when adjusting the concentration of piperidine in the de-protection solution in accordance with the above ranges.

[0113] 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 optionally contains a side-chain protection group.

[0114] In other words, according to a preferred embodiment of the present disclosure, the method according to the third aspect of the present disclosure and / or the method according to the sixths aspect of the present disclosure comprises the following stages of the stepwise solid-phase peptide synthesis:

[0115] According to a preferred embodiment of the present disclosure, in a step c1 ), Fmoc- Arg(PG2)-OH is added to the H-L-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. 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).

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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).

[0121] According to another preferred embodiment of the present disclosure, Fmoc- Asn(PG3)-OH is Fmoc-Asn(Boc)-OH.

[0122] According to another preferred embodiment of the present disclosure, Fmoc- Asn(PG3)-OH is Fmoc-Asn(Acm)-OH.

[0123] 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.

[0124] 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.

[0125] 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).

[0126] 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. 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.

[0127] 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.

[0128] 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.

[0129] 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).

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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 .

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] According to a preferred embodiment of the present disclosure, PG5 is tert-butyl (tBu) or trityl (Trt) or 2-chlorotrityl (2-CITrt).

[0143] 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.

[0144] 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.

[0145] 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.

[0146] According to a preferred embodiment of the present disclosure, in a step c14), Fmoc- Pro-OH added to the 13thintermediate provided in step d 3) 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. 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.

[0147] According to a preferred embodiment of the present disclosure, in a step c16), Fmoc- Gly-OH added to the 15thintermediate provided in step c15) 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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. 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.

[0155] 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.

[0156] 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 .

[0157] 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.

[0158] 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.

[0159] According to a preferred embodiment of the present disclosure, in a step c29), Fmoc- Ser(PG6)-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.

[0160] According to a preferred embodiment of the present disclosure, PG6 is tert-butyl (tBu) or trityl (Trt).

[0161] According to another preferred embodiment of the present disclosure Fmoc- Ser(PG6)-OH is Fmoc-Ser(tBu)-OH. Fmoc-Ser(tBu)-OH is also known under CAS number 71989-33-8.

[0162] According to another preferred embodiment of the present disclosure Fmoc- Ser(PG6)-OH is Fmoc-Ser(Trt)-OH. Fmoc-Ser(Trt)-OH is also known under CAS number 111061-56-4.

[0163] 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. 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] Optionally, further amino acids may be coupled to the 37thintermediate provided in step c37).

[0171] 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.

[0172] It is understood that all Fmoc-deprotection steps as disclosed herein are performed under the conditions as disclosed under step b) as disclosed herein. Releasinq the peptide from the resin and deprotection

[0173] In step d) of the method according to the third aspect of the present invention and / or according to the sixths aspect of the present invention, the peptide is released from the solid resin by cleaving a bond between said solid resin and Cys(PG1 ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain a composition comprising compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3 and 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 third aspect of the present invention and / or according to the sixths aspect of the present invention yields a composition according to the fourth aspect of the present disclosure and / or the fifth aspect of the present disclosure.

[0174] 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 Cys38and the solid resin. In other words, the solid resin is adapted in that the bond between Cys38and the solid resin is hydrolyzed upon addition of TFA.

[0175] Furthermore, it is a preferred embodiment of the present disclosure that all side chain protection groups, in particular PG1 , PG2, PG3, PG4, PG5, PG6, Trt, and tBu-groups, are cleaved off the respective amino acids upon addition of trifluoroacetic acid (TFA).

[0176] The inventors of the present disclosure have surprisingly found that a composition comprising a high excess of compound C comprising or consisting of an amino acid SEQ ID No.: 3 (such as Si-544-linear) over compound D comprising or consisting of an amino acid SEQ ID No.: 4 (such as D-Cys38-Si-544-linear) can thus be obtained. Thereby, Cys38was not susceptible to epimerization during step d).

[0177] Hence, the inventors of the present disclosure have surprisingly found that the epimerization of Cys38can be efficiently prevented even under standard conditions.

[0178] According to another preferred embodiment of the present disclosure, sted d) further comprises a step of purifying the composition comprising compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3 and compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4.

[0179] 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). According to another preferred embodiment of the present disclosure, the step of purifying further comprises a lyophilizing step to yield a purified composition comprising compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3 and compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4. Oxidizing treatment

[0180] In step e) of the method according to the third aspect of the present invention, the composition comprising compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3 and 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 third aspect of the present disclosure is subjected to an oxidizing treatment to obtain the composition according to the first aspect of the present disclosure. In other words, step e) of the method according to the third aspect of the present invention comprises performing an oxidizing treatment to yield a composition according to the first aspect of the present disclosure.

[0181] 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.

[0182] In other words, compound C comprising or consisting of an amino acid of SEQ ID No.: 3 is reacted to compound A comprising or consisting of an amino acid of SEQ ID No. 1 . Compound D comprising or consisting of an amino acid of SEQ ID No.: 4 is reacted to compound B comprising or consisting of an amino acid of SEQ ID No. 2.

[0183] According to a preferred embodiment of the present disclosure, the oxidizing treatment in step e) of the method according to the third aspect of the present disclosure is performed using a mixture of cysteine and cystine.

[0184] Adding a mixture of cysteine and cystine 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.

[0185] The concentrations and ratio of cysteine to cystine are not particularly limited.

[0186] According to another preferred embodiment of the present disclosure, step e) further comprises a step of purifying the composition according to the first aspect of the present disclosure comprising compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 and compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2.

