Dosage form for peptide agents

Abstract

A subject matter of the invention is a pharmaceutical single dosage form for oral delivery consisting of two capsules, an outer enteric capsule, and an inner capsule, wherein said inner capsule comprises at least one physiologically active peptide agent and wherein said outer capsule contains inner capsule and an organic acid, wherein said outer capsule contains an absorption enhancer which is a carnitine compound in the form of optionally coated pellets. A subject matter of the invention is also use of the pharmaceutical single dosage form as a dietary supplement.

Description

[0001] DOSAGE FORM FOR PEPTIDE AGENTS

[0002] DESCRIPTION OF THE RELATED ART

[0003] Many peptides with biological activity (peptide agents) are widely known. Therapeutically effective amounts of such biologically relevant peptides may be administered to a patient in a variety of ways. However, the most preferred oral administration is very difficult to achieve with this type of compounds.

[0004] Peptide agents used in the prior art have been frequently administered by injection or by nasal administration (e.g., Insulin). Oral administration is problematic as peptide agents are very susceptible to degradation in the environment of the digestive system as in the stomach or intestine. Proteolytic enzymes of both stomach and intestine degrade peptides, leading to their inactivity before they are absorbed into the bloodstream. Any amount of a peptide that survives proteolytic degradation by proteases in stomach (typically having acidic pH optima) is later confronted with proteases of the small intestine and enzymes secreted by pancreas (typically having neutral to basic pH optima). The problem is even more evident when very active peptide agents are administered in a very small amount per single dosage form. In such cases, protection of the peptide agents become a key issue.

[0005] The prior art teaches how to overcome the problem with degradation of the peptide agents in the stomach after oral administration.

[0006] International publication WO2019193204 describes a pharmaceutical composition for transmucosal administration, comprising a peptide or protein drug in combination with an excipient with a pKa value of 12 or higher (e.g., arginine free base, EDTA tetrasodium salt, trisodium phosphate, tris(hydroxymethyl)aminomethane, lysine, or calcium hydroxide).

[0007] Furthermore, international publication WO2019058273 describes a pharmaceutical composition including: a pharmaceutically effective amount of at least one peptide; and a pharmaceutically acceptable amount of a combination of: (a) at least one metal in form of any or a combination of a salt thereof and a complex thereof; and (b) at least one reducing agent, wherein, the at least one metal is selected from any or a combination of: vanadium, chromium and manganese, and wherein the combination of (a) at least one metal in form of any or a combination of a salt and a complex and (b) at least one reducing agent affords protection, at least in part, to the at least one peptide from proteolytic degradation upon ingestion thereof. Vanadium, chromium, and manganese are metals that can be toxic when administered in higher dosages or for prolonged time. Moreover, international publication WO2020183318 describes a method of preventing or treating disease or disorder comprising administration of oral pharmaceutical composition comprising therapeutically effective amount of PTH analog and at least one degradation preventing agent, wherein said composition provides relative bioavailability of at least 0.5%, compare to subcutaneous administration. The present invention also relates to the oral pharmaceutical composition comprising therapeutically effective amount of PTH analog and least one degradation preventing agent in physically separated dosage form and wherein said PTH analogue is in enteric coated form. At least one degradation preventing agent includes combination of at least one metal containing compound and at least one reducing agent where in at least one metal containing compound is selected for group consisting of vanadium containing compound, chromium containing compound and manganese containing compound. Preferably PTH analogue are teriparatide or abaloparatide. Here again, it is required that the pharmaceutical composition contains vanadium containing compound, chromium containing compound and manganese containing compound which can be detrimental to the human body.

[0008] In our unpublished patent application no. EP 22156902.3, we disclose a new pharmaceutical single dosage form for oral delivery consisting of two capsules, an outer enteric capsule, and an inner capsule, wherein said inner capsule comprises at least one physiologically active peptide agent and at least one absorption enhancer, and wherein said outer capsule contains inner capsule and an organic acid. This single dosage form is simple to manufacture, does not contain heavy metals, prevents better against undesirable effects of acids on the peptide agent, provides better protease inactivation in intestine and maintains good dissolution profile. Furthermore, all the ingredients of the single dosage form are released in the intestine in a close time proximity enhancing bioavailability.

[0009] In our previous application, an absorption enhancer, which may be a solubility enhancer and / or transport enhancer, aids transport of the peptide agent from the intestine to the blood. Preferable compounds to be used as the absorption enhancer, acyl carnitines are mentioned. More preferably, the acyl L-carnitine is lauroyl L-carnitine.

[0010] It was noted that acyl L-carnitine purchased from suppliers contains moisture due to its hygroscopic properties.

[0011] It was found that hygroscopic properties of acyl L-carnitine, preferably lauroyl L-carnitine, reduce a shelf-life of the said single dosage form. Specifically, it was observed during storage of the single dosage form that acyl L-carnitine draws moisture from the acid located outside the inner capsule and / or capsules, and consequently causes the walls of the inner capsule to dissolve. In addition, the process of capsule’s wall disintegration increases the amount of moisture contained in the carnitine purchased from suppliers. Ultimately, the peptide comes into contact with the acid and degrades.

[0012] L-Carnitine as well as its derivatives (collectively called carnitine compounds herein) such as acyl carnitines are highly hygroscopic compounds and render compositions comprising carnitine compounds typically sticky. This property makes the use of carnitine compositions more difficult, and leads to both loss of valuable carnitine composition, and issues during manufacturing of the compositions. In addition, the hygroscopic nature of carnitine may complicate packaging and proportioning of carnitine, for example when preparing a composition comprising other relevant nutrients, such as essential vitamins and minerals. Furthermore, carnitine is unstable, especially at elevated temperatures that are typically required to load it on a suitable carrier. Decomposition of carnitine releases ammonia, which has an unpleasant smell and is corrosive, thus providing a health risk to those working with it.

[0013] To minimize the effects of these properties, L-camitine is converted into several salts and derivatives.

[0014] L-Carnitine in the pure base form is a highly hygroscopic compound which is difficult to formulate as tablets and the objective is generally attained using for example L-Carnitine L-Tartarate. Many other salts and derivatives of carnitine are known to be less hygroscopic compared to L-carnitine, nevertheless so far, neither derivative nor salt thereof has been found that eliminates this property completely.

[0015] For instance EP 0 434088 discloses the use of the non-hygroscopic L(-)carnitine L(+)tartrare (2:1) (the preparation and physio-chemical characterization of which were, however, described by D. Miller and E. Strack in Hoppe Seyler's Z. Physiol. Chem 353, 618-622, April 1972) for the preparation of solid forms suitable for oral administration. In fact, it is perhaps less hygroscopic than pure L-carnitine, but it is still considered hygroscopic. This salt presents, however, some drawbacks, such as e.g. the release, after prolonged storage, of traces of trimethylamine which give the product an unpleasant fishy odor. Moreover, L(-) -carnitine L(+)tartrate (2:1) becomes deliquescent at relative humidity slightly exceeding 60%. Furthermore, L-(+)-tartaric acid is unable to give non-hygroscopic salts with the alkanoyl-L- carnitines, such as e.g. acetyl-L-carnitine. It should, furthermore, be noticed that tartaric anion is unable by itself to enhance the therapeutic / nutritional value of L-carnitine.

[0016] WO03066573 Al discloses stable non-hygroscopic carnitine zinc citrates.

[0017] US 4602039 discloses the acid fumarate of L-carnitine, acetyl-L-carnitine and propionyl-L-carnitine. While the acid fumarate of L-carnitine is strongly non-hygroscopic and withstands even higher values of relative humidity than L-carnitine tartrate does, this ability seems to decline as the weight of the alkanoyl group linked to the L-carnitine backbone increases.

[0018] Over time, many other specific alkanoyl derivatives of carnitine have been developed to find a compound of reduced hygroscopicity.

[0019] EP0979224 Aldiscloses a salt of L-carnitine or alkanoyl-L-carnitine of formula (I). The aforesaid object of the disclosure, i.e. to provide novel, pharmacologically acceptable salts of both L-carnitine and lower alkanoyl-L-carnitines which not only are stable and non-hygroscopic but also possess a higher therapeutic and / or nutritional value than the corresponding inner salts. Although many salts and derivatives have reduced hygroscopicity in comparison to pure carnitine, these derivatives are still considered hygroscopic and pose problems during the manufacture and storage of pharmaceutical preparations containing them.

[0020] According to the WO02058693 Al, the problems of storage and processing brought about by the high hygroscopicity of L-carnitine and alkanoyl L-carnitine inner salts have long since been known. This high hygroscopicity renders the manufacture and storage of orally administrable solid presentation forms particularly troublesome. This disclosure proposes stable, non-hygroscopic zinc salts of carnitines with galactaric acid as an alternative to other known salts.

[0021] Many of known salts or derivatives of L-carnitine even if less hygroscopic than pure carnitine, are difficult to access or expensive, and their therapeutic function is unknown.

[0022] Another way to deal with the hygroscopicity of carnitine is to use stabilizers in the preparations.

[0023] It was disclosed in WO22258631 Al that in the presence of a stabilizer one or more of the drawbacks of the prior art related to L-carnitine or its derivatives has been overcome. Accordingly, the disclosure relates to a carnitine formulation, comprising carnitine or a derivative thereof and a stabilizer selected from an inorganic salt, a C1-C3 carboxylic acid or a salt thereof, and combinations thereof. However, then another ingredient must be added to the preparation, which involves determining its impact on health and is economically unjustified.

[0024] There are known methods of separating hygroscopic ingredients from other ingredients of preparations to avoid moisture absorption during storage which may have an adverse effect on the form of the preparation and its other ingredients. However, it is difficult in pharmacy due to the need to provide a specific dosage form and limited possibilities of separation.

[0025] For example, EP1469743B1 discloses methods for providing hygroscopic substances in a form which is stable in a moist environment. More specifically, the disclosure relates to methods for stabilizing hygroscopic bioactive ingredients, such as choline chloride or lysine hydrochloride, by encapsulating said hygroscopic ingredient with a lipid coating, in an animal feed composition and also providing significant rumen protection for the ingredient.

[0026] SUMMARY OF THE INVENTION

[0027] The object of the invention is to provide a peptide delivery system which is in the form of a pharmaceutical single dosage form for oral delivery consisting of two capsules, an outer enteric capsule, and an inner capsule, wherein said inner capsule comprises at least one physiologically active peptide agent and wherein said outer capsule contains inner capsule and an organic acid, characterized in that said outer capsule contains an absorption enhancer which is a carnitine compound in the form of optionally coated pellets.

[0028] The peptide delivery system is simple to manufacture, better prevents against undesirable effects of acids on the peptides and maintains good dissolution profile. Furthermore, it is free from metals like chromium, manganese, or vanadium, and provides a better bioavailability in comparison with formulation with strongly alkaline agents as protease inhibitors.

[0029] Surprisingly, it was noticed that the single dosage form of the invention is stable during storage. It was observed that the inner capsule does not dissolve during storage keeping its integrity over the shelf-life time. Additionally, all the ingredients of the single dosage form are released in the intestine in a close time proximity, with the acid released somewhat (couple of minutes) earlier, thus enhancing bioavailability.

[0030] In one particularly preferable embodiment, the organic acid can be also in the form of optionally coated pellets. This further facilitate loading the organic acid into the outer capsule and also can further increase stability of the pharmaceutical composition.

[0031] Preferably, the carnitine compound is an acyl L-carnitine, preferably lauroyl L-carnitine.

[0032] Preferably, the peptide agent is composed of 3-20 amino acids, preferably 3-16 amino acids.

[0033] More preferably, the peptide agent is selected from GHK-Cu, Epithalon, ARG*BPC-157, Selank, Semax, Cortagen, Ac-BPC-157-Arg-NH2 having a sequence of Ac-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro- Ala-Asp-Asp-Ala-Gly-Leu-Val-Arg-NHz (SEQ. ID 1); Ac-Epithalon-Arg-NFL having a sequence of Ac- Ala-Glu-Asp-Gly-Arg-NH2 (SEQ.. ID 2); Ac-GHK-Arg-NEL having a sequence of Ac-Gly-His-Lys-Arg- NH2 (SEQ. ID 3); Ac-Scmax-Arg-NEL having a sequence of Ac-Met-Glu-His-Phe-Pro-Gly-Pro-Arg-NFL (SEQ. ID 4), Ac-Selank-Arg-NH2 having a sequence of Ac-Thr-Lys-Pro-Arg-Pro-Gly-Pro-Arg-NFL (SEQ. ID 5), Ac-Cortagen-Arg-NH2 having a sequence of Ac-Ala-Glu-Asp-pro-ARG-NFL (SEQ. ID 6).

[0034] All amino acids indicated in the sequences above are L-amino acids. Three letter codes for amino acids are in line with IUPAC nomenclature and are as follows:

[0035] Three letter

[0036] Amino acid code

[0037] Ala Alanine

[0038] Cys Cysteine

[0039] Asp Aspartic Acid Glutamic

[0040] Glu

[0041] Acid

[0042] Phe Phenylalanine

[0043] Gly Glycine

[0044] His Histidine

[0045] He Isoleucine

[0046] Lys Lysine

[0047] Leu Leucine

[0048] Met Methionine

[0049] Asn Asparagine

[0050] Pro Proline

[0051] Gin Glutamine

[0052] Arg Arginine

[0053] Ser Serine

[0054] Thr Threonine

[0055] Vai Valine

[0056] Trp Tryptophan

[0057] Tyr Tyrosine

[0058] Preferably, the inner capsule is enteric capsule or non-enteric capsule, most preferably it is non-enteric capsule.

[0059] Preferably, organic acid is selected from the group of carboxylic acids such as acetylsalicylic, acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glyoxylic, isocitric, isovaleric, lactic, maleic, oxaloacetic, propionic, pyruvic, succinic, tartaric, valeric acid, or a mixture thereof.

[0060] More preferably, the organic acid is citric acid or ascorbic acid or a mixture thereof. Preferably, the amount of peptide agent is 0.2mg - 20mg, the amount of absorption enhancer is 50mg - 150mg and the amount of organic acid is lOOmg - 500mg per single dosage form.

[0061] The object of the invention is also a use of the dosage form defined above as a dietary supplement. The aim of the dietary supplement is to improve the condition of the body.

[0062] In accordance with the invention, patients in need of treatment with peptide agents (active ingredients) are provided with a stable oral pharmaceutical dosage form. When the single dosage form of the invention is administered to a patient, the organic acid is released in the small intestine in a close time proximity with release of the peptide agent and the absorption enhancer. This reduces the activity of neutral to basic-acting proteases (e.g., luminal or digestive protease and proteases of the brush border membrane) by lowering pH below the optimal activity range of these proteases. Thus, the peptide active agents are less vulnerable to proteolytic degradation and can be successfully transported into the bloodstream.