[0187] 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). According to another preferred embodiment of the present disclosure, the step of purifying further comprises a lyophilizing step to yield a composition according to the first aspect of the present disclosure comprising compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 and compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2.

[0188] According to another preferred embodiment of the present disclosure, the step of purifying further comprises a step of ion exchange to yield a composition according to the first aspect of the present disclosure comprising a pharmaceutically acceptable salt of compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 and a pharmaceutical acceptable salt of 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.

[0189] According to another preferred embodiment of the present disclosure, step e) of the method according to the third aspect of the present disclosure is a random oxidizing treatment mediated by cysteine / cystine.

[0190] Further steps

[0191] According to another preferred embodiment of the present disclosure, step d) further comprises a step of purifying the composition comprising compound C and compound D.

[0192] 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 composition comprising compound C and compound D.

[0193] According to another preferred embodiment of the present disclosure, the step of purifying further comprises a lyophilizing step to yield a lyophilized purified composition comprising compound C and compound D.

[0194] According to another preferred embodiment of the present disclosure, step e) further comprises a step of purifying the composition comprising compound A and compound B.

[0195] 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 composition comprising compound A and compound B.

[0196] According to another preferred embodiment of the present disclosure, the step of purifying further comprises a lyophilizing step to yield a lyophilized purified composition comprising compound A and compound B.

[0197] According to another preferred embodiment of the present disclosure, the step e) further comprises a step of ion exchange to yield a composition comprising a pharmaceutically acceptable salt of compound A and a pharmaceutically acceptable salt of compound B. Composition comprising linear peptides

[0198] According to a fourth aspect, the present invention relates to a composition comprising a) compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3 (such as, Si-544-linear) or a pharmaceutically acceptable salt thereof; and b) compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4 (D-Cys38-Si-544-linear), or a pharmaceutically acceptable salt thereof; characterized in that c) the composition comprises compound C in an amount of at least about 95 % (n / n) based on the total combined amount of substance of compound C and compound D.

[0199] It is understood that the composition according to the fourth aspect of the present invention is an important intermediate in the preparation of the composition according to the first aspect of the present disclosure. However, the composition of the fourth aspect of the present disclosure is different from the composition of the first aspect of the present disclosure in that compounds C and D comprised in the composition according to the third aspect of the present disclosure do not have intramolecular disulfide bonds.

[0200] In other words, the present invention in a fourth aspect relates to a composition comprising a) compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3, wherein Cys38is L-cysteine, and wherein compound C has no intramolecular disulfide bonds, or a pharmaceutically acceptable salt thereof; and b) 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; characterized in that c) the composition comprises compound C in an amount of at least about 95 % (n / n) based on the total combined amount of substance of compound C and compound D.

[0201] The inventors of the present disclosure have surprisingly found that Cys38of compound C according to the present disclosure may be prone to epimerization during preparation, that is the interconversion of one epimer to the other epimer (compound D). This is particularly true if compound C is to be prepared by solid-phase peptide synthesis: It is understood the Cys38is the “first” amino acid that is bound to a solid resin for the synthesis. Thus, Cys38not only experiences a high chemical stress during all subsequent coupling and de-protection steps, but must also be cleaved from the resin in the last step under harsh conditions.

[0202] However, it is of utmost importance to provide epimer-pure compounds for clinical trials for several the same reasons as discussed with regard to the first aspect of the present disclosure.

[0203] According to a preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of at least about 97 % (n / n) based on the total combined amount of substance of compound C and compound D. According to another preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of at least about 98 % (n / n) based on the total combined amount of substance of compound C and compound D. According to a further preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof more preferably in an amount of more than about 98 % (n / n) based on the total combined amount of substance of compound C and compound D.

[0204] According to a preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of about 5 % (n / n) or less based on the total combined amount of substance of compound C and compound D. According to another, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of about 3 % (n / n) or less based on the total combined amount of substance of compound C and compound D. According to another preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of about 2 % (n / n) or less based on the total combined amount of substance of compound C and compound D. According to a further preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof more preferably in an amount of less than about 2 % (n / n) based on the total combined amount of substance of compound C and compound D.

[0205] The inventors of the present disclosure have surprisingly found that compositions comprising compound C (such as, Si-544-1 in ear) in a high purity, in particular in high excess of compound C (such as Si-544-linear) over the epimer compound D (such as D-Cys38-Si-544-linear) can be prepared. Said compositions overcome the need for epimer- pure compositions of compound C and, hence, enable studying compound A (such as Si-544), that is a highly potent and selective inhibitor of Kv1 .3, in clinical trials.

[0206] According to a preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of from about 95 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound C and compound D. According to another preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of from about 97 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound C and compound D. According to a further preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of from about 98 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound C and compound D.

[0207] According to a preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 % (n / n) to about 5 % (n / n) based in the total combined amount of substance of compound C and compound D. According to another preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 % (n / n) to about 3 % (n / n) based in the total combined amount of substance of compound C and compound D. According to a further preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 % (n / n) to about 2 % (n / n) based in the total combined amount of substance of compound C and compound D.

[0208] The above disclosed ratio of the amounts of compound C and compound D comprised in the composition according to the fourth aspect of the present disclosure may also be expressed as a weight ratio. In this, it is understood that the anion of a pharmaceutical salt of compound C is the same as the anion of a pharmaceutical salt of compound D.