[0063] Without intending to be bound by theory, the materials and structure of the single dosage form of the present invention reduces hygroscopicity of the carnitine compound as well as the whole single dosage form without compromising bioavailability of the peptide agent. The single dosage form are stable during storage and do not promote degradation of the peptide agent.

[0064] DETAILED DESCRIPTION OF THE INVENTION

[0065] As mentioned above, the present invention provides a pharmaceutical single dosage form for oral delivery consisting of two capsules, an outer enteric capsule, and an inner capsule, wherein said inner capsule comprises at least one physiologically active peptide agent and wherein said outer capsule contains inner capsule and an organic acid, characterized in that said outer capsule contains an absorption enhancer which is a carnitine compound in the form of optionally coated pellets.

[0066] Capsules

[0067] The single dosage form of the invention is composed of two capsules of an ordinary size known in the pharmaceutical industry. One of the capsules is an inner capsule and has a smaller size than the outer capsule, wherein the outer capsules contains / encapsules the inner capsule closed in the outer capsule forming a kind of a capsule-in-capsule system or double capsule system.

[0068] For example, but without limitation, the inner capsule is of size 5, 4 or 3, and, for example, but without limitation, the outer capsule is of size 0, 00 or 000. Preferably, the inner capsule is of size 5, and the outer capsule is of size 0. Also preferably when inner capsule is of size 4, and the outer capsule is 00. Alternatively preferably, when inner capsule is of size 3, and the outer capsule is 000. The person skilled in the art would know how to choose capsules available on the market to be able to put one smaller, inner capsule into the other, bigger, outer capsule so that there is still space in the outer capsule for an organic acid.

[0069] The outer capsule must be made of an enteric material. The inner capsule may be made of an enteric or non-enteric material. Capsules made of enteric material and capsules made of non-enteric material are known in the art, and commercially available, for example, capsules available from ADOX Sp. z o. o. (https: / / kapsulki.com.pl / ) or Lonza (https: / / pharma.lonza.com / offerings / eapsule-delivery-solutions).

[0070] As used herein, „enteric” relates to a material that does not dissolve or disintegrate in the gastric environment, and it helps to protect active agents, such as peptide agents, from the acidity of the stomach by their release after the stomach (usually in the upper tract of the intestine).

[0071] Any enteric capsules that protect the content of the single dosage form against stomach proteases and releases peptide agents in the intestine is suitable. Many enteric capsules are known in the art and are useful in the present invention. Examples include capsules made of cellulose acetate phthalate, hydroxypropylmethylethylcellulose succinate, hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, and methacrylic acid-methyl methacrylate copolymer.

[0072] As used herein, „non-enteric” relates to a material that can dissolve or disintegrate in the gastric environment.

[0073] Non-enteric capsule may be made of a water-soluble material. Many water-soluble capsules are known in the art and are useful in the present invention. Examples include capsule made of gelatine, hydroxypropylmethylcellulose, hydroxypropylcellulose and methylcellulose.

[0074] According to the present invention, the inner capsule comprises at least one physiologically active peptide agent and at least one absorption enhancer.

[0075] Peptide agents

[0076] According to the invention, all known physiologically active peptides may be used as the peptide agents, components of the single dosage form of the invention, comprised in the inner capsule.

[0077] Preferably, the peptide agent is a peptide composed of 3-20 amino acids, preferably 3-16 amino acids.

[0078] Preferably, the peptide agent is selected from the following list:

[0079] GHK-Cu having a sequence of

[0080] Gly-His-Lys *Cu(II) (CAS No.: 89030-95-5 for Cu complex)

[0081] Epithalon having a sequence of

[0082] Ala-Glu-Asp-Gly (CAS No.: 307297-39-8) ARG*BPC-157 being an arginine salt of BPC-157 peptide having a sequence of

[0083] Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val (CAS No.: 137525-51-0 for BPC-157)

[0084] Selank having a sequence of

[0085] Thr-Lys-Pro-Arg-Pro-Gly-Pro (CAS No. 129954-34-3)

[0086] Semax having a sequence of

[0087] Met-Glu-His-Phe-Pro-Gly-Pro (CAS No.: 80714-61-0), and

[0088] Cortagen having a sequence of

[0089] Ala-Glu-Asp-Pro

[0090] Both man-made and natural peptides can be orally delivered in accordance with the invention.

[0091] The peptide is to be understood by the person skilled in the art as short chain of amino acids linked by peptide bonds. Simple derivatives of the peptides are also in the scope of the invention. A person skilled in the art would know what simple derivatives are. These are compounds which come under the definition of the peptide known in the art after the transformation into a derivative.

[0092] The exemplary derivatives are chosen, but not limiting, from the group of metal complexes, N-terminal modified peptides, C-terminal modified peptides, or peptides containing unnatural amino acids.

[0093] The metal complexes can be, for example, copper complexes.

[0094] The C-terminal modified peptides can be amides, alkylamides, anilides, aldehydes, or esters, preferably amides.

[0095] The N-terminal modified peptides can be N-acylated compound (e.g., acetyl, formyl, pyroglutamyl, fatty acids), ureas, carbamates, sulfonamides, or N-alkylated compounds, preferably, N-acylated compounds.

[0096] The peptides containing unnatural amino acids can be peptides containing D-amino acids, homoamino acids, N-methyl amino acids, or alpha-methyl amino acids or unusual amino acids such as citrulline or naphtylalanine.

[0097] Such modifications are customary in the art, and can be provided by external suppliers such as Pepscan (see, for example, https: / / www.pepscan.com / custom-peptide-synthesis / peptide-modifications / unusual- non-natural-amino-acids / , https: / / www.pepscan.com / custom-peptide-synthesis / peptide- modifications / n-terminal- modifications / c-terminal-modifications / ). The applicant performed also synthesis of derivatives of the peptides listed above, and obtained derivatives having both N-acetyl and C-Arg-amide in theirs molecules that led to formation of new compounds having very favorable stability in comparison with the compounds above. The derivatives - new peptide agents can be obtained by a simple synthesis known in the art.

[0098] For example, reference is made to: Fields, Gregg B, and Janelle L Lauer-Fields, 'Principles and Practice of Solid-Phase Peptide Synthesis', Synthetic Peptides: A User's Guide (New York, 2002; online edn, Oxford Academic, 12 Nov. 2020), Handbook of Reagents for Organic Synthesis. Reagents for Glycoside, Nucleotide and Peptide Synthesis Edited by David Crich. John Wiley & Sons Ltd., Chichester, West Sussex, England. 2005, Principles of Peptide Synthesis, Miklos Bodanszky, Springer, September 1993, Methods of Enzymology, 289, Solid Phase peptide Synthesis, (G. B. Fields Ed.) Academic Press, 1997. Chemical Approaches to the Synthesis of Peptides and Proteins, P. Lloyd- Williams, F. Albericio, and E. Giralt Eds), CRC Press, 1997. Fmoc Solid Phase Peptide Synthesis, A Practical Approach, (W. C. Chan, P. D. White Eds), Oxford University Press, 2000. Solid Phase Synthesis, A Practical Guide, (S. F. Kates, F Albericio Eds), Marcel Dekker, 2000. P. Seneci, Solid- Phase Synthesis and Combinatorial Technologies, John Wiley & Sons, 2000. Houben-Weyl E22a, Synthesis of Peptides and Peptidomimetics (M. Goodman, Editor-in-chief, A. Felix, L. Moroder, C. Tmiolo Eds), Thieme, 2002, p. 665ff. N. L. Benoiton, Chemistry of Peptide Synthesis, CRC Press, 2005. J. Howl, Methods in Molecular Biology, 298, Peptide Synthesis and Applications, (J. Howl Ed) Humana Press, 2005.

[0099] Here, we use solid-phase synthesis on chlorotrityl resin using Fmoc protected amino acids. Outline of syntheses is presented in the Examples below.

[0100] Alternatively, and more preferably, peptide agents are selected from the following list:

[0101] Ac-BPC-157-Arg-NH2 having a sequence of

[0102] Ac-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val-Arg-NHz (SEQ. ID 1)

[0103] Ac-Epithalon-Arg-NH2 having a sequence of

[0104] Ac-Ala-Glu-Asp-Gly-Arg-NH2 (SEQ. ID 2)

[0105] Ac-GHK-Arg-NH2 having a sequence of

[0106] Ac-Gly-His-Lys-Arg-NH2 (SEQ.. ID 3)

[0107] Ac-Semax-Arg-NH2 having a sequence of

[0108] Ac-Met-Glu-His-Phe-Pro-Gly-Pro-Arg-NH2 (SEQ.. ID 4),

[0109] Ac-Selank-Arg-NH2 having a sequence of Ac-Thr-Lys-Pro-Arg-Pro-Gly-Pro-Arg-NHz (SEQ. ID 5),

[0110] Ac-Cortagen-Arg-NHz having a sequence of

[0111] Ac-Ala-Glu-Asp-Pro-Arg-NH2 (SEQ. ID 6)

[0112] Synthesis of this derivatives is presented in the examples.

[0113] All sequences are written according to the rules known in the art. That means, are presented from left to right from N-terminus to C-terminus. All amino acids are L-amino acids.

[0114] Term “-NH2” means that C-terminus is amidated to form primary amide forming -CONH2 group at C- terminus.

[0115] Term “Ac-” means that N-terminus is acetylated (-C(0)CH3) therefore forming a CH3-C(0)-NH- group at N-terminus. According to the invention, the peptide agent can be a single peptide agent or a mixture of different peptide agent.

[0116] Absorption enhancer

[0117] According to the invention, an absorption enhancer is comprised in the outer capsule together with the organic acid. Furthermore, according to the invention, the absorption enhancer is a carnitine compound.

[0118] The enhancer, which may be a solubility enhancer and / or transport enhancer, aids transport of the peptide agent from the intestine to the blood and may promote the process so that it better occurs during the time of reduced intestinal pH and reduced intestinal proteolytic activity. Many surface-active agents may act as both solubility enhancers and transport (uptake) enhancers. Again, without intending to be bound by theory, it is believed that enhancing solubility desirably provides (i) a more simultaneous release of the peptide agent of the invention into the aqueous portion of the intestine, (ii) better solubility of the peptide agent in, and transport through, a mucous layer along the intestinal walls. Once the peptide agent reaches the intestinal walls, an uptake enhancer provides better transport through the brush border membrane of the intestine into the blood, via either transcellular or paracellular transport. As discussed in more detail below, many preferred compounds may provide both functions. In those instances, preferred embodiments utilizing both functions may do so by adding only one additional compound to the pharmaceutical composition. In other embodiments, separate absorption enhancers may provide the two functions separately.

[0119] Carnitine compounds are particularly suitable and preferable absorption enhancers. Especially, acyl carnitines (e.g., lauroyl carnitine) are particularly good absorption enhancers. It is also preferred that the absorption enhancer is soluble at acid pH, particularly in the pH range of 3.0 to 5.0.

[0120] An absorption enhancer of the invention, that is, a carnitine compound, must be in a particulate form. It was surprisingly discovered that when it is in such a form like pellet or granulate, optionally coated, hygroscopicity of the carnitine compound is lowered so that it does not attract moisture and as a result does not lead to degradation of the inner capsule and the peptide agent contained therein.

[0121] Preferably, the absorption enhancer of the invention, that is, a carnitine compound, is in the form of coated pellets.

[0122] Term “pellets” as used herein is intended to mean small (diameter: 0.5-2.0 mm) spheres, also called microspheres, in which the drug is contained together with excipients. The pellets are produced by pelletizing. Pelletizing can be performed by coating the cores, by wet granulation, by hot-melt granulation, by extrusion-spheronization. Production of pellets is well-known in the art.

[0123] As mentioned before, pellets can have a coating. The coating can be either functional or non-functional, so it can be suitable for immediate release or modified release. Preferably, however, as it is advantageous that the contents of inner capsule should be release at the same time as the absorption enhancer, they should be suitable to immediate release, so the coating should have immediate release characteristics, like in the case of cosmetic coatings.

[0124] Most preferably, however, pellets are uncoated. It turned out that the unit dosage form of the invention work well when pellets are uncoated and there is no need to modify release of a substance from the pellets. Nevertheless, cosmetic coatings may be of benefit when the pellets are to have special visual features, like different colors for pellets with different substances.

[0125] Pellets of different characteristics may be commercially available or can be customized. Preferably, such pellets can be obtained for example from Allium pharma or Nutrifirst Biotech Inc in the form of Immediate Release Micropellet. Pellets can be also obtained using customary formulation techniques for formulation of pellets as described in the review by Prabhakaran, L. & Prushothaman, M. & Sriganesan, P.. (2009). Pharmaceutical micropellets: An overview. Pharmaceutical Reviews. 7, or by Bhowmik, D., Sampath Kumar, K.P., Bhanot, R. Pelletization Techniques used in Pharmaceutical Industry, LAP LAMBERT Academic Publishing (July 9, 2018).

[0126] Mixtures of different pellets may also be used. Person skilled in the art would know which absorption enhancer form may be blended to provide preferable conditions for absorption of the peptide agents.

[0127] Preferably, the absorption enhancers are selected from L-carnitine and acyl L-carnitine. Most preferably, the absorption enhancer is lauroyl L-carnitine.

[0128] Organic acid

[0129] Organic acid is a component of the single dosage form of the invention comprised in the outer capsule.

[0130] The acid is believed to lower the local intestinal pH (where the active agent has been released) to levels below the optimal range for many intestinal proteases. Decrease in pH reduces the proteolytic activity of the intestinal proteases, thus affording protection to the peptide from potential degradation. The activity of these proteases is diminished by the temporarily acidic environment provided by the organic acid.

[0131] It is preferred that the organic acid (to be used in such single dosage form) lowers local intestinal pH transiently to 5.5 or below, preferably 5.0, or 4.7 or below and more preferably 3.5 or below. Person skilled in the art would know what amounts of the organic acid should be used in the capsule to provide the preferred pH values.

[0132] Preferably, conditions of lowered intestinal pH last for a time sufficient to protect the peptide agent from proteolytic degradation and until at least some of the peptide agent has had an opportunity to cross the intestinal wall into the bloodstream. Absorption enhancers may synergistically promote peptide absorption into the blood while conditions of lowered proteolytic activity prevail. Preferred absorption enhancers were discussed above.

[0133] The organic acid of the invention is a pharmaceutically acceptable organic acid. Organic acid acidify basic environment of the intestine to provide the local pH below 6. Examples of the pharmaceutically acceptable acids include but are not limited to carboxylic acids such as acetylsalicylic, acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glyoxylic, isocitric, isovaleric, lactic, maleic, oxaloacetic, propionic, pyruvic, succinic, tartaric, valeric, and the like.

[0134] Mixtures of organic acids may also be used. Person skilled in the art would know which organic acids may be mixed to provide preferable acidic conditions.

[0135] Most preferably, the organic acid is in the form of pellets as defined in the case of absorption enhancer.

[0136] As mentioned before, pellets can have a coating. The coating can be either functional or non-functional, so it can be suitable for immediate release or modified release. Preferably, however, as it is advantageous that the contents of inner capsule should be release at the same time as the organic acid, they should be suitable to immediate release, so the coating should have immediate release characteristics, like in the case of cosmetic coatings. It is of utmost importance that the organic acid is release immediately, as it assures suitable environment for the peptide agents.

[0137] Most preferably, however, pellets are uncoated. It turned out that the unit dosage form of the invention work well when pellets are uncoated and there is no need to modify release of a substance from the pellets. Nevertheless, cosmetic coatings may be of benefit when the pellets are to have special visual features, like different colors for pellets with different substances.

[0138] Pellets of different characteristics may be commercially available or can be customized. Preferably, such pellets can be obtained for example from Allium pharma or Nutrifirst Biotech Inc in the form of Immediate Release Micropellet. Pellets can be also obtained using customary formulation techniques for formulation of pellets as described in the review by Prabhakaran, L. & Prushothaman, M. & Sriganesan, P.. (2009). Pharmaceutical micropellets: An overview. Pharmaceutical Reviews. 7, or by Bhowmik, D., Sampath Kumar, K.P., Bhanot, R. Pelletization Techniques used in Pharmaceutical Industry, LAP LAMBERT Academic Publishing (July 9, 2018).

[0139] Mixtures of different pellets may also be used. Person skilled in the art would know which organic acid form may be blended to provide preferable acidic conditions for absorption of the peptide agents.

[0140] Preferably, the organic acid is citric acid or ascorbic acid. Preferably the organic acid id citric acid. Preferably the organic acid id ascorbic acid.

[0141] Other components

[0142] The single dosage form of the invention may optionally also include, in outer as well as in inner capsule, typical pharmaceutical excipients. These ingredients suitable for pharmaceutical formulations are not crucial for the invention.

[0143] Selection of the excipients lies within the routine activities of one skilled in the art.

[0144] Use

[0145] According to the invention, patients in need of use of pharmaceutical peptide agent are provided with an oral pharmaceutical dosage form. The dosages and frequency of dosing the products are discussed in more detail below. Patients who may benefit from the use are those suffering from disorders that respond favorably to increased levels of a peptide agent.

[0146] It should be stressed that the way the use is performed will depend on the type of peptide agent used in the single dosage form of the invention.

[0147] For example, a single dosage form of the invention containing Epithalon may be used for slowing the aging of the cell population by stimulating the renewal and elongation of telomeres, slowing down aging / exhibiting anti-aging effect, prevention of cancer and age-related diseases, restoration and normalization of the production of melatonin, strong acting as an antioxidant that protects against oxidative stress, or improvement of sleep, and its quality (for details, see: Khavinson VKh, Bondarev IE, Butyugov AA. Epithalon peptide induces telomerase activity and telomere elongation in human somatic cells. Bull Exp Biol Med. 2003 Jun;135(6):590-2. doi: 10.1023 / a: 1025493705728. PMID: 12937682; Goncharova ND, Khavinson BK, Lapin BA. Regulatory effect of Epithalon on production of melatonin and cortisol in old monkeys. Bull Exp Biol Med. 2001 Apr;131(4):394-6. doi: 10.1023 / a:1017928925177. PMID: 11550036; Vanhee C, Moens G, Van Hoeck E, Deconinck E, De Beer JO. Identification of the small research tetra peptide Epithalon, assumed to be a potential treatment for cancer, old age and Retinitis Pigmentosa in two illegal pharmaceutical preparations. Drug Test Anal. 2015 Mar;7(3):259-64. doi: 10.1002 / dta.l771. Epub 2014 Dec 22. PMID: 25535022.) For example, a single dosage form of the invention containing GHK-Cu may be used for skin treatment / skin condition improvement, influencing lung function, Alzheimer's disease, and for gene regulation (for details, see Pickart, L., Vasquez-Soltero, J. M., & Margolina, A. (2015). GHK Peptide as a Natural Modulator of Multiple Cellular Pathways in Skin Regeneration. BioMed research international, 2015, 648108. https: / / doi.org / 10.1155 / 2015 / 648108; Pickart L, Vasquez-Soltero JM, Margolina A. The human tripeptide GHK-Cu in prevention of oxidative stress and degenerative conditions of aging: implications for cognitive health. Oxid Med Cell Longev. 2012;2012:324832. doi:10.1155 / 2012 / 324832).

[0148] For example, a single dosage form of the invention containing BPC-157 may be used for diseases, disorders or conditions for which neuroprotective effect, antidepressant effect, effect of alleviating diseases of the digestive system, help in the regeneration of the bones and joints, help with thrombosis, arterial hypertension, or help with skin diseases is necessary (for details, see: Gjurasin M, Miklic P, Zupancic B, Perovic D, Zarkovic K, Brcic L, Kolenc D, Radic B, Seiwerth S, Sikiric P. Peptide therapy with pentadecapeptide BPC 157 in traumatic nerve injury. Regul Pept. 2010 Feb 25 ; 160( 1 -3):33-41. doi: 10.1016 / j.regpep.2009.11.005. Epub 2009 Nov 10. PMID: 19903499; Knezevic, Mario et al. “Occluded Superior Mesenteric Artery and Vein. Therapy with the Stable Gastric Pentadecapeptide BPC 157.” Biomedicines vol. 9,7 792. 8 Jul. 2021, doi: 10.3390 / biomedicines9070792; Hsieh, Ming-Jer et al. “Modulatory effects of BPC 157 on vasomotor tone and the activation of Src-Caveolin-1- endothelial nitric oxide synthase pathway.” Scientific reports vol. 10,1 17078. 13 Oct. 2020, doi:10.1038 / s41598-020-74022-y; Perovic, Darko et al. “Stable gastric pentadecapeptide BPC 157 can improve the healing course of spinal cord injury and lead to functional recovery in rats.” Journal of orthopaedic surgery and research vol. 14,1 199. 2 Jul. 2019, doi:10.1186 / sl3018-019-1242-6; Takeo M, Lee W, Ito M. Wound healing and skin regeneration. Cold Spring Harb Perspect Med. 2015 Jan 5;5(l):a023267. doi: 10.1101 / cshperspect.a023267. PMID: 25561722; PMCID: PMC4292081).

[0149] Semax may be used for improvement of memory and concentration [Shadrina M, Kolomin T, Agapova T, Agniullin Y, Shram S, Slominsky P, Lymborska S, Myasoedov N, Comparison of the temporary dynamics of NGF and BDNF gene expression in rat hippocampus, frontal cortex, and retina under Semax action. 2010 DGI:10.1007 / sl2031-009-9270-z.]. It may be used also to support the therapy of brain stroke [Ekaterina V Medvedeva, BMC Genomics, 15, Article number: 228 (2014); doi: 10.1186 / 1471-2164-15-228]. Semax is known from prevention the death of tyrosine hydroxylasepositive neurons in a mixed neuroglial cell culture derived from the embryonic rat mesencephalon in a model of 6-hydroxydopamine-induced neurotoxicity [O.Dolotov, K.O.Eremin, Semax prevents the death of tyrosine hydroxylase-positive neurons in a mixed neuroglial cell culture derived from the embryonic rat mesencephalon in a model of 6-hydroxydopamine-induced neurotoxicity. 2015, D01:10.1134 / S1819712415040066], Selank is known to act on relaxation, calming down and better mood [A Volkova, M Shadrina, TKolomin, L Andreeva, S Limborska, N Myasoedov, P Slominsky, Selank Administration Affects the Expression of Some Genes Involved in GABAergic Neurotransmission. 2016. DOI: 10.3389 / fphar.2016.00031]. Selank administration affects the expression of some genes involved in GABAergic neurotransmission [VE Miedwiediew, ON Tereshchenko, NV Kost, Yu Ter-Izraelczyk, EV Gushanskaya, IK Chobanu, O Yu Sokolow, NF Miasojedow, Optimization of the treatment of anxiety disorders with selank. 2015. DOI: 10.17116 / jnevro20151156133-40].

[0150] Cortagen is known from regenerative function for the brain [Kolosova LI, Moiseeva AB, Turchaninova LN, Malinin VV, Polyakov EL, Nozdrachev AD, Khavinson VKh, The delayed effect of cortagen on the restoration of injured nerve function. 2002. DOI: 10.1023 / a: 1016098302564.]. Cortagen is also used as correcting agents in functional and metabolic disorders in the brain in chronic ischemia [V Zarubina, P D Shabanov, Eksp Klin Farmakol., 2011 ;74(2):8-15. PMID: 21476278]

[0151] Furthermore, the peptide agents that are N-Ac and C-Arg-NFE have the same advantages and uses as the peptides cited above. They differ just in their stability.

[0152] For example, N-Ac and C-Arg-NEL derivative prepared based on Epithalon sequence (SEQ. ID 2) will act as the Epithalon.

[0153] For example, the N-Ac and C-Arg-NEL derivative prepared based on GHK sequence (SEQ.. ID 3) will act as a GHK or its Cu salt (GHK-Cu).

[0154] For example, the N-Ac and C-Arg-NEL derivative prepared based on BPC sequence (SEQ. ID 1) will act as a BPC.

[0155] For example, the N-Ac and C-Arg-NEL derivative prepared based on Semax sequence (SEQ. ID 4) will act as a Semax.

[0156] For example, the N-Ac and C-Arg-NEL derivative prepared based on Selank sequence (SEQ. ID 5) will act as a Selank.

[0157] For example, the N-Ac and C-Arg-NEL derivative prepared based on Cortagen sequence (SEQ. ID 6) will act as a Cortagen.

[0158] As the single dosage form of the invention for oral delivery protects the peptide agent from proteolytic degradation and organic acid attack, it is expected that it provides bioavailability of a wide range of therapeutic peptide agents.

[0159] A person skilled in the art will know what amount of the peptide agent is to be used in the single dosage form. The amount should provide a therapeutic effect after administration. The amount of peptide agent and other ingredients is limited by the size of the capsules used. When a higher amount of peptide agent is to be administered to a patient, it is obvious that two or more single dosage forms can be administered at the same time.

[0160] The major advantage of the invention is to provide a single dosage form using commercially available capsules. The present approach does not require any special techniques or expensive equipment to manufacture the single dosage form as this was in the prior art, especially, in the case of coated acid particles. The single dosage form of the invention as a capsule-in-capsule system does not require tableting process, not to mention multilayer tableting. The approach of the invention separates spatially the peptide agent and the organic acid so that there is no contact between them at all. Even in the multilayer tablets of the prior art there it is still some contact causing degradation of the peptide agents.

[0161] As mentioned above, simple tableting itself carries many risks connected to peptide agent stability. For example, contacting a peptide agent with water and subjecting it to drying steps at elevated temperatures may lead to peptide degradation.

[0162] The single dosage form of the invention is simple and ensures a good bioavailability of the peptide agent.

[0163] The invention is further illustrated by the following non-limiting examples.

[0164] BRIEF DESCRIPTION OF THE DRAWINGS

[0165] Fig. 1 presents a comparison of the pharmacokinetic profiles of a peptide Epithalon for inventive single dosage form and two comparative dosages.

[0166] EXAMPLES:

[0167] Materials and methods

[0168] Enteric capsule of size 0, 00, 000 (made of pharmaceutical cellulose derivatives [HPMC-AS, HPMC]), enteric capsule of size 5, 4, 3 (made of pharmaceutical cellulose derivatives [HPMC-AS, HPMC]) or gelatin capsule (made of gelatine) was purchased from Capsugel.

[0169] Peptides was purchased from Lipopharm (http: / / www.lipopharm.pl / pP), N-acyl, C-Arg-NIL peptides were obtained as described below.

[0170] Powders of citric acid, ascorbic acid and magnesium stearate were purchased from Sigma Aldrich.

[0171] Lauroyl L-carnitine micropellets, citric acid micropellets, ascorbic acid micropellets all were obtained from Nutrifirst Biotech Inc. in the form of Immediate Release Micropellet having size of 0.6mm-1.2mm. All the micropellets are uncoated. The lauroyl L-carnitine micropellets consisted of lauroyl L-carinitine (50wt. %) and the further excipients were Dextrin, Starch, Microcrystalline cellulose, HPMC.

[0172] Micropellets with ascorbic acid or citric acid consisted of the respective acid (80wt. %) and Dextrin, Microcrystalline Cellulose, HPMC.

[0173] Microcrystalline cellulose was purchased from Bart (https: / / bart.p1 / substanc e-pomocmcze. / )

[0174] Preparation ofN-acyl, C-Arg-NPp peptides

[0175] For clarity reasons, for all examples concerning preparation of N-acetyl, C-Arg-NFL peptides the A- prefix was used.

[0176] EXAMPLE A-l - Synthesis of the new peptide agents

[0177] Equipment and reagents:

[0178] Equipment used in the synthesis and analysis of the final peptides was as follows: Reactor for industrial peptide synthesis: model PSR 1000C (NOV Biotech Ltd.), High Performance Liquid Chromatographs: 1. Preparative HPLC: CXTH P1001L i. 70 x 150 mm Vydac® column, 10 mm filter; 2. Preparative HPLC: AKTATM Pure 25 L / M System (GE Healthcare), fraction collector: Frac F9-R (Cat. No. 29011362) i. Kromasil® 100-13-C18 column 50 x 250mm (pore size: 100 A, bed grains: 13 run, phase: Cl 8, diam. internal: 50mm); 3. Analytical HPLC: UHPLC (UltiMate 3000 Thermo Scientific / Dionex) with diode detector coupled to QTOF-MS i. Kromasil® 100-4-C18 column 4.6 x 250mm (pore size: 100 A, bed grain size: 4 pm, chamfer: C18, ID: 4.6 mm); QTOF-MS mass spectrometer: Impact HD, Bruker; Lyophilizer: Christ Beta 2-8 LSCplus Lyophilizer 102125; Vacuum pump Pfeiffer Vacuum D- 35614 Asslar,: Mod. DUO 10M, M. No.: PK D62 712D; V. Rotary evaporator R-1001-VN Rotary Evaporator, Greatwall.

[0179] All reagents were obtained from commercial sources like Sigma-Aldrich, Chempur, Poch, Merk. All protected amino acids were obtained from NOV Biotech Ltd. 2 -Chlorotrityl amine resin, 100-200 mesh was obtained from Sigma-Aldrich, Fmoc-Pro-CTC resin was obtained from NOV Biotech Ltd. BPC- 157 (in the form of Arg salt), GHK, Epithalon, Semax, Selank and Cortagen were obtained from Lipopharm, Poland.