[0209] In a further embodiment of the fourth aspect, the present invention relates to a composition comprising a) Compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3 (such as “Si-544-linear”) or a pharmaceutically acceptable salt thereof; and b) 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; characterized in that c) the composition comprises compound C in an amount of at least about 95 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof.

[0210] According to a preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of at least about 97 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of at least about 98 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof. According to a further preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof more preferably in an amount of more than about 98 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof.

[0211] According to a preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of about 5 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of about 3 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of about 2 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof. According to a further preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof more preferably in an amount of less than about 2 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof.

[0212] According to a preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of from about 95 wt.-% to about 99.5 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of from about 97 wt.-% to about 99.5 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof. According to a further preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound C or a pharmaceutically acceptable salt thereof in an amount of from about 98 wt.-% to about 99.5 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof.

[0213] According to a preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 wt.-% to about 5 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof. According to another preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 wt.-% to about 3 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof. According to a further preferred embodiment, the composition according to the fourth aspect of the present disclosure comprises compound D or a pharmaceutically acceptable salt thereof in an amount of from about 0.5 wt.-% to about 2 wt.-% based on the total combined weight of compound C or the pharmaceutically acceptable salt thereof and compound D or the pharmaceutically acceptable salt thereof.

[0214] The inventors of the present disclosure have surprisingly found that compositions comprising compound C (such as Si-544-1 in ear) in a high purity, in particular in high excess of compound C (such as Si-544-linear) over the epimer compound D (such as D-Cys38-Si-544-linear) can be prepared. Said compositions overcome the need for epimer- pure compositions of compound C and, hence, enable studying compound A (such as Si-544), that is a highly potent and selective inhibitor of Kv1 .3, in clinical trials.

[0215] In that, the composition according to the fourth aspect of the present disclosure may be prepared in a convenient manner by solid-phase peptide synthesis (SPPS) which is often preferred in the pharmaceutical industry over recombinant methods for several reasons as explained herein.

[0216] Therefore, the composition of the first aspect of the present disclosure solves the object of providing a composition comprising the highly potent and highly selective Kv1 .3 inhibitor compound A (such as si-544) with high purity including high enantiomeric purity and high epimeric excess by convenient and scalable SPPS. Thus, compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 (such as si-544) can now be studied in clinical trials and — pending the outcome of such trials — advanced to regulatory approval.

[0217] Composition comprising linear peptides prepared by SPPS

[0218] According to a fifth aspect, the present invention relates to a composition of the fourth aspect of the present invention that was prepared by solid-phase peptide synthesis (SPPS). It is understood that the composition according to the fifth aspect of the present invention is characterized by the same features as the composition of the fourth aspect of the present invention. In particular, the composition according to the fifth aspect of the invention comprises, preferably consists of, the same components in the same amounts as the composition according to the fourth aspect of the present disclosure.

[0219] Method of preparation of linear peptides

[0220] According to a sixths aspect, the present invention relates to a solid-phase peptide synthesis method of preparing the composition according to the fourth aspect of the present invention, the method comprising the steps of a) providing a solid resin having Fmoc- L-Cys(PGI) bound to said solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-L-Cys(PGI ) bound to said resin and washing to provide H-L-Cys(PGI) bound to said resin; c) subsequent coupling, by stepwise solid-phase peptide synthesis, each 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; or building blocks consisting of said amino acids, wherein each of said amino acids optionally comprises a side-chain protection group, and wherein each of said subsequent couplings comprises adding a respective Fmoc-protected amino acid, washing, Fmoc-deprotection, and washing to obtain a peptide bound to said solid resin; d) cleaving a bond between said solid resin and Cys(PG1 ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain a composition according to the fourth aspect of the present invention.

[0221] It is understood that the method according to the sixths aspect of the present invention is a method for preparing the important intermediate according to the fourth aspect of the present disclosure. However, the method of the sixths aspect of the present disclosure is different from the method of the third aspect of the present disclosure in that the method of the sixths aspect of the present disclosure does not comprise a method step of performing an oxidizing treatment.

[0222] Therefore, it is understood that all features discussed under steps a), b), c), and d) of the method according to the third aspect of the present disclosure are the same for the method according to the sixths aspect of the present disclosure. In other words, steps a), b), c), and d) of the method according to the sixths 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 third aspect of the present disclosure. Composition prepared by method of third aspect or method of sixths aspect

[0223] According to a seventh aspect, the present invention relates to a composition prepared by the method according to the third aspect of the present invention, or prepared by the method of the sixths aspect of the present invention.

[0224] It is understood that the compositions according to the seventh aspect of the present invention are characterized by the same features as the composition of the first and second, or fourth and fifth aspect of the present invention, respectively. In particular, the composition according to the seventh aspect of the invention comprises, preferably consists of, the same components in the same amounts as the composition according to the first and second aspect of the present disclosure or the fourth and fifth aspect of the present disclosure, respectively.

[0225] Pharmaceutical composition

[0226] According to an eighth aspect, the present invention relates to a pharmaceutical composition comprising a composition according to the present invention, in particular a composition according to the first, second, fourth, fifth, or seventh aspect of the present invention.

[0227] It is understood that the pharmaceutical compositions according to the eights aspect of the present disclosure solves the object of providing a pharmaceutical composition comprising the highly potent and highly selective Kv1 .3 inhibitor compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 (such as Si-544) with high purity including high enantiomeric purity and high epimeric excess by convenient and scalable SPPS. Thus, compound A (such as Si-544) can now be studied in clinical trials and — pending the outcome of such trials — advanced to regulatory approval.