[0180] Abbreviations:

[0181] CS (cleavage solution): TFA:EDT:Phenol:TIS:Water = 86:4:4:4:2

[0182] CTC-resin: 2-chlorotrityl chloride resin DCM: dichloromethane

[0183] DIC: N,N'-diisopropylcarbodiimide

[0184] DMF: N,N-dimethylformamide

[0185] DP: 20% piperidine + 80% N,N-dimethylformamide

[0186] EDT: 1.2-ethanedithiol

[0187] Fmoc: 9-fluorenylmethoxycarbonyl

[0188] HOBt: 1 -hydroxybenzotriazole

[0189] MS: Mass Spectrometry

[0190] MW: Molecular Weight

[0191] Ninhydrin: 2.2-dihydroxyindan- 1 ,3-dione

[0192] RE: Resin

[0193] RP-HPLC: Reverse Phase High Performance Liquid Chromatography (Reversed-Phase High-Performance Liquid Chromatography)

[0194] TFA: trifluoroacetic acid

[0195] TIS: thioanisole

[0196] EXAMPLE A-1,1

[0197] Preparation of Ac-BPC-157-Arg-NHz (Ac-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala- Gly-Leu-Val-Arg-NH2) (SEQ. ID 1).

[0198] Outline of the synthesis

[0199] Peptide synthesis was performed using a solid support. Amino acids with a blocked quaternary 9- fluorenylmethoxycarbonyl group were used for the synthesis (Fmoc). During the course of the first step of the synthesis, the first Fmoc -protected amino acid was attached directly to the P-linker of the solid phase support. During the further reaction of cyclic deprotection and condensation of peptide bonds, the remaining amino acids were attached starting from the C -terminal end and ending with the N-terminal part of the peptide. In this way, a peptide-carrier complex was obtained (Ac-Gly-Glu-Pro-Pro-Pro-Gly- Lys-ProAla-Asp-Asp-Ala-Gly-Leu-Val-Arg-Pro-C-H-Resin). In the next step of the synthesis, the peptide was detached from the support using trifluoroacetic acid (TFA). Amidation of the C-terminal amino acid occurs during cleavage of the peptide from the support. This resulted in a peptide of the assumed sequence Ac-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro- Ala-Asp- Asp- Ala-Gly-Leu- V alArg-amide (SEQ.. ID 1). To obtain a peptide with an assumed purity of >95%, the Reverse Phase High Performance Liquid Chromatography (RP-HPLC) technique was used. The analytical stage was completed with ultra-high- performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (QTOF-MS), which confirmed the molar mass of the synthesized peptide.

[0200] Detailed description of the synthesis

[0201] 1. Peptide synthesis

[0202] The peptide was synthesized on a scale of 2 g

[0203] 1.Preparation of Fmoc-Pro-CTC-resin

[0204] A weight of the Fmoc-Pro-CTC-resin carrier was placed in a chemical reactor, then DCM was added and left for 30 min. The DCM was removed and then the resin was rinsed with DMF under constant stirring. The operation was repeated twice, each time using DMF. After rinsing, the DMF solution was thoroughly removed.

[0205] 2. Attachment of Arg to solid support Fmoc-Pro-CTC-Resin.

[0206] After preparing the solid phase, arginine was attached to it. Then, in the deprotection step, the Fmoc group was removed. DP was added with continuous stirring under a nitrogen atmosphere. After 5 min, DP was removed, then the carrier was rinsed once with DMF, which was completely removed in the next step. The deprotection step was repeated again with the addition of DP under continuous stirring under a continuous flow of nitrogen. DP was completely removed after 10 minutes. The carrier was washed 6 times, each time using DMF. Fmoc-Arg-OH (3-times the amount of the support used) and HOBt (3-times the amount of the support used) were weighed out and then dissolved in DMF in the reactor. In the next step of the synthesis, DIC was added (3-times the amount of the support used, v:v) with constant stirring under a constant supply of nitrogen for 2 hours. At the end of the step, a ninhydrin test was performed to ensure that Fmoc-Arg-OH is fully attached to the support Fmoc-Pro-CTC-Resin. A negative result of the nihydrin reaction means that there are no free NH2 groups, and thus the reaction is considered complete. The liquid was removed from the reactor then it washed with DMF 3 times. From this point on, the amino acid in the form of Fmoc-Arg-OH is linked to the support. Upon completion of the step, Fmoc-Arg-Pro-CTC-Resin was obtained.

[0207] 3. Attachment of Vai to -Arg-Pro-CTC-Resin.

[0208] DP was added with constant stirring under a constant supply of nitrogen. Removed DP after 5 min then rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 minutes then rinsed resin 6 times with DMF each time. Fmoc-Val-OH (3-times the amount of the support used) and HOBt (3- times the amount of the support used) were weighed out then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. At the end of the step, a ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times using DMF each time. From then on, the amino acid in the form Fmoc-Val-OH was attached to Arg-Pro-CTC-Resin. Fmoc-Val-Arg-Pro-CTC-Resin was obtained.

[0209] 4. Attachment Leu to -Val-Arg-Pro-CTC-Resin.

[0210] DP was added with constant stirring under a constant supply of nitrogen and then the DP was removed after 5 min and rinsed once with DMF which was removed in the next step. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. Removed DP after 10 minutes. The support was then washed 6 times, each time using DMF. Fmoc-Leu-OH (3-times the amount of the support used) and HOBt (3-times the amount of the support used) were weighed out which was dissolved in DMF and fed into the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed from the reactor then washed 3 times with DMF. From then on, an amino acid in the form of Fmoc-Leu-OH was attached to Val-Arg-Pro-CTC-Resin. Fmoc-Leu- Vai- Arg-Pro-CTC-Resin was obtained.

[0211] 5. Attachment of Gly to -Leu- Vai- Arg-Pro-CTC-Resin.

[0212] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Gly-OH (3-times the amount of the support used) and HOBt (3-times the amount of the support used) were weighed out, then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Gly-OH was attached to -Leu- Vai- Arg-Pro- CTC-Resin. As a result, Fmoc-Gly-Leu-Val-Arg-Pro-CTC-Resin was obtained.

[0213] 6. Attachment of Ala to -Gly-Leu- Vai- Arg-Pro-CTC-Resin.

[0214] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Ala-OH (3-times the amount of the support used) and HOBt (3-times the amount of the support used) were weighed out, then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Ala-OH was attached to -Gly-Leu-Val-Arg- Pro-CTC-Resin. As a result, Fmoc-Ala-Gly-Leu-Val-Arg-Pro-CTC-Resin was obtained.

[0215] 7. Attachment Asp do Ala-Gly-Leu-Val-Arg-Pro-CTC-Resin.

[0216] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Asp-OH was weighed out (3- times the amount of the support used) and HOBt (3-times the amount of the support used), then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring with a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Asp-OH was attached to -Ala-Gly-Leu-Val- Arg-Pro-CTC-Resin. As a result, Fmoc-Asp-Ala-Gly-Leu-Val-Arg-Pro-CTC-Resin was obtained.

[0217] 8. Attachment of Asp to -Asp-Ala-Gly-Leu-Val-Arg-Pro-CTC-Resin.

[0218] Step 7 was repeated. After the stage was completed, Fmoc-Asp-Asp-Ala-Gly-Leu-Val-Arg-Pro-CTC- Resin was obtained.

[0219] 9. Attachment of Ala to -Asp-Asp-Ala-Gly-Leu-Val-Arg-Pro-CTC-Resin was obtained.

[0220] Step 6 was repeated. After the stage was completed, Fmoc-Ala-Asp-Asp-Ala-Gly-Leu-Val-Arg-Pro- CTC-Resin was obtained.

[0221] 10. Attachment Pro to -Ala-Asp-Asp-Ala-Gly-Leu-Val-Arg-Pro-CTC-Resin.

[0222] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Pro-OH was weighed out (3- times the amount of the support used) and HOBt (3-times the amount of the support used), then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Pro-OH was attached to -Ala- Asp- Asp- Ala- Gly-Leu-Val-Arg-Pro-CTC-Resin. As a result, was obtained Fmoc-Pro-Ala-Asp-Asp-Ala-Gly-Leu- Val-Arg-Pro-CTC-Resin.

[0223] 11. Attachment Lys to - Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val-Arg-Pro-CTC-Resin.

[0224] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Lys-OH was weighed out (3- times the amount of the support used) and HOBt (3 -times the amount of the support used), then it was dissolved in DMF and charged to the reactor. DIC was then added (3 -times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Lys-OH was attached to -Pro- Ala- Asp- Asp- Ala-Gly-Leu-Val-Arg-Pro-CTC-Resin. As a result, was obtained Fmoc-Lys-Pro-Ala-Asp-Asp-Ala- Gly-Leu- V al- Arg-Pro-CTC-Resin.

[0225] 12. Attachment Gly to -Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val-Arg-Pro-CTC-Resin.

[0226] Step 5 was repeated. After the stage was completed, Fmoc-Gly-Lys-Pr- Ala- Asp- Asp- Ala-Gly-Leu- Val- Arg-Pro-CTC-Resin was obtained.

[0227] 13. Attachment of Pro to -Asp- Asp- Ala-Gly-Leu- Vai- Arg-Pro-CTC-Resin.

[0228] Step 10 was repeated. After the stage was completed, Fmoc-Pro-Pro-Pro-AlaAsp-Asp-Ala-Gly-Leu- Val- Arg-Pro-CTC-Resin was obtained.

[0229] 14. Attachment of Glu to -Pro-Pro-Pro-Gly-Lys-Pro- Ala- Asp- Asp- Ala-Gly-Leu- ValArg-Pro-CTC- Resin.

[0230] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Glu-OH was weighed out (3- times the amount of the support used) and HOBt (3 -times the amount of the support used), then it was dissolved in DMF and charged to the reactor. DIC was then added (3 -times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Glu-OH was attached to -Pro-Pro-Pro-Gly- LysPro- Ala- Asp- Asp- Ala-Gly-Leu- Vai- Arg-Pro-CTC-Resin. As a result, Fmoc-GluPro-Pro-Pro-Gly- Lys-Pro- Ala- Asp- Asp- Ala-Gly-Leu- V al- Arg-Pro-CTC-Resin was obtained.

[0231] 15. Attachment of Gly to -Glu-Pro-Pro-Pro-Gly-Lys-Pro- Ala- Asp- Asp- Ala-Gly-Leu Vai- Arg-Pro-CTC- Resin.

[0232] Step 5 was repeated. After the stage was completed, Fmoc-Gly-Glu-Pro-Pro-Pro-Gly-LysPro-Ala-Asp- Asp- Ala-Gly-Leu- Vai- Arg-Pro-CTC-Resin was obtained.

[0233] 16. Acetylation of the N-terminal amino acid DP was added with constant stirring under a constant supply of nitrogen, then DP was removed after 5 min and rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min. The support was washed 6 times, each time using DMF. Prepared were acetic acid (3-times the amount of the support used) and HOBt (3-times the amount of the support used), dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The liquid was removed from the reactor and then washed 3 times using DMF each time. Ac-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val-Arg-Pro- CTC-Resin was obtained .

[0234] II. Detachment of the peptide from the support

[0235] After the amino acid attaching steps are completed, all peptide side chains are blocked and the peptide itself is attached to the support. In the next stage of the synthesis, the peptide has to be detached from the support and the side chains deblocked to obtain a crude peptide. Thanks to the use of a modified resin, spontaneous amidation of the C-terminal amino acid occurs during the cleavage of the peptide from the support. The obtained peptide -resin complex together with CS (cleaving solution) was then stirred for 3 hours in the reactor. At this stage, the peptide is released from the support and the side groups are deprotected. Upon completion, the solution was filtered discarding the solid phase and retaining the liquid phase containing the peptide. In the next step, the liquid was cooled using diethyl ether with constant stirring. The crude peptide precipitated as a white precipitate. The precipitate was filtered and then washed with cooled diethyl ether 4 times using diethyl ether each time. The white precipitate was dried in a vacuum oven R-1001-VN. This completes the synthesis of the crude peptide of the sequence Ac-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-GlyLeu-Val-Arg-amide (SEQ.. ID 1).

[0236] III. Purification of the crude peptide by RP-HPLC

[0237] Two preparative liquid chromatographs were used to separate and purify the synthesis product: 1) AKTATM Pure 25 with Frac F9-R fraction collector. The Kromasil® 100- 13-C 18 50 x 250 mm column allows the purification of up to 1-2 g of product in one process at maximum flow 25 mL / min. 2) CXTH P1001L. Vydac column 70 x 150 mm, 10 mm filter allows to purify up to 50 g of product in one process at a maximum flow of 500 mL / min.

[0238] 3 Analysis parameters:

[0239] Sample: purified peptide dissolved in water to concentration ~5 g / L;

[0240] Mobile phase: AC fFLO (17:83 v / v) with 0,1% TFA;

[0241] Flow: 25 mL / min / 500 mL / min

[0242] Analysis time: 30 min; Column temperature: 25 °C;

[0243] Detection conditions: X = 220 nm.

[0244] IV. Lyophilization of the peptide

[0245] For lyophilization of the peptide, the solvent obtained during the HPLC paging process was evaporated and dried in a lyophilizer (Christ Beta 2-8) within 48-72 hours. The collected white solid phase was the purified peptide.

[0246] V. Synthesis quality analysis

[0247] Peptide purity was checked by analytical HPLC, molar mass was confirmed by mass spectrometry as described below.

[0248] 1. HPLC analysis to evaluate the purity of the obtained peptide.

[0249] To assess the purity of the purified peptide, HPLC-MS was measured using a liquid chromatograph UHPLC (UltiMate 3000 Thermo Scientific / Dionex) with a fast (200 Hz) diode detector coupled to QTOF-MS (Impact HD, Bruker)

[0250] Analysis parameters:

[0251] Sample: 10 pl dissolved in water (to a concentration of ~5 g / L) of the purified peptide;

[0252] Mobile phase: AC hHzO (17:83 v / v) with 0,1% TFA;

[0253] Flow: 1 mL / min;

[0254] Analysis time: 30 min;

[0255] Column temperature: 25 °C;

[0256] Detection conditions: I = 220 nm.

[0257] Purity obtained was 99%.

[0258] 2. QTOF-MS analysis to identify the peptide

[0259] To accurately determine the molecular weight of the peptide, a high-resolution QTOF mass spectrometer, a hybrid quadrupole mass spectrometer with time-of-flight analysis was used - TOF, and ionization at atmospheric pressure - ESI (Impact HD, Bruker) The sample was fed to the source by the syringe pump of the chromatograph. A small amount of the peptide was dissolved in MeOH.