[0228] According to a preferred embodiment, the pharmaceutical composition according to the eighth aspect of the present disclosure is suitable for oral, rectal, nasal, sublingual, percutaneous, topic, intravenous, or intramuscular administration.

[0229] According to a preferred embodiment, the pharmaceutical composition according to the eighth 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.

[0230] According to a preferred embodiment, the pharmaceutical composition according to the eighth aspect of the present disclosure is suitable for rectal administration, for example in the form of a suppository.

[0231] According to a preferred embodiment, the pharmaceutical composition according to the eighth 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 eighth 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.

[0232] According to a preferred embodiment, the pharmaceutical composition according to the eighth aspect of the present disclosure is suitable for intranasal application. Thus, the pharmaceutical composition according to the eighth 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.

[0233] According to a preferred embodiment, the pharmaceutical composition according to the eighth aspect of the present disclosure is suitable for sublingual application. Thus, the pharmaceutical composition according to the eighth 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.

[0234] According to a preferred embodiment, the pharmaceutical composition according to the eighth aspect of the present disclosure is suitable for percutaneous application. Thus, the pharmaceutical composition according to the eighth 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.

[0235] According to a preferred embodiment, the pharmaceutical composition according to the eighth aspect of the present disclosure is suitable for intravenous application. Thus, the pharmaceutical composition according to the eighth aspect of the present disclosure is preferably in the form of a solution, an emulsion, or a suspension.

[0236] According to a preferred embodiment, the pharmaceutical composition according to the eighth aspect of the present disclosure is suitable for intramuscular application. Thus, the pharmaceutical composition according to the eighth aspect of the present disclosure is preferably in the form of a solution, an emulsion, a suspension, or a depot-injection.

[0237] 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.

[0238] In some embodiments of the present disclosure, the pharmaceutical composition may be a sustained release formulation.

[0239] 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.

[0240] First medical use

[0241] According to a ninth aspect, the present invention relates to a composition according to the invention, in particular a composition according to the first, second, fourth, fifth, or seventh aspect of the present invention, or a pharmaceutical composition according to the eighth aspect of the invention for use in medicine.

[0242] Second medical use

[0243] According to a tenth aspect, the present invention relates to a composition according to the invention, in particular a composition according to the first, second, fourth, fifth, or seventh aspect of the present invention, or a pharmaceutical composition according to the eighth aspect of the invention for use in a method of treatment or prevention of an auto immune disease, diabetes, obesity, parodontitis and / or tissue transplant rejection.

[0244] 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.

[0245] In a particularly preferred embodiment the present invention relates to the use of a composition or pharmaceutical composition according to the invention or a pharmaceutical composition according to the invention for use in the treatment or prevention of an autoimmune disease.

[0246] 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

[0247] T cell mediated autoimmune disorder.

[0248] 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.

[0249] 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).

[0250] The same applies to obesity (Xu, Human Molecular Genetics, 2003, vol. 12, pp 551 ).

[0251] 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.

[0252] In 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.

[0253] Preferably the auto immune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, psoriasis, type-1 diabetes and vasculitis. Definitions and general embodiments

[0254] As used herein, the term “compound A” refers to a compound or peptide comprising or consisting of an amino acid sequence of SEQ ID No.: 1 that is represented by the following formula:

[0255] 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.

[0256] According to a further preferred embodiment of the present disclosure, the term “compound A” refers to a compound or peptide consisting of an amino acid sequence of SEQ ID No.: 1 . As used herein, the terms “compound A consisting of an amino acid sequence of SEQ ID No.: 1”, “Si-544”, and “cgtx-544” are used interchangeably and refer to a compound or peptide consisting of an amino acid sequence of SEQ ID No.: 1 .

[0257] As used herein, the term “compound B” refers to a peptide comprising or consisting of an amino acid sequence of SEQ ID No.: 2 that is represented by the following formula:

[0258] 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 D-Cys38.

[0259] According to a further preferred embodiment of the present disclosure, the term “compound B” refers to a compound or peptide consisting of an amino acid sequence of SEQ ID No.: 2. As used herein, the terms “compound B consisting of an amino acid sequence of SEQ ID No.: 2”, “D-Cys38-Si-544”, and “D-Cys38-cgtx-544" are used interchangeably and refer to a peptide consisting of an amino acid sequence of SEQ ID No.: 2.

[0260] Hence, it is understood that compound A and compound B as disclosed herein differ in the configuration of one amino, that is Cys38. Thus, compound A and compound B are epimers. Both compound A and compound B as disclosed herein have the same molecular formula C173H297N55O49S9 (net) and the same molecular mass of 4212.1 g / mol (net).

[0261] As used herein, the term “compound C” refers to a compound or peptide comprising or consisting of an amino acid sequence of 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.

[0262] According to a further preferred embodiment of the present disclosure, the term “compound C” refers to a compound or peptide consisting of an amino acid sequence of SEQ ID No.: 3. As used herein, the terms “compound C consisting of an amino acid sequence of SEQ ID No.: 3”, “Si-544-linear”, and “cgtx-544-linear” are used interchangeably and refer to a peptide consisting of an amino acid sequence of SEQ ID No.: 3.

[0263] As used herein, the term “compound D” refers to a compound or peptide comprising or consisting of an amino acid sequence of SEQ ID No.: 4 that is represented by the following formula:

[0264] 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 D has no intramolecular disulfide bonds.

[0265] According to a further preferred embodiment of the present disclosure, the term “compound D” refers to a compound or peptide consisting of an amino acid sequence of SEQ ID No.: 4. As used herein, the terms “compound D consisting of an amino acid sequence of SEQ ID No.: 4”, “D-Cys38-Si-544-linear”, and “D-Cys38-cgtx-544-linear" are used interchangeably and refer to a compound or peptide consisting of an amino acid sequence of SEQ ID No.: 4.

[0266] Hence, it is understood that compound C and compound D as disclosed herein differ in the configuration of one amino, that is Cys38. Thus, compound C and compound D are epimers. Both compound C and compound D as disclosed herein have the same molecular formula C173H297N55O49S9 (net) and the same molecular mass of 4220.1 g / mol (net).

[0267] As used herein, the term “fragment A” refers to a compound of formula H-Ans-Arg- Cys-OH, wherein Cys is L-Cys.

[0268] As used herein, the term “fragment B” refers to a compound of formula H-Ans-Arg- Cys-OH, wherein Cys is D-Cys.

[0269] As used herein, the term “pharmaceutically acceptable salt” refers to a salt of the respective compound that retains the desired biological activity of the respective compound and include pharmaceutically acceptable acid addition salts. Suitable pharmaceutically acceptable acid addition salts of the respective compound 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, trifluoro acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic, and arylsulfonic acid.

[0270] As used herein, any reference to an amino acid refers to the respective L-amino acid unless specified otherwise.

[0271] As used herein, the term “oxyma” relates to ethyl cyanohydroxyiminoacetate, also known under CAS number 3849-21-6.

[0272] As used herein, the term “DIC” relates to N,N- diisopropylcarbodiimide, also known under CAS number 693-13-0.

[0273] As used herein, the term “TBTU” relates to 2-(1 / 7-benzotriazole-1-yl)-1 ,1 ,3,3- tetramethylaminium tetrafluoroborate, also known under CAS number 125700-67-6.

[0274] As used herein, the term “DIPEA" relates to A / ,A / -diisopropylethylamine, also known as Hunig’s base and under CAS number 7087-68-5.

[0275] As used herein, the term “Fmoc-protected” refers to a compound in which at least one, preferably all, amine groups are protected with a fluorenylmethoxycarbonyl protection group. In other words, an Fmoc-protected compound comprises the following structural element:

[0276] 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 % (VA / ) 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).

[0277] 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.

[0278] As used herein, the term “room temperature” means 25 °C. 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.

[0279] Assay (I):

[0280] As used herein, the amount of compound A comprised in a composition according to the present invention is determined by Assay (I) comprising, preferably consisting of, the following steps:

[0281] A sample of 2.5 mg of a composition comprising compound A or a pharmaceutically acceptable salt thereof and compound B or a pharmaceutically acceptable salt thereof as disclosed herein is accurately weighed and dissolved in 1 mL of a 20 mM ammonium bicarbonate buffer adjusted to pH 7.0 to provide a test stock solution.

[0282] A solution of tris(2-carboxyethyl)phosphine (TCEP) at 1 mg / mL in 20 mM ammonium bicarbonate buffer adjusted to pH 7.0 is prepared to provide a TCEP solution.

[0283] A solution of a rLys-C endoproteinase in resuspension buffer is provided. Preferably, the rLys-C endoproteinase is adapted to hydrolyse peptide bonds of lysine in position 6, 11 , 18, 27, 32, and 34 on its C-terminal side; for example, Endoproteinase rLysC, Mass Spec Grade from Promega (catalog number V1671 ) may be advantageously used.

[0284] 225 pL of the TCEP solution, 200 pL of the test stock solution, and 560 pL of 20 mM ammonium bicarbonate buffer adjusted to pH 7.0 are mixed and added to 15 pL of a solution of rLys-C endoproteinase and mixed well. The resulting mixture is incubated for 60 min at 31 °C. For stopping the reaction, 5 pL TFA are added and mixed to provide a digested solution.

[0285] During digestion, the rLys-C endoproteinase cleaves compound A and compound B. Thereby, compound A forms a fragment of amino acids 35 to 38, i.e. a compound of formula H-Ans-Arg-Cys-OH, wherein Cys is L-Cys (herein also referred to as “fragment A”). Compound B forms a fragment of amino acids 35 to 38, i.e. a compound of formula H-Ans- Arg-Cys-OH, wherein Cys is D-Cys (herein also referred to as “fragment B”).

[0286] 20 pL of the digested solution are subjected to RP-HPLC as follows:

[0287] HPLC is performed on a RP C18 column, such as Waters Acquity HSS T3 C18, 1 .7 pm, 150 x 2.1 mm (Waters, Germany) at a column temperature of 40 °C, a flow rate of 0.3 mL / min with the following gradient profile as shown in Table 1 : Table 1 Gradient profile of RP-HPLC as used in Assay (I)

[0288] With the following Eluents A and B: Eluent A: 0.10 % (VAX) Pentafluoropropionic acid (PFPA) and 5 % (V / V) acetonitrile in water; Eluent B: 0.10 % (VAX) PFPA and 10 % (VAX) water in acetonitrile.

[0289] A chromatogram is detected at a wavelength of 220 nm.

[0290] The amount of fragment B is determined according to formula:

[0291] , , „ A (fragment B) amount of fragment B (% (n / n) = — - - r - 7- - * 100% (n / n)

[0292] 6A(fragment A) + A(fragment B)Jwith “A (fragment B)” being the peak area of fragment B in the chromatogram and “A (fragment A)” being the peak area of fragment A in the chromatogram.