[0260] Analysis parameters:

[0261] Sample: 10 pl dissolved in methanol (to a concentration of ~5 g / L) of the purified peptide;

[0262] Ionization: nitrogen laser beam, I = 337 nm

[0263] Matrix: a-cyano-4-hydroxy cinnamic acid (CCA). The obtained BPC-157 derivative of (SEQ. ID 1) was identified by the QTOF MS: Mass Calc, for C70H113N21O23: 1616.77 g / mol, Found: 1617.99 [M+H]+;

[0264] EXAMPLE A-1.2

[0265] Preparation of N-Ac-Epithalon-Arg-NHz (N-Ac-Ala-Glu-Asp-Gly-Arg-NHj) (SEQ.. ID 2)

[0266] Outline of the synthesis

[0267] Peptide synthesis was performed using a solid support. Amino acids with a blocked quaternary 9- fluorenylmethoxycarbonyl group (Fmoc) were used for the synthesis. During the first stage of the synthesis, the first Fmoc protected amino acid was attached directly to the p-linker of the solid support. During the further cyclic deprotection and condensation reaction of peptide bonds, the remaining amino acids were attached starting from the C-terminus and ending at the N-terminal part of the peptide. In this way, the peptide-support complex was obtained (Ac-Ala-Glu-Asp-Gly-Arg-Pro-C-H-Resin). In the next step of the synthesis, the peptide was cleaved from the support using trifluoroacetic acid (TFA). When cleaving the peptide from the support, amidation of the C-terminal peptide occurs. As a result, a peptide with the assumed sequence was obtained Ac-Ala-Glu-Asp-Gly-Arg-amide (SEQ. ID 2).

[0268] To obtain the peptide of assumed purity> 95%, the RP-HPLC technique was used. The analytical stage was finished with the ultra-high-performance liquid chromatography (UHPLC) technique and the high- resolution mass spectrometry (QTOF-MS) technique to confirm molar mass of the obtained final peptide.

[0269] I. Peptide synthesis

[0270] The peptide was synthesized on a scale of 10 g

[0271] 1. Preparation of Fmoc-Pro-CTC-resin

[0272] The Fmoc-Pro-CTC resin support was weighed out in a chemical reactor, then DCM was added and left for 30 min. DCM was removed and the resin was washed with DMF with constant stirring. The operation was repeated twice, each time using DMF. After washing, the DMF solution was completely removed.

[0273] 2. Attachment of Fmoc-Arg to Pro-CTC-Resin on solid support.

[0274] After preparation of the solid phase, arginine was attached in the following way. The Fmoc group from the Fmoc-Pro-CTC-Resin was removed in the deprotection step. DP was added with constant stirring under nitrogen atmosphere. After 5 min, DP was removed, and the solid support was rinsed once with DMF, and the DMF was completely removed. The deprotection step was repeated by adding DP under a constant flow of nitrogen. DP was completely removed after 10 minutes. The solid support was rinsed 6 times, each time with DMF. Fmoc-Arg-OH (3-times the amount of the solid support used) and HOBt (3-times the amount of the solid support used) was weighted out and dissolved in DMF in a reactor. In the next step of the synthesis, DIC was added (3 times the amount of support used, v:v) constantly stirring under a constant flow of nitrogen for 2 hours. At the end of this step, a ninhydrin test was performed to ensure that Fmoc-Arg-OH was completely attached to the solid support. A negative result of the ninhydrin test means that no NH2 groups are present and therefore the reaction is deemed complete. The liquid was removed from the reactor then washed with DMF three times. As a result, the amino acid in the form Fmoc-Arg-OH is attached to the support. At the end of the stage, Fmoc-Arg-Pro- CTC-Resin was obtained.

[0275] 3. Attachment of Fmoc-Gly to Arg-Pro-CTC-Resin.

[0276] DP was added to the Fmoc-Arg-Pro-CTC-Resin with constant stirring under constant flow of nitrogen. DP was removed after 5 min followed by rinsing once with DMF and its removal. The deprotection was repeated by adding DP with constant stirring under constant flow of nitrogen. DP was removed after 10 minutes then the resin was rinsed 6 times with DMF (4 mL). Fmoc-Gly-OH (3-times the amount of the solid support used) and HOBt (3-times the amount of the solid support used) was weighted out and dissolved in DMF and charged to the reactor. Next, DIC (3-times the amount of the solid support used) was added constantly stirring under a constant flow of nitrogen for 2 hours. At the end of this step, a ninhydrin test was performed to ensure that the reaction was completed. The liquid was removed and then the reactor was washed 3-times using DMF each time (4 mL). As a result, Fmoc-Gly-OH was attached to Arg-Pro-CTC-Resin. Fmoc-Gly- Arg-Pro-CTC-Resin was obtained.

[0277] 4. Attachment of Asp to -Gly-Arg-Pro-CTC-Resin.

[0278] DP was added constantly stirring under a constant flow of nitrogen, and then the DP was removed after 5 min and rinsed once with DMF, which was removed in the next step. The deprotection was repeated with the addition of DP constantly stirring under a constant flow of nitrogen. DP was removed after 10 minutes. The solid support was then rinsed 6 times with DMF. Fmoc-Asp-OH (3-times the amount of the solid support used) and HOBt (3-times the amount of the solid support used) was weighted out and dissolved in DMF and charged to the reactor. Next, DIC (3-times the amount of the solid support used) was added constantly stirring under a constant flow of nitrogen for 2 hours. The ninhydrin test was performed to ensure the reaction was completed. The liquid was removed from the reactor, and it was then washed 3 times with DMF. As a result, Fmoc-Asp-OH was attached to Gly-Arg-Pro-CTC-Resin. Fmoc-Asp-Gly-Arg-Pro-CTC-Resin was obtained.

[0279] 5. Attachment of Glu to -Asp-Gly- Arg-Pro-CTC-Resin.

[0280] DP was added constantly stirring under a constant flow of nitrogen, and then DP was removed after 5 min and rinsed once with DMF which was then removed from the reactor. The deprotection was repeated with the addition of DP constantly stirring under a constant flow of nitrogen. The DP was removed after 10 min, and then the resin was rinsed 6 times with DMF. Fmoc-Glu-OH (3-times the amount of the solid support used) and HOBt (3-times the amount of the solid support used) was weighted, and then dissolved

[0281] T1 in DMF and charged to the reactor. Next, DIC (3 -times the amount of the solid support used) was added constantly stirring under a constant flow of nitrogen for 2 hours. The ninhydrin test was performed to ensure the reaction was completed. The liquid was removed and then the reactor content was washed 3 times with DMF. As a result, Fmoc-Glu-OH was attached to Asp-Gly-Arg-Pro-CTC-Resin. As a result, Fmoc-Glu-Asp-Gly-Arg-Pro-CTC-Resin was obtained.

[0282] 6. Attachment of Ala to -Glu-Asp-Gly-Arg-Pro-CTC-Resin.

[0283] DP was added constantly stirring under a constant flow of nitrogen, then the DP removed after 5 min and rinsed once with DMF, which was then removed. The deprotection was repeated with the addition of DP constantly stirring under a constant flow of nitrogen. DP was removed after 10 minutes. The solid support was rinsed 6 times with DMF. Fmoc-Ala-OH (3-times the amount of the solid support used) and HOBt (3-times the amount of the solid support used) was weighted out and dissolved in DMF and charged to the reactor. Next, DIC (3-times the amount of the solid support used) was added constantly stirring under a constant flow of nitrogen for 2 hours. The ninhydrin test was performed to ensure the reaction was completed. The liquid was removed from the reactor and then washed 3 times using DMF each time. As a result, Fmoc-Ala-OH was attached to -Glu-Asp-Gly-Arg-Pro-CTC-Resin. Fmoc-Ala- Glu-Asp-Gly-Arg-Pro-CTC-Resin was obtained.

[0284] 7. Acetylation of the N-terminal amino acid.

[0285] DP was added constantly stirring under a constant flow of nitrogen then the DP removed after 5 min and rinsed once with DMF, which was then removed. The deprotection was repeated with the addition of DP constantly stirring under a constant flow of nitrogen. DP was removed after 10 minutes. The solid support was rinsed 6 times with DMF. Acetic acid (3-times the amount of the solid support used) and HOBt (3-times the amount of the solid support used) dissolved in DMF and charged to the reactor. Next, DIC (3-times the amount of the solid support used) was added constantly stirring under a constant flow of nitrogen for 2 hours. The liquid was removed from the reactor and then washed 3 times using DMF each time. Ac-Ala-Glu-Asp-Gly-Arg-Pro-CTC-Resin was used.

[0286] II. Cleavage of the peptide from the solid support

[0287] After completion of the peptide formation, all side chains of the peptide are protected and the peptide itself is attached to the support. Next, the peptide must be cleaved from the solid support and the protection of side chains has to be removed to obtain a crude peptide. Thanks to the use of a modified resin, spontaneous amidation of the C-terminal amino acid takes place during the cleavage of the peptide from the solid support.

[0288] The peptide -resin complex obtained above was placed in a reactor with CS, and then stirred for 3 hours. At this stage, the peptide releases from the solid support and the protection groups of the side chains are deprotected. Upon completion, the solution was filtered discarding the solid phase and retaining the liquid phase containing the peptide. Next, the liquid was cooled using diethyl ether with constant stirring. The crude peptide precipitates as a white solid. The precipitate was filtered, and then washed with cooled diethyl ether 4 times. The white solid was dried in a vacuum oven R-1001-VN. As a result, the synthesis of the crude peptide Ac-Ala-Glu-Asp-Gly-Arg-amide (SEQ. ID 2) was completed.

[0289] III. Purification of crude peptide by RP-HPLC

[0290] To separate and purify the synthesis product, two preparative liquid chromatographs were used: 1) AKTATM Pure 25 with Frac F9-R fraction collector. The Kromasil® 100- 13-C 18 50 x 250 mm column allows the purification of up to 1 -2 g of product in one process with a maximum flow of 25 mL / min; 2) CXTH P1001L. The Vydac® 70 x 150 mm column, 10 mm filter, allows up to 50 g of product to be purified in one process at a maximum flow of 500 mL / min.

[0291] Analysis parameters:

[0292] Sample: purified peptide dissolved in water to a concentration ~5 g / L;

[0293] Mobile phase: ACbLFLO (17:83 v / v) with addition of 0.1% TFA;

[0294] Flow: 25 mL / min / 500 mL / min

[0295] Time of analysis: 30 min;

[0296] Column temperature: 25 °C;

[0297] Detection conditions: X = 220 nm.

[0298] IV. Peptide lyophilization

[0299] To lyophilize the peptide, the solvent obtained during the HPLC purification process was evaporated and dried in a freeze dryer (Christ Beta 2-8) for 48-72 hours. The collected white solid was purified peptide.

[0300] V. Analysis of the synthesis quality

[0301] Peptide purity was checked by analytical HPLC, the molar mass was confirmed by mass spectrometry as described below.

[0302] 1. HPLC analysis to evaluate the purity of the obtained peptide.

[0303] To assess the purity of the purified peptide, the measurement was carried out using HPLC-MS methods using a UHPLC liquid chromatograph (UltiMate 3000 Thermo Scientific / Dionex) with a fast (200 Hz) diode detector coupled with QTOF-MS (Impact HD, Bruker).

[0304] Analysis parameters:

[0305] Sample: 10 pl of the purified peptide dissolved in water (to a concentration of ~5 g / L)

[0306] Mobile phase: ACN:H O (17:83 v / v) with addition of 0.1% TFA;

[0307] Flow: 1 mL / min; Time of analysis: 30 min;

[0308] Column temperature: 25 °C;

[0309] Detection conditions: X = 220 nm.

[0310] Purity obtained was 96%.

[0311] 2. QTOF-MS analysis for peptide identification

[0312] To accurately determine the molecular weight of the peptide, a QTOF high-resolution mass spectrometer was used, a hybrid quadrupole mass spectrometer with time-of-flight analysis - TOF, and atmospheric pressure ionization - ESI (Impact HD, Bruker). The sample was fed to the source using a syringe pump of the chromatograph. A small amount of the peptide was dissolved in MeOH.

[0313] Analysis parameters:

[0314] Sample: 10 pl of the purified peptide dissolved in methanol (to a concentration of ~5 g / L);

[0315] Ionization: nitrogen laser beam, I = 337 nm

[0316] Matrix: a-cyano-4-hydroxy cinnamic acid (CCA).

[0317] The obtained Epithalon derivative of (SEQ. ID 2) was identified by the QTOF MS: Mass Calc, for C22H37N9O10: 587.5835 g / mol, Found: 588.61 [M+H]+;

[0318] EXAMPLE A-1,3

[0319] Preparation of Ac-GHK-Arg-NHj (Ac-Gly-His-Lys-Arg-NHj) (SEQ.. ID 3)

[0320] Outline of the synthesis

[0321] Peptide synthesis was performed using a solid support. Amino acids with a blocked quaternary 9- fluorenylmethoxycarbonyl group were used for the synthesis (Fmoc). During the course of the first step of the synthesis, the first Fmoc -protected amino acid was attached directly to the P-linker of the solid phase support. During the further reaction of cyclic deprotection and condensation of peptide bonds, the remaining amino acids were attached starting from the C -terminal end and ending with the N-terminal part of the peptide.

[0322] In this way, a peptide-carrier complex was obtained (Ac-Gly-His-Lys-Arg-Pro-CTC-Resin). In the next step of the synthesis, the peptide was detached from the support using trifluoroacetic acid (TFA). During cleavage of the peptide from the support, amidation of the C-terminal peptide takes place. As a result, a peptide of the assumed sequence was obtained Ac-Gly-His-Lys-Arg-amide (SEQ. ID 3).

[0323] To obtain a peptide with an assumed purity of >95%, the Reverse Phase High Performance Liquid Chromatography (RP-HPLC) technique was used. The analytical stage was completed with ultra-high- performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (QTOF-MS), which confirmed the molar mass of the synthesized peptide.

[0324] Detailed description of the synthesis

[0325] 1. Peptide synthesis

[0326] The peptide was synthesized on a scale of 10 g

[0327] 1.Charging of the resin Fmoc-Pro-CTC

[0328] A weight of the Fmoc-Pro-CTC-resin carrier was placed in a chemical reactor, then DCM was added and left for 30 min. The DCM was removed and then the resin was rinsed with DMF under constant stirring. The operation was repeated twice, each time using DMF. After rinsing, the DMF solution was thoroughly removed.

[0329] 2. Attachment of Arg to solid support Fmoc-Pro-CTC-Resin.