[0293] It is understood that the ..amount of fragment B” is identical to the amount of compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2, or a pharmaceutically acceptable salt thereof in a composition as disclosed herein. Thus, the amount of compound B comprising or consisting of an amino acid sequence of SEQ ID

[0294] No.: 2, or a pharmaceutically acceptable salt thereof in a composition as disclosed herein may be determined according to formula (I)

[0295] A (fragment B) amount of compound B (% (n / n) = 100% (n / n)

[0296] A(fragment A)+A(fragment B) (I) with “A (fragment B)” being the peak area of fragment B in the chromatogram and

[0297] “A (fragment A)” being the peak area of fragment A in the chromatogram.

[0298] It is understood that the ..amount of fragment B" is identical to the amount of compound B comprising or consisting of an amino acid sequence of SEQ ID No.: 2, or a pharmaceutically acceptable salt thereof in a composition as disclosed herein. Thus, the amount of compound B comprising or consisting of an amino acid sequence of SEQ ID

[0299] No.: 2, or a pharmaceutically acceptable salt thereof in a composition as disclosed herein may be determined according to formula (II):

[0300] A (fragment B) amount of compound B (% (n / n) = 100% (n / n)

[0301] A(fragment A)+A(fragment B) (H) with “A (fragment B)” being the peak area of fragment B in the chromatogram and “A (fragment A)” being the peak area of fragment A in the chromatogram.

[0302] The amount of fragment A is determined according to formula (III): A (fragment A) amount of fragment A (% (n / n) = 100% (n / n)

[0303] A(fragment A)+A(fragment B) (HI) with “A (fragment B)” being the peak area of fragment B in the chromatogram and “A (fragment A)” being the peak area of fragment A in the chromatogram.

[0304] It is understood that the ..amount of fragment A” is identical to the amount of compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 , or a pharmaceutically acceptable salt thereof in a composition as disclosed herein. Thus, the amount of compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 , or a pharmaceutically acceptable salt thereof in a composition as disclosed herein may be determined according to formula (IV): amount of compound A (% (n / n) = — -A(frag A(fragment A)+mAe(nft (n / n) (IV) ragment B)v J' with “A (fragment B)” being the peak area of fragment B in the chromatogram and

[0305] “A (fragment A)” being the peak area of fragment A in the chromatogram.

[0306] In other words, Assay (I) is performed as follows: i) mixing a 200 pL of a solution of the composition comprising compound A or a pharmaceutically acceptable salt thereof and compound B or a pharmaceutically acceptable salt thereof as disclosed herein (2.5 mg / mL in 20 mM ammonium bicarbonate buffer, pH 7.0), 225 pL of TCEP solution (1 mg / mL in 20 mM ammonium bicarbonate buffer, pH 7.0), 560 pL of buffer (20 mM ammonium bicarbonate buffer, pH 7.0), and 15 pL of a solution of rCys-C endoproteinase to provide a reaction mixture; ii) incubating said reaction mixture for about 60 min at about 31 °C, followed by adding 5 pL of TFA to provide a digested solution; iii) subjecting 20 pL of the digested solution to a HPLC to determine a chromatogram, wherein the HPLC method is performed on a RP C18 column, such as Waters Acquity HSS T3 C18, 1.7 pm, 150 x 2.1 mm (Waters, Germany) at a column temperature of 40 °C, at a flow rate of 0.3 mL / min with the following gradient profile as shown in Table 1 and Eluents A and B as disclosed herein, wherein the chromatogram is detected at a wavelength of 220 nm; and iv) determining the peak area of fragment A and the peak area of fragment B in said chromatogram, and v) determining the amount of the amount of compound A comprised in a composition according to the present invention using formula (IV), and / or or determining the amount of the amount of compound B comprised in a composition according to the present invention using formula (II).

[0307] It is understood that Assay (I) as disclosed herein may be used to determine the amount of compound C or a pharmaceutically acceptable salt thereof and the amount of compound D or a pharmaceutically acceptable salt thereof comprised in a composition according to the present invention with the following modification:

[0308] A sample of 2.5 mg of a composition comprising compound C or a pharmaceutically acceptable salt thereof and compound D or a pharmaceutically acceptable salt thereof as disclosed herein is accurately weighed and dissolved in 1 mL of a 20 mM ammonium bicarbonate buffer adjusted to pH 7.0 to provide a test stock solution.

[0309] The ..amount of fragment A” is identical to the amount of compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3, or a pharmaceutically acceptable salt thereof in a composition as disclosed herein. Thus, the amount of compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3, or a pharmaceutically acceptable salt thereof in a composition as disclosed herein may be determined according to formula (V):

[0310] A (fragment A) amount of compound C (% (n / n) = 100% (n / n)

[0311] A(fragment A)+A(fragment B) (V)

[0312] The ..amount of fragment B” is identical to the amount of compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4, or a pharmaceutically acceptable salt thereof in a composition as disclosed herein. Thus, the amount of compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4, or a pharmaceutically acceptable salt thereof in a composition as disclosed herein may be determined according to formula (VI): amount of compound