[0330] After preparing the solid phase, arginine was attached to it. Then, in the deprotection step, the Fmoc group was removed. DP was added with continuous stirring under a nitrogen atmosphere. After 5 min, DP was removed, then the carrier was rinsed once with DMF, which was completely removed in the next step. The deprotection step was repeated again with the addition of DP under continuous stirring under a continuous flow of nitrogen. DP was completely removed after 10 min. The support was washed 6 times, each time using DMF. Fmoc-Arg-OH (3-times the amount of the support used) and HOBt (3- times the amount of the support used) were weighed out and then dissolved in DMF in the reactor. In the next step of the synthesis, DIC was added (3-times the amount of the support used, v:v) with constant stirring under a constant supply of nitrogen for 2 hours. At the end of the step, a ninhydrin test was performed to ensure that Fmoc-Arg-OH is fully attached to the support Fmoc-Pro-CTC-Resin. A negative result of the nihydrin reaction means that there are no free NH2 groups, and thus the reaction is considered complete. The liquid was removed from the reactor then it washed with DMF 3 times. From this point on, the amino acid in the form of Fmoc-Arg-OH is linked to the support. Upon completion of the step, Fmoc-Arg-Pro-CTC-Resin was obtained.

[0331] 3. Attachment Lys to -Arg-Pro-CTC-Resin.

[0332] DP was added with constant stirring under a constant supply of nitrogen. Removed DP after 5 min then rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 minutes then rinsed resin 6 times with DMF each time. Fmoc-Lys-OH was weighed out (3-times the amount of the support used) and HOBt (3-times the amount of the support used) then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. At the end of the step, a ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times using DMF each time. From then on, the amino acid in the form Fmoc-Lys-OH was attached to Arg-Pro-CTC-Resin.

[0333] Fmoc-Lys-Arg-Pro-CTC-Resin was obtained.

[0334] 4. Attachment His to -Lys-Arg-Pro-CTC-Resin.

[0335] DP was added with constant stirring under a constant supply of nitrogen and then the DP was removed after 5 min and rinsed once with DMF which was removed in the next step. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. Removed DP after 10 minutes. The support was then washed 6 times, each time using DMF. Fmoc-His-OH (3-times the amount of the support used) and HOBI (3-times the amount of the support used) were weighed out, dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed from the reactor then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-His-OH was attached to Lys-Arg-ProCTC-Resin. Fmoc-His-Lys-Arg-Pro-CTC-Resin was obtained.

[0336] 5. Attachment Gly to - His-Lys- Arg-Pro-CTC-Resin.

[0337] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Gly-OH was weighed out (3- times the amount of the support used) and HOBI (3-times the amount of the support used), then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Gly-OH was attached to -His-Lys-Arg-Pro- CTC-Resin. As a result, was obtained Fmoc-Gly-His-Lys-Arg-Pro-CTC-Resin.

[0338] 6. Acetylation of the N-terminal amino acid

[0339] DP was added with constant stirring under a constant supply of nitrogen, then DP was removed after 5 min and rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min. The support was washed 6 times, each time using DMF. Prepared were acetic acid (3-times the amount of the support used) and HOBt (3-times the amount of the support used), dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The liquid was removed from the reactor and then washed 3 times using DMF each time, was obtained Ac-Gly-His-Lys-Arg-Pro-CTC-Resin.

[0340] II. Detachment of the peptide from the support After the amino acid attaching steps are completed, all peptide side chains are blocked and the peptide itself is attached to the support. In the next stage of the synthesis, the peptide has to be detached from the support and the side chains deblocked to obtain a crude peptide. Thanks to the use of a modified resin, spontaneous amidation of the C-terminal amino acid occurs during the cleavage of the peptide from the support. The obtained peptide -resin complex together with CS (cleaving solution) was then stirred for 3 hours in the reactor. At this stage, the peptide is released from the support and the side groups are deprotected. Upon completion, the solution was filtered discarding the solid phase and retaining the liquid phase containing the peptide. In the next step, the liquid was cooled using diethyl ether with constant stirring. The crude peptide precipitated as a white precipitate. The precipitate was filtered and then washed with cooled diethyl ether 4 times using diethyl ether each time. The white precipitate was dried in a vacuum oven R-1001-VN. This completes the synthesis of the crude peptide of the sequence Ac-Gly-His-Lys-Arg-amide (SEQ. ID 3).

[0341] III. Purification of the crude peptide by RP-HPLC

[0342] Two preparative liquid chromatographs were used to separate and purify the synthesis product: 1) AKTATM Pure 25 with Frac F9-R fraction collector. Kolumna Kromasil® 100-13-C18 50 x 250 mm allows to purify up to 1-2 g of product in one process at maximum flow 25 mL / min. 2) CXTH P1001L. Vydac column 70 x 150 mm, 10 mm filter allows to purify up to 50 g of product in one process at a maximum flow of 500 mL / min.

[0343] Analysis parameters:

[0344] Sample: purified peptide dissolved in water to concentration ~5 g / L;

[0345] Mobile phase: AC fFLO (17:83 v / v) with 0,1% TFA;

[0346] Flow: 25 mL / min / 500 mL / min

[0347] Analysis time: 30 min;

[0348] Column temperature: 25 °C;

[0349] Detection conditions: I = 220 nm.

[0350] IV. Lyophilization of the peptide

[0351] For lyophilization of the peptide, the solvent obtained during the HPLC paging process was evaporated and dried in a lyophilizer (Christ Beta 2-8) within 48-72 hours.

[0352] The collected white solid phase was the purified peptide.

[0353] V. Synthesis quality analysis

[0354] Peptide purity was checked by analytical HPLC, molar mass was confirmed by mass spectrometry as described below. 1. HPLC analysis to evaluate the purity of the obtained peptide.

[0355] To assess the purity of the purified peptide, HPLC-MS was measured using a liquid chromatograph UHPLC (UltiMate 3000 Thermo Scientific / Dionex) with a fast (200 Hz) diode detector coupled to QTOF-MS (Impact HD, Bruker)

[0356] Analysis parameters:

[0357] Sample: 10 pl dissolved in water (to a concentration of ~5 g / L) of the purified peptide;

[0358] Mobile phase: ACbkHzO (17:83 v / v) with 0,1% TFA;

[0359] Flow: 1 mL / min;

[0360] Analysis time: 30 min;

[0361] Column temperature: 25 °C;

[0362] Detection conditions: I = 220 nm.

[0363] Purity obtained was 95%.

[0364] 2. QTOF-MS analysis to identify the peptide

[0365] To accurately determine the molecular weight of the peptide, a high-resolution QTOF mass spectrometer, a hybrid quadrupole mass spectrometer with time-of-flight analysis was used - TOF, and ionization at atmospheric pressure - ESI (Impact HD, Bruker) The sample was fed to the source by the syringe pump of the chromatograph. A small amount of the peptide was dissolved in MeOH.

[0366] Analysis parameters:

[0367] Sample: 10 pl dissolved in methanol (to a concentration of ~5 g / L) of the purified peptide;

[0368] Ionization: nitrogen laser beam, = 337 nm

[0369] Matrix: a-cyano-4-hydroxycinnamic acid (CCA)

[0370] The obtained GHK derivative of (SEQ. ID 3) was identified by the QTOF MS: Mass Calc, for C22H39N11O5: 537.61 g / mol, Found: 538.60 [M+H]+;

[0371] EXAMPLE A-1.4

[0372] Preparation of Ac-Semax-NH2(Ac-Met-Glu-His-Phe-Pro-Gly-Pro-Arg-NH2)_(SEQ. ID 4)

[0373] Outline of the synthesis

[0374] Peptide synthesis was performed using a solid support. Amino acids with a blocked quaternary 9- fluorenylmethoxycarbonyl group were used for the synthesis (Fmoc). During the course of the first step of the synthesis, the first Fmoc -protected amino acid was attached directly to the P-linker of the solid phase support. During the further reaction of cyclic deprotection and condensation of peptide bonds, the remaining amino acids were attached starting from the C -terminal end and ending with the N-terminal part of the peptide. In this way, a peptide-carrier complex was obtained (Ac-Met-Glu-His-Phe-Pro-Gly- Pro-ArgPro-C-H-Resin). In the next step of the synthesis, the peptide was detached from the support using trifluoroacetic acid (TFA). Amidation of the C-terminal amino acid occurs during cleavage of the peptide from the support. This resulted in a peptide of the assumed sequence Ac-Met-Glu-His-Phe-Pro- Gly-Pro-Arg-amide (SEQ. ID 4).

[0375] To obtain a peptide with the assumed purity of >80%, High Performance Liquid Chromatography in the Reverse Phase System was used (RP-HPLC). The analytical stage was completed with ultra-high- performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (QTOF-MS), which confirmed the molar mass of the synthesized peptide.

[0376] Detailed description of the synthesis

[0377] I. Peptide synthesis

[0378] The peptide was synthesized on a scale of 10 g

[0379] 1. Preparation of Fmoc-Pro-CTC-resin

[0380] A weight of the Fmoc-Pro-CTC-resin carrier was placed in a chemical reactor, then DCM was added and left for 30 min. The DCM was removed and then the resin was rinsed with DMF under constant stirring. The operation was repeated twice, each time using DMF. After rinsing, the DMF solution was thoroughly removed.

[0381] 2. Attachment of Arg to solid support Fmoc-Pro-CTC-Resin.

[0382] After preparing the solid phase, arginine was attached to it. Then, in the deprotection step, the Fmoc group was removed. DP was added with continuous stirring under a nitrogen atmosphere. After 5 min, DP was removed, then the carrier was rinsed once with DMF, which was completely removed in the next step. The deprotection step was repeated again with the addition of DP under continuous stirring under a continuous flow of nitrogen. DP was completely removed after 10 min. The support was washed 6 times, each time using DMF. Fmoc-Arg-OH (3-times the amount of the support used) and HOBt (3- times the amount of the support used) were weighed out and then dissolved in DMF in the reactor. In the next step of the synthesis, DIC was added (3-times the amount of the support used, v:v) with constant stirring under a constant supply of nitrogen for 2 hours. At the end of the step, a ninhydrin test was performed to ensure that Fmoc-Arg-OH is fully attached to the support Fmoc-Pro-CTC-Resin. A negative result of the nihydrin reaction means that there are no free NH2 groups, and thus the reaction is considered complete. The liquid was removed from the reactor then it washed with DMF 3 times. From this point on, the amino acid in the form of Fmoc-Arg-OH is linked to the support. Upon completion of the step, Fmoc-Arg-Pro-CTC-Resin was obtained. 3. Attachment Pro to -Arg-Pro-CTC-Resin.

[0383] DP was added with constant stirring under a constant supply of nitrogen. Removed DP after 5 min then rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 minutes then rinsed resin 6 times with DMF each time. Fmoc-Pro-OH was weighed out (3-times the amount of the support used) and HOBt (3-times the amount of the support used) then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. At the end of the step, a ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times using DMF each time. From then on, the amino acid in the form Fmoc-Pro-OH was attached to Arg-Pro-CTC-Resin. was obtained Fmoc-Pro- Arg-Pro-CTC-Resin.

[0384] 4. Attachment Gly to -Pro-Arg-Pro-CTC-Resin.

[0385] DP was added with constant stirring under a constant supply of nitrogen and then the DP was removed after 5 min and rinsed once with DMF which was removed in the next step. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. Removed DP after 10 minutes. The support was then washed 6 times, each time using DMF. Odwazono Fmoc-Gly-OH (3- times the amount of the support used) and HOBt (3-times the amount of the support used), dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed from the reactor then washed 3 times with DMF.

[0386] From then on, the amino acid in the form Fmoc-Gly-OH was attached to Pro-Arg-ProCTC-Resin. Fmoc- Gly-Pro- Arg-Pro-CTC-Resin was obtained.

[0387] 5. Attachment Pro to -Gly-Pro- Arg-Pro-CTC-Resin.

[0388] Step 3 was repeated three times. After the stage was completed, was obtained Fmoc-Pro-Gly-Pro-Arg- ProCTC-Resin.

[0389] 6. Attachment Phe to Pro-Gly-Pro- Arg-Pro-CTC-Resin.

[0390] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Phe-OH (3-times the amount of the support used) and HOBt (3-times the amount of the support used) were weighted out, then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Phe-OH was attached to -Pro-Gly-Pro-Arg- Pro-CTC-Resin. As a result, was obtained Fmoc-Phe-Pro-Gly-Pro-Arg-Pro-CTC-Resin.

[0391] 7. Attachment His to -Phe-Pro-Gly-Pro-Arg-Pro-CTC-Resin.

[0392] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Przygotowano nawazkc Fmoc-His-OH (3-times the amount of the support used) and HOBt (3-times the amount of the support used), then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-His-OH was attached to -Phe-Pro- Gly-Pro-ArgPro-CTC-Resin. As a result, was obtained Fmoc-His-Phe-Pro-Gly-Pro-Arg-Pro-CTC- Resin.

[0393] 8. Attachment Glu to His-Phe-Pro-Gly-Pro-Arg-Pro-CTC-Resin.

[0394] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Glu-OH was weighed out (3- times the amount of the support used) and HOBt

[0395] (3-times the amount of the support used), then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Glu-OH was attached to His-Phe-Pro-Gly-ProArg-Pro-CTC-Resin. As a result, was obtained Fmoc-Glu-His-Phe-Pro-Gly-Pro-Arg-Pro-CTC-Resin.

[0396] 9. Attachment Met to -Phe-Pro-Gly-Pro-Arg-Pro-CTC-Resin.

[0397] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Met-OH (3-times the amount of the support used) and HOBt (3-times the amount of the support used) were weighted out, then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Met-OH was attached to Glu-His-Phe-Pro- Gly-Pro-Arg-Pro-CTC-Resin. As a result, was obtained Fmoc-Met-Glu-His-Phe-Pro-Gly-Pro-Arg-Pro- CTC-Resin.

[0398] 10. Acetylation of the N-terminal amino acid

[0399] DP was added with constant stirring under a constant supply of nitrogen, then DP was removed after 5 min and rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min. The support was washed 6 times, each time using DMF. Prepared were acetic acid (3-times the amount of the support used) and HOBt (3-times the amount of the support used), dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The liquid was removed from the reactor and then washed 3 times using DMF each time, was obtained Ac-Met-Glu-His-Phe-Pro-Gly-Pro-Arg-Pro-CTC-Resin.

[0400] 11. Detachment of the peptide from the support

[0401] After the amino acid attaching steps are completed, all peptide side chains are blocked and the peptide itself is attached to the support. In the next stage of the synthesis, the peptide has to be detached from the support and the side chains deblocked to obtain a crude peptide. Thanks to the use of a modified resin, spontaneous amidation of the C-terminal amino acid occurs during the cleavage of the peptide from the support. The obtained peptide -resin complex together with CS (cleaving solution) was then stirred for 3 hours in the reactor. At this stage, the peptide is released from the support and the side groups are deprotected. Upon completion, the solution was filtered discarding the solid phase and retaining the liquid phase containing the peptide. In the next step, the liquid was cooled using diethyl ether with constant stirring. The crude peptide precipitated as a white precipitate. The precipitate was filtered and then washed with cooled diethyl ether 4 times using diethyl ether each time. The white precipitate was dried in a vacuum oven R-1001-VN. This completes the synthesis of the crude peptide of the sequence Ac-Met-Glu-His-Phe-Pro-Gly-Pro-Arg-amide (SEQ. ID 4).

[0402] III. Purification of the crude peptide by RP-HPLC

[0403] Two preparative liquid chromatographs were used to separate and purify the synthesis product: 1) AKTA™ Pure 25 with Frac F9-R fraction collector. The Kromasil® 100-13-C18 50 x 250 mm column allows the purification of up to 1-2 g of product in one process at maximum flow 25 mL / min; 2) CXTH P 100 IL. Vydac® column 70 x 150 mm, 10 mm filter allows to purify up to 50 g of product in one process at maximum flow 500 mL / min.

[0404] Analysis parameters:

[0405] Sample: purified peptide dissolved in water to concentration ~5 g / L; Mobile phase: ACNiHzO (17:83 v / v) with 0,1% TFA;

[0406] Flow: 25 mL / min / 500 mL / min

[0407] Analysis time: 30 min;

[0408] Column temperature: 25 °C;

[0409] Detection conditions: X = 220 nm.

[0410] IV. Lyophilization of the peptide

[0411] For lyophilization of the peptide, the solvent obtained during the HPLC paging process was evaporated and dried in a lyophilizer (Christ Beta 2-8) within 48-72 hours.

[0412] The collected white solid phase was the purified peptide.

[0413] V. Synthesis quality analysis

[0414] Peptide purity was checked by analytical HPLC, molar mass was confirmed by mass spectrometry as described below.

[0415] 1. HPLC analysis to evaluate the purity of the obtained peptide.

[0416] To assess the purity of the purified peptide, HPLC-MS was measured using a liquid chromatograph UHPLC (UltiMate 3000 Thermo Scientific / Dionex) with a fast (200 Hz) diode detector coupled to QTOF-MS (Impact HD, Bruker)

[0417] Analysis parameters:

[0418] Sample: 10 pl dissolved in water (to a concentration of ~5 g / L) of the purified peptide;

[0419] Mobile phase: AC hHzO (17:83 v / v) with 0,1% TFA;

[0420] Flow: 1 mL / min;

[0421] Analysis time: 30 min;

[0422] Column temperature: 25 °C;

[0423] Detection conditions: I = 220 nm.

[0424] Purity obtained was 92%.

[0425] 2. QTOF-MS analysis to identify the peptide

[0426] To accurately determine the molecular weight of the peptide, a high-resolution QTOF mass spectrometer, a hybrid quadrupole mass spectrometer with time-of-flight analysis was used - TOF, and ionization at atmospheric pressure - ESI (Impact HD, Bruker)

[0427] The sample was fed to the source by the syringe pump of the chromatograph. A small amount of the peptide was dissolved in MeOH. Analysis parameters:

[0428] Sample: 10 pl dissolved in methanol (to a concentration of ~5 g / L) of the purified peptide;

[0429] Ionization: nitrogen laser beam, X = 337 nm

[0430] Matrix: a-cyano-4-hydroxy cinnamic acid (CCA).

[0431] The obtained Semax derivative of (SEQ. ID 4) was identified by the QTOF MS: Mass Calc, for C45H66N14O11S1: 1011.16 g / mol, Found: 1012.51 [M+H]+

[0432] EXAMPLE A-1.5

[0433] Preparation of Ac-Selank-Arg-NHz (Ac-Thr-Lys-Pro-Arg-Pro-Gly-Pro-Arg-NHz) (SEQ.. ID 5).

[0434] Outline of the synthesis

[0435] Peptide synthesis was performed using a solid support. Amino acids with a blocked quaternary 9- fluorenylmethoxycarbonyl group were used for the synthesis (Fmoc). During the course of the first step of the synthesis, the first Fmoc -protected amino acid was attached directly to the P-linker of the solid phase support. During the further reaction of cyclic deprotection and condensation of peptide bonds, the remaining amino acids were attached starting from the C-terminal end and ending with the N-terminal part of the peptide.

[0436] In this way, a peptide-carrier complex was obtained (Ac-Thr-Lys-Pro-Arg-Pro-Gly-Pro-ArgPro-C-H- Resin). In the next step of the synthesis, the peptide was detached from the support using trifluoroacetic acid (TFA). Amidation of the C-terminal amino acid occurs during cleavage of the peptide from the support. This resulted in a peptide of the assumed sequence Ac-Thr-Lys-Pro-Arg-Pro-Gly-Pro-Arg- amide (SEQ. ID 5).

[0437] To obtain a peptide with the assumed purity of >80%, High Performance Liquid Chromatography in the Reverse Phase System was used (RP-HPLC). The analytical stage was completed with ultra-high- performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (QTOF-MS), which confirmed the molar mass of the synthesized peptide.

[0438] Detailed description of the synthesis

[0439] I. Peptide synthesis

[0440] The peptide was synthesized on a scale of 10 g

[0441] 1. Preparation of Fmoc-Pro-CTC-resin

[0442] A weight of the Fmoc-Pro-CTC-resin carrier was placed in a chemical reactor, then DCM was added and left for 30 min. The DCM was removed and then the resin was rinsed with DMF under constant stirring. The operation was repeated twice, each time using DMF. After rinsing, the DMF solution was thoroughly removed.

[0443] 2. Attachment of Arg to solid support Fmoc-Pro-CTC-Resin.

[0444] After preparing the solid phase, arginine was attached to it. Then, in the deprotection step, the Fmoc group was removed. DP was added with continuous stirring under a nitrogen atmosphere. After 5 min, DP was removed, then the carrier was rinsed once with DMF, which was completely removed in the next step. The deprotection step was repeated again with the addition of DP under continuous stirring under a continuous flow of nitrogen. DP was completely removed after 10 min. The support was washed 6 times, each time using DMF. Fmoc-Arg-OH (3-times the amount of the support used) and HOBt (3- times the amount of the support used) were weighed out and then dissolved in DMF in the reactor. In the next step of the synthesis, DIC was added (3-times the amount of the support used, v:v) with constant stirring under a constant supply of nitrogen for 2 hours. At the end of the step, a ninhydrin test was performed to ensure that Fmoc-Arg-OH is fully attached to the support Fmoc-Pro-CTC-Resin. A negative result of the nihydrin reaction means that there are no free NH2 groups, and thus the reaction is considered complete. The liquid was removed from the reactor then it washed with DMF 3 times. From this point on, the amino acid in the form of Fmoc-Arg-OH is linked to the support. Upon completion of the step, Fmoc-Arg-Pro-CTC-Resin was obtained.

[0445] 3. Attachment Pro to -Arg-Pro-CTC-Resin.

[0446] DP was added with constant stirring under a constant supply of nitrogen. Removed DP after 5 min then rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 minutes then rinsed resin 6 times with DMF each time. Fmoc-Pro-OH was weighed out (3-times the amount of the support used) and HOBt (3-times the amount of the support used) then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. At the end of the step, a ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times using DMF each time. From then on, the amino acid in the form Fmoc-Pro-OH was attached to Arg-Pro-CTC-Resin. was obtained Fmoc-Pro- Arg-Pro-CTC-Resin.

[0447] 4. Attachment Gly to -Pro-Arg-Pro-CTC-Resin.

[0448] DP was added with constant stirring under a constant supply of nitrogen and then the DP was removed after 5 min and rinsed once with DMF which was removed in the next step. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. Removed DP after 10 minutes. The support was then washed 6 times, each time using DMF. Odwazono Fmoc-Gly-OH (3- times the amount of the support used) and HOBt (3-times the amount of the support used), dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed from the reactor then washed 3 times with DMF.

[0449] From then on, the amino acid in the form Fmoc-Gly-OH was attached to Pro-Arg-Pro-CTC-Resin. Fmoc-Gly-Pro-Arg-Pro-CTC-Resin was obtained.

[0450] 5. Attachment Pro to -Gly-Pro-Arg-Pro-CTC-Resin.

[0451] Step 3 was repeated. After the stage was completed, was obtained Fmoc-Pro-Gly-Pro-Arg-Pro-CTC- Resin.

[0452] 6. Attachment Arg do Pro-Gly-Pro-Arg-Pro-CTC-Resin.

[0453] Step 2 was repeated. After the stage was completed, was obtained Fmoc-Arg-Pro-Gly-Pro-Arg-Pro- CTC-Resin.

[0454] 7. Attachment Pro to -Gly-Pro-Arg-Pro-CTC-Resin.

[0455] Step 3 was repeated. After the stage was completed, was obtained Fmoc-Pro-Arg-Pro-Gly-Pro-Arg- ProCTC-Resin.

[0456] 8. Attachment Lys to -Pro-Arg-Pro-CTC-Resin.

[0457] DP was added with constant stirring under a constant supply of nitrogen and then the DP was removed after 5 min and rinsed once with DMF which was removed in the next step. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. Removed DP after 10 minutes. The support was then washed 6 times, each time using DMF. Fmoc-Lys-OH (3-times the amount of the support used) and HOBt (3-times the amount of the support used) were weighted out, dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed from the reactor then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Lys-OH was attached to Pro-Arg-Pro-Gly-Pro-Arg-Pro-CTC-Resin. Fmoc-Lys-Pro-Arg-Pro-Gly-Pro-Arg-Pro-CTC-Resin was obtained.

[0458] 9. Attachment Thr to -Pro-Arg-Pro-CTC-Resin.

[0459] DP was added with constant stirring under a constant supply of nitrogen and then the DP was removed after 5 min and rinsed once with DMF which was removed in the next step. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. Removed DP after 10 minutes. The support was then washed 6 times, each time using DMF. Fmoc-Thr-OH (3-times the amount of the support used) and HOBt (3-times the amount of the support used) were weighted out, dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed from the reactor then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Thr-OH was attached to Lys-Pro-Arg-Pro-Gly-Pro-Arg-Pro-CTC-Resin. Fmoc-Thr-Lys-Pro-Arg-Pro-Gly-Pro-Arg-Pro-CTC- Resin was obtained.

[0460] 10. Acetylation of the N-terminal amino acid

[0461] DP was added with constant stirring under a constant supply of nitrogen, then DP was removed after 5 min and rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min. The support was washed 6 times, each time using DMF. Prepared were acetic acid (3-times the amount of the support used) and HOBt (3-times the amount of the support used), dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The liquid was removed from the reactor and then washed 3 times using DMF each time, was obtained Ac-Thr-Lys-Pro-Arg-Pro-Gly-Pro-Arg-Pro-CTC-Resin.

[0462] 11. Detachment of the peptide from the support

[0463] After the amino acid attaching steps are completed, all peptide side chains are blocked and the peptide itself is attached to the support. In the next stage of the synthesis, the peptide has to be detached from the support and the side chains deblocked to obtain a crude peptide. Thanks to the use of a modified resin, spontaneous amidation of the C-terminal amino acid occurs during the cleavage of the peptide from the support. The obtained peptide -resin complex together with CS (cleaving solution) was then stirred for 3 hours in the reactor. At this stage, the peptide is released from the support and the side groups are deprotected. Upon completion, the solution was filtered discarding the solid phase and retaining the liquid phase containing the peptide. In the next step, the liquid was cooled using diethyl ether with constant stirring. The crude peptide precipitated as a white precipitate. The precipitate was filtered and then washed with cooled diethyl ether 4 times using diethyl ether each time. The white precipitate was dried in a vacuum oven R-1001-VN. This completes the synthesis of the crude peptide of the sequence Ac-Thr-Lys-Pro-Arg-Pro-Gly-Pro-Arg-amide (SEQ. ID 5).

[0464] III. Purification of the crude peptide by RP-HPLC

[0465] Two preparative liquid chromatographs were used to separate and purify the synthesis product: 1) AKTA™ Pure 25 with Frac F9-R fraction collector. The Kromasil® 100-13-C18 50 x 250 mm column allows the purification of up to 1-2 g of product in one process at maximum flow 25 mL / min; 2) CXTH P 100 IL. Vydac® column 70 x 150 mm, 10 mm filter allows to purify up to 50 g of product in one process at maximum flow 500 mL / min.

[0466] Analysis parameters: Sample: purified peptide dissolved in water to concentration ~5 g / L;

[0467] Mobile phase: AC hHzO (17:83 v / v) with 0,1% TFA;

[0468] Flow: 25 mL / min / 500 mL / min

[0469] Analysis time: 30 min;

[0470] Column temperature: 25 °C;

[0471] Detection conditions: X = 220 nm.

[0472] IV. Lyophilization of the peptide

[0473] For lyophilization of the peptide, the solvent obtained during the HPLC paging process was evaporated and dried in a lyophilizer (Christ Beta 2-8) within 48-72 hours.

[0474] The collected white solid phase was the purified peptide.

[0475] V. Synthesis quality analysis

[0476] Peptide purity was checked by analytical HPLC, molar mass was confirmed by mass spectrometry as described below.

[0477] 1. HPLC analysis to evaluate the purity of the obtained peptide.

[0478] To assess the purity of the purified peptide, HPLC-MS was measured using a liquid chromatograph UHPLC (UltiMate 3000 Thermo Scientific / Dionex) with a fast (200 Hz) diode detector coupled to QTOF-MS (Impact HD, Bruker)

[0479] Analysis parameters:

[0480] Sample: 10 pl dissolved in water (to a concentration of ~5 g / L) of the purified peptide;

[0481] Mobile phase: AC hHzO (17:83 v / v) with 0,1% TFA;

[0482] Flow: 1 mL / min;

[0483] Analysis time: 30 min;

[0484] Column temperature: 25 °C;

[0485] Detection conditions: I = 220 nm.

[0486] Purity obtained was 84%.

[0487] 2. QTOF-MS analysis to identify the peptide

[0488] To accurately determine the molecular weight of the peptide, a high-resolution QTOF mass spectrometer, a hybrid quadrupole mass spectrometer with time-of-flight analysis was used - TOF, and ionization at atmospheric pressure - ESI (Impact HD, Bruker) The sample was fed to the source by the syringe pump of the chromatograph. A small amount of the peptide was dissolved in MeOH.

[0489] Analysis parameters:

[0490] Sample: 10 pl dissolved in methanol (to a concentration of ~5 g / L) of the purified peptide;

[0491] Ionization: nitrogen laser beam, X = 337 nm

[0492] Matrix: a-cyano-4-hydroxy cinnamic acid (CCA).