[0313] In other words, Assay (I) is performed as follows: mixing a 200 pL of a solution of the composition comprising compound C or a pharmaceutically acceptable salt thereof and compound D or a pharmaceutically acceptable salt thereof as disclosed herein (2.5 mg / mL in 20 mM ammonium bicarbonate buffer, pH 7.0), 225 pL of TCEP solution (1 mg / mL in 20 mM ammonium bicarbonate buffer, pH 7.0), 560 pL of buffer (20 mM ammonium bicarbonate buffer, pH 7.0), and 15 pL of a solution of rCys-C endoproteinase to provide a reaction mixture; incubating said reaction mixture for about 60 min at about 31 °C, followed by adding 5 pL of TFA to provide a digested solution; subjecting 20 pL of the digested solution to a HPLC to determine a chromatogram, wherein the HPLC method is performed on a RP C18 column, such as Waters Acquity HSS T3 C18, 1.7 pm, 150 x 2.1 mm (Waters, Germany) at a column temperature of 40 °C, at a flow rate of 0.3 mL / min with the following gradient profile as shown in Table 1 and Eluents A and B as disclosed herein, wherein the chromatogram is detected at a wavelength of 220 nm; and iv) determining the peak area of fragment A and the peak area of fragment B in said chromatogram, and v) determining the amount of the amount of compound C comprised in a composition according to the present invention using formula (V), and / or or determining the amount of the amount of compound D comprised in a composition according to the present invention using formula (VI).

[0314] Examples

[0315] 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.

[0316] All chemicals and materials used herein were obtained from commercial suppliers unless stated otherwise. All reactions were carried out at room temperature unless indicated otherwise.

[0317] Example 1 : Preparation of a composition comprising linear peptides

[0318] A composition comprising Si-544-linear (compound C) and D-Cys38-Si-544-linear (compound D) was prepared according to the method as shown in Fig. 1.

[0319] For that, a Fmoc-Cys(PG1)-OH was coupled to a 2-chlorotirtyl chloride resin in the presence of DIC / Oxyma or TBTU / DIPEA, wherein PG1 is given in in Table 2. Subsequently, the Fmoc-protection group was removed in accordance with the conditions given in Table 2, and the resulting Cys.

[0320] Table 2 PG1 and conditions of Fmoc deprotection solutions

[0321] 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 removed under the conditions indicated in Table 2 revealing a new N-terminal amine to which a further amino acid may be attached, followed by washing.

[0322] After subsequently coupling all 38 amino acids forming the peptide of SEQ ID No. 3, the peptide was cleaved from the solid resin and all side-chain protection groups were - M - removed by treatment with trifluoroacetic acid (TFA). The crude products were 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.

[0323] The linear peptides were obtained with a purity of > 90 % determined by RP-HPLC.

[0324] Example 2: Oxidative treatment and determination of amount of D-Cys38epimer

[0325] The linear peptides as prepared in Example 1 were subjected to an oxidation treatment by adding a mixture of cysteine and cysteine for forming disulfide bonds and folding.

[0326] After oxidation treatment, the crude peptides were purified by preparative RP-HPLC as described in Example 1 . The resulting fractions were subjected to lyophilization. The folded peptides were obtained with a purity of > 95 % determined by RP-HPLC.

[0327] Then, the content of compound B was determined according to assay (I) as disclosed herein:

[0328] Table 3 Amount of compound B determined after preparation

[0329] The results show that all methods yield a composition comprising only low amounts of compound B that is an impurity resulting from epimerization of Cys38during preparation.

[0330] In particular, reducing the amount of piperidine in the Fmoc-deprotection solution to 10 % (VAX) and / or using Mmt as side chain protection group during synthesis reduces the undesired epimerization of Cys38.

[0331] Example 3: Confirmation experiment at elevated temperature and determination of amount of D-Cys38epimer

[0332] As a confirmation for the robustness of the method, the synthesis of linear peptides was repeated as described in example 1 with the following modifications: PG1 was 4-(Methoxyphenyl)diphenylmethyl (Mmt), the Fmoc-deprotection solution was 10 % (V / V) of piperidine in DMF, and all steps were carried out at 35 °C.

[0333] The content of compound D in the purified linear peptide thus obtained as determined according to assay (I) as disclosed herein was found to be 2.8 % (n / n). This final experiment confirmed that the adapted manufacturing parameters (10 % (V / V) Pip / DMF instead of 20 % (V / V) Pip / DMF, and using Cys(Mmt) for Cys38) also result in a low amount of D-Cys38- epimer compound D under harsh SPPS conditions (35 °C). Thus, the method is robust. Example 4: Scalability

[0334] A composition comprising Si-544-linear (compound C) and D-Cys38-Si-544-linear (compound D) was prepared with standard SPPC. The batch size was adjusted to yield about 60 g of the composition.

[0335] In essence, a peptide synthesis was carried out on an insoluble 2-CT resin having a L-Cys(Trt) or L-Cys(Mmt) bound to said resin. For subsequent couplings, an F-moc strategy was used.

[0336] The free N-terminal amine of a solid-phase attached peptide was coupled to a single N-protected amino acid unit. This unit is then deprotected (F-moc groups are cleaved at basic pH by piperidine in DMF as shown in Table 4), revealing a new N-terminal amine to which a further amino acid may be attached.

[0337] Side-chain protection groups were cleaved after the peptide synthesis is finished by treatment with TFA (trifluoroacetic acid), thereby also cleaving the peptide from the resin. Table 4 PG1 and conditions of Fmoc deprotection solutions

[0338] After completion of peptide synthesis the peptide is deprotected and cleaved from the polystyrol resin. At this point the raw peptide product displays a purity of about 50% followed by analytical UPLC and LC-MS. In a subsequent step the raw peptide is purified by preparative HPLC resulting in a linear peptide product with a purity of > 90%.

[0339] The linear peptides thus obtained were subjected to an oxidation treatment by adding a mixture of cysteine and cysteine for forming disulfide bonds and folding. After oxidation treatment, the crude peptides were purified by preparative RP-HPLC as described in Example 1. The resulting fractions were subjected to lyophilization. The folded peptides were obtained with a purity of > 95 % determined by RP-HPLC.

[0340] Then, the content of compound B was determined according to assay (I) as disclosed herein.

[0341] Table 5 Amount of compound B determined after preparation

[0342] The results show that the composition comprises only low amounts of compound B when using Mmt as PG1 and / or low amount of piperidine of 10 % (V / V) during Fmoc deprotection. The method according to the present disclosure is also scalable and can be used in the preparation of large batches.

Claims

Claims1 . A composition comprising a) a compound A comprising or consisting of an amino acid sequence of SEQ ID No.: 1 , 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 L-Cys38, or a pharmaceutically acceptable salt thereof; and b) a 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 D-Cys38, or a pharmaceutically acceptable salt thereof; characterized in that c) the composition comprises compound A in an amount of at least about 95 % (n / n) based on the total combined amount of substance of compound A and compound B.

2. The composition according to claim 1 , wherein the composition comprises compound A or a pharmaceutically acceptable salt thereof in an amount of at least about 97 % (n / n) based on the total combined amount of substance of compound A and compound B, preferably in an amount of at least about 98 % (n / n) based on the total combined amount of substance of compound A and compound B, more preferably in an amount of more than about 98 % (n / n) based on the total combined amount of substance of compound A and compound B.

3. The composition according to claim 1 or 2, wherein the composition comprises compound A or a pharmaceutically acceptable salt thereof in an amount of from about 95 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound A and compound B, preferably in an amount of from about 97 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound A and compound B, more preferably in an amount of from about 98 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound A and compound B.

4. A solid-phase peptide synthesis method of preparing the composition according to any one of claims 1 to 3, the method comprising the steps of a) Providing a solid resin having Fmoc-L-Cys(PGI ) bound to said solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-L-Cys(PGI ) bound to said solid resin and washing to provide H-L-Cys(PGI ) bound to said solid resin;c) Subsequent coupling, by stepwise solid-phase peptide synthesis, each 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; or building blocks consisting of said amino acids, wherein each of said subsequent couplings comprises adding a respective Fmoc- protected amino acid or building block, washing, Fmoc-deprotection, and washing to obtain a peptide bound to said solid resin; d) cleaving a bond between said solid resin and Cys(PG1 ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain a composition comprising compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3 and compound D comprising or consisting of an amino acid sequence of SEQ ID No.: 4; and e) Performing an oxidizing treatment to obtain the composition according to claim 1 to 3.

5. The method according to claim 4, wherein step e) is a random oxidizing treatment mediated by cysteine / cystine.

6. A composition comprising a) Compound C comprising or consisting of an amino acid sequence of SEQ ID No.: 3, wherein Cys38is L-cysteine, and wherein compound C has no intramolecular disulfide bonds, or a pharmaceutically acceptable salt thereof; and b) 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; characterized in that c) the composition comprises compound C in an amount of at least about 95 % (n / n) based on the total combined amount of substance of compound C and compound D.

7. The composition according to claim 6, wherein the composition comprises compound C or a pharmaceutically acceptable salt thereof in an amount of at least about 97 % (n / n) based on the total combined amount of substance of compound C and compound D, preferably in an amount of at least about 98 % (n / n) based on the total combined amount of substance of compound C and compound D, more preferably in an amount of more than about 98 % (n / n) based on the total combined amount of substance of compound C and compound D.

8. The composition according to claim 6 or 7, wherein the composition comprises compound C or a pharmaceutically acceptable salt thereof in an amount of from about 95 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound C and compound D, preferably in an amount of from about 97 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound Cand compound D, more preferably in an amount of from about 98 % (n / n) to about 99.5 % (n / n) based in the total combined amount of substance of compound C and compound D.

9. A solid-phase peptide synthesis method of preparing the composition according to any one of claims 6 to 8, the method comprising the steps of a) Providing a solid resin having Fmoc-L-Cys(PGI ) bound to said solid resin, wherein PG1 is a side-chain protecting group; b) Fmoc-deprotection from the Fmoc-L-Cys(PGI ) bound to said solid resin and washing to provide H-L-Cys(PGI ) bound to said solid resin; c) Subsequent coupling, by stepwise solid-phase peptide synthesis, each 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; or building blocks consisting of said amino acids, wherein each of said subsequent couplings comprises adding a respective Fmoc- protected amino acid, washing, Fmoc-deprotection, and washing to obtain a peptide bound to said solid resin; d) cleaving a bond between said solid resin and Cys(PG1 ) bound to said solid resin, and cleaving off all side-chain protection groups to obtain the composition according to any one of claims 5 to 8.

10. The method according to any one of claims 4, 5, or 9, 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 tetrahydropyranyl (Thp); more preferably wherein PG1 is (4-methoxyphenyl)diphenylmethyl (Mmt).11 . The method according to any one of claims 4, 5, 9, or 10, 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 about 8 % (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.

12. The method according to any one of claims 4, 5, or 9 to 11 , 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.

13. A composition prepared by the method according to any one of claims 4, 5, or 9 to 12.

14. A pharmaceutical composition comprising the composition according to any one of claims 1 to 3 or 13.

15. A composition according to any one of claims 1 to 3 or 13 or a pharmaceutical composition according to claim 14 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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