[0493] The obtained Selank derivative of (SEQ. ID 5) was identified by the QTOF MS: Mass Calc, for C41H72N16O10: 949.11 g / mol, Found: 950.25 [M+H]+

[0494] EXAMPLE A-1.6

[0495] Preparation of Ac-Cortagen-Arg-NHj (Ac-Ala-Glu-Asp-Pro-Arg-NHz) (SEQ.. ID 6)

[0496] Outline of the synthesis

[0497] Peptide synthesis was performed using a solid support. Amino acids with a blocked quaternary 9- fluorenylmethoxycarbonyl group were used for the synthesis (Fmoc). During the course of the first step of the synthesis, the first Fmoc -protected amino acid was attached directly to the P-linker of the solid phase support. During the further reaction of cyclic deprotection and condensation of peptide bonds, the remaining amino acids were attached starting from the C-terminal end and ending with the N-terminal part of the peptide.

[0498] In this way, a peptide-carrier complex was obtained (Ac-Ala-Glu-Asp-Pro-Arg-Pro-CH-Resin). In the next step of the synthesis, the peptide was detached from the support using trifluoroacetic acid (TFA). Amidation of the C-terminal amino acid occurs during cleavage of the peptide from the support. This resulted in a peptide of the assumed sequence Ac-Ala-Glu-Asp-Pro-Arg-amide (SEQ. ID 6).

[0499] To obtain a peptide with the assumed purity of >80%, High Performance Liquid Chromatography in the Reverse Phase System was used (RP-HPLC). The analytical stage was completed with ultra-high- performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (QTOF-MS), which confirmed the molar mass of the synthesized peptide.

[0500] Detailed description of the synthesis

[0501] I. Peptide synthesis

[0502] The peptide was synthesized on a scale of 10 g

[0503] 1. Preparation of Fmoc-Pro-CTC-resin A weight of the Fmoc-Pro-CTC-resin carrier was placed in a chemical reactor, then DCM was added and left for 30 min. The DCM was removed and then the resin was rinsed with DMF under constant stirring. The operation was repeated twice, each time using DMF. After rinsing, the DMF solution was thoroughly removed.

[0504] 2. Attachment of Arg to solid support Fmoc-Pro-CTC-Resin.

[0505] After preparing the solid phase, arginine was attached to it. Then, in the deprotection step, the Fmoc group was removed. DP was added with continuous stirring under a nitrogen atmosphere. After 5 min, DP was removed, then the carrier was rinsed once with DMF, which was completely removed in the next step. The deprotection step was repeated again with the addition of DP under continuous stirring under a continuous flow of nitrogen. DP was completely removed after 10 min. The support was washed 6 times, each time using DMF. Fmoc-Arg-OH (3-times the amount of the support used) and HOBt (3- times the amount of the support used) were weighed out and then dissolved in DMF in the reactor. In the next step of the synthesis, DIC was added (3-times the amount of the support used, v:v) with constant stirring under a constant supply of nitrogen for 2 hours. At the end of the step, a ninhydrin test was performed to ensure that Fmoc-Arg-OH is fully attached to the support Fmoc-Pro-CTC-Resin. A negative result of the nihydrin reaction means that there are no free NH2 groups, and thus the reaction is considered complete. The liquid was removed from the reactor then it washed with DMF 3 times. From this point on, the amino acid in the form of Fmoc-Arg-OH is linked to the support. Upon completion of the step, Fmoc-Arg-Pro-CTC-Resin was obtained.

[0506] 3. Attachment Pro to -Arg-Pro-CTC-Resin.

[0507] DP was added with constant stirring under a constant supply of nitrogen. Removed DP after 5 min then rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 minutes then rinsed resin 6 times with DMF each time. Fmoc-Pro-OH was weighed out (3-times the amount of the support used) and HOBt (3-times the amount of the support used) then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. At the end of the step, a ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times using DMF each time. From then on, the amino acid in the form Fmoc-Pro-OH was attached to Arg-Pro-CTC-Resin. was obtained Fmoc-Pro- Arg-Pro-CTC-Resin.

[0508] 4. Attachment Asp to -Pro- Arg-Pro-CTC-Resin.

[0509] DP was added with constant stirring under a constant supply of nitrogen and then the DP was removed after 5 min and rinsed once with DMF which was removed in the next step. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. Removed DP after 10 minutes. The support was then washed 6 times, each time using DMF. Fmoc-Asp-OH was weighed out (3-times the amount of the support used) and HOBt (3-times the amount of the support used), dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed from the reactor then washed 3 times with DMF.

[0510] From then on, the amino acid in the form Fmoc-Asp-OH was attached to Pro-Arg-ProCTC-Resin. Fmoc- Asp-Pro-Arg-Pro-CTC-Resin was obtained.

[0511] 5. Attachment Glu to Asp-Pro-Arg-Pro-CTC-Resin.

[0512] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Glu-OH was weighed out (3- times the amount of the support used) and HOBt (3-times the amount of the support used), then it was dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Glu-OH was attached to -Asp-Pro-Arg-Pro- CTC-Resin. As a result, was obtained Fmoc-Glu-Asp-Pro-Arg-Pro-CTC-Resin.

[0513] 6. Attachment Ala to -Glu-Asp-Pro-Arg-Pro-CTC-Resin.

[0514] DP was added with constant stirring under a constant supply of nitrogen, next DP was removed after 5 min and rinsed once with DMF, which was then removed from the reactor. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min, the resin was then rinsed 6 times, each time using DMF. Fmoc-Ala-OH was weighed out (3- times the amount of the support used) and HOBt (3-times the amount of the support used), then it was dissolved in DMF and charged to the reactor.

[0515] DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. A ninhydrin test was performed to ensure that the reaction was complete. The liquid was removed and then washed 3 times with DMF. From then on, the amino acid in the form Fmoc-Ala-OH was attached to -Glu-Asp-Pro-Arg-ProCTC-Resin. As a result, was obtained Fmoc-Ala-Glu-Asp-Pro-Arg-Pro-CTC-Resin.

[0516] 7. Acetylation of the N-terminal amino acid

[0517] DP was added with constant stirring under a constant supply of nitrogen, then DP was removed after 5 min and rinsed once with DMF which was then removed. The deprotection was repeated by adding DP with constant stirring under a constant supply of nitrogen. DP was removed after 10 min. The support was washed 6 times, each time using DMF. Prepared were acetic acid (3-times the amount of the support used) and HOBt (3-times the amount of the support used), dissolved in DMF and charged to the reactor. DIC was then added (3-times the amount of the support used) with constant stirring under a constant supply of nitrogen within 2 hours. The liquid was removed from the reactor and then washed 3 times using DMF each time, was obtained Ac-Ala-Glu-Asp-Pro-Arg-Pro-CTC-Resin.

[0518] II. Detachment of the peptide from the support

[0519] After the amino acid attaching steps are completed, all peptide side chains are blocked and the peptide itself is attached to the support. In the next stage of the synthesis, the peptide has to be detached from the support and the side chains deblocked to obtain a crude peptide. Thanks to the use of a modified resin, spontaneous amidation of the C-terminal amino acid occurs during the cleavage of the peptide from the support. The obtained peptide -resin complex together with CS (cleaving solution) was then stirred for 3 hours in the reactor. At this stage, the peptide is released from the support and the side groups are deprotected. Upon completion, the solution was filtered discarding the solid phase and retaining the liquid phase containing the peptide. In the next step, the liquid was cooled using diethyl ether with constant stirring. The crude peptide precipitated as a white precipitate. The precipitate was filtered and then washed with cooled diethyl ether 4 times using diethyl ether each time. The white precipitate was dried in a vacuum oven R-1001-VN. This completes the synthesis of the crude peptide of the sequence Ac-Ala-Glu-Asp-Pro-Arg-amide (SEQ. ID 6).

[0520] III. Purification of the crude peptide by RP-HPLC

[0521] Two preparative liquid chromatographs were used to separate and purify the synthesis product: 1) AKTA™ Pure 25 with Frac F9-R fraction collector. The Kromasil® 100-13-C18 50 x 250 mm column allows the purification of up to 1-2 g of product in one process at maximum flow 25 mL / min; 2) CXTH P1001L. Vydac® column 70 x 150 mm, 10 mm filter allows to purify up to 50 g of product in one process at maximum flow 500 mL / min.

[0522] Analysis parameters:

[0523] Sample: purified peptide dissolved in water to concentration ~5 g / L;

[0524] Mobile phase: AC fFLO (17:83 v / v) with 0,1% TFA;

[0525] Flow: 25 mL / min / 500 mL / min

[0526] Analysis time: 30 min;

[0527] Column temperature: 25 °C;

[0528] Detection conditions: I = 220 nm. IV. Lyophilization of the peptide

[0529] For lyophilization of the peptide, the solvent obtained during the HPLC paging process was evaporated and dried in a lyophilizer (Christ Beta 2-8) within 48-72 hours.

[0530] The collected white solid phase was the purified peptide.

[0531] V. Synthesis quality analysis

[0532] Peptide purity was checked by analytical HPLC, molar mass was confirmed by mass spectrometry as described below.

[0533] 1. HPLC analysis to evaluate the purity of the obtained peptide.

[0534] To assess the purity of the purified peptide, HPLC-MS was measured using a liquid chromatograph UHPLC (UltiMate 3000 Thermo Scientific / Dionex) with a fast (200 Hz) diode detector coupled to QTOF-MS (Impact HD, Bruker)

[0535] Analysis parameters:

[0536] Sample: 10 pl dissolved in water (to a concentration of ~5 g / L) of the purified peptide;

[0537] Mobile phase: AC hHzO (17:83 v / v) with 0,1% TFA;

[0538] Flow: 1 mL / min;

[0539] Analysis time: 30 min;

[0540] Column temperature: 25 °C;

[0541] Detection conditions: I = 220 nm.

[0542] Purity obtained was 86%.

[0543] 2. QTOF-MS analysis to identify the peptide

[0544] To accurately determine the molecular weight of the peptide, a high-resolution QTOF mass spectrometer, a hybrid quadrupole mass spectrometer with time-of-flight analysis was used - TOF, and ionization at atmospheric pressure - ESI (Impact HD, Bruker)

[0545] The sample was fed to the source by the syringe pump of the chromatograph. A small amount of the peptide was dissolved in MeOH.

[0546] Analysis parameters:

[0547] Sample: 10 pl dissolved in methanol (to a concentration of ~5 g / L) of the purified peptide;

[0548] Ionization: nitrogen laser beam, I = 337 nm

[0549] Matrix: a-cyano-4-hydroxy cinnamic acid (CCA). The obtained Cortagen derivative of (SEQ. ID 6) was identified by the QTOF MS: Mass Calc, for C25H41N9O10: 627.65 g / mol, Found: 628.71 [M+H]+

[0550] Preparation of the capsules of the invention

[0551] The single dosage form of the invention containing a peptide agent was prepared by manually.

[0552] General Procedure:

[0553] A peptide agent and a carrier (microcrystalline cellulose) were weighted out, blended and packed into a gelatine capsule.

[0554] Further, the organic acid (powder or micropellets) and the absorption enhancer (micropellets) were weighted out, blended, and packed together with the gelatine capsule into outer enteric capsule.

[0555] Table 1: Examples of single dosage form Preparation of the comparative capsules

[0556] Two sets of comparative capsules were prepared.

[0557] The first set of comparative compounds was prepared as above but taking organic acid and carnitine compound in non-micropelleted form.

[0558] Table 2

[0559] The second set of comparative examples has been taken from our previous unpublished European application EP 22156902.3 describing capsule-in-capsule formulations, but in the form two capsules, an outer enteric capsule, and an inner capsule, wherein said inner capsule comprises at least one physiologically active peptide agent and at least one absorption enhancer, and wherein said outer capsule contains inner capsule and an organic acid.

[0560] Table 3

[0561] Test description

[0562] All capsules of the invention as well as comparative capsules were subjected to storage stability test.

[0563] The aim of this test was to assess the physio-chemical and organoleptic stability of all the products.

[0564] Methodology. The products were stored for 2, 4 and 6 (T2, T4 and T6) weeks after production at room temperature (20 °C, 50% RH), at refrigeration temperature (4°C), at -18°C (97% RH), and at 40° C, 20% RH in PET bottles.

[0565] The physio-chemical analysis was performed as follows.

[0566] Dry matter content - dry matter content was determined in accordance with the Polish Standard PN-A- 88027:1984.

[0567] Determination of dry matter content was performed by drying method at 105 °C. A clean, dry weighing bottle with a lid was placed in a dryer heated to 105 °C ± 2°C and dried for about 40 minutes to constant weight. After this time, the vessel was closed with a lid, transferred to a desiccator, cooled to laboratory temperature, and weighed. 5.0 g ± 0.001 g of the sample was weighed into the weighed weighing bottle. The weight of the sample cup was recorded. The cup was placed in a dryer heated to 105°C ± 2°C, the lid was removed and dried for 3 h. After 3 h of drying, the cup was closed with the lid and transferred to a desiccator to cool down to laboratory temperature (cooling time 30-45 min.). The lid of the sample cup was opened briefly, then weighed and recorded. The cup was put back into the dryer and dried for 30 min. After this time, the vessel with the sample was cooled in a desiccator and weighed again. This was done until two consecutive determinations differed by more than 0.004 g.

[0568] The dry matter content (X) is calculated in % according to the formula: wherein: a - mass of the cup with the test sample before drying (g); b - mass of the cup with the test sample after drying (g); c - mass of the empty cup (g).

[0569] Moisture content was calculated as a difference between dry matter content and the actual mass taken for the dry matter test.

[0570] Visual inspection of capsules

[0571] The consumer evaluation was performed using the ten-point hedonic scale method and was carried out in a group of 12 people in a sensory laboratory meeting the requirements of the PN-ISO 8589:1998 standard. The evaluation sheet was prepared based on the study by J. Gawccka T. Jqdryk “Analiza sensory czna. Wybrane metody i przyklady zastosowari”, with own modifications. The distinguishing features that the evaluators considered were color, taste, smell, and general desirability of a given sample. The assessment was carried out on a ten-point scale on a panel of 12 men aged 35-40., wherein 1 is the lowest rating of respondents, and 10 is the highest rating of respondents.

[0572] Results:

[0573] 1. Comparison of Moisture content, dry matter content, for Examples of the inventions as well as for Comparative Examples 1 and 2 of table 1 : Table. 4. Example 1 of Table 1 (N-Ac-GHK-Arg-NH2) (SEQ. ID 3):

[0574] Table. 5. Example 2 of Table 1 (N-Ac-Epithalon-Arg-NJE) (SEQ.. ID 2):

[0575] Moreover, all the examples mentioned in the Table 1 performed analogously giving moisture contents around 0% and dry matter content around 100%.

[0576] Table 6. Comparative Example 1 (CE-1, N-Ac-GHK-Arg-NHz) (SEQ. ID 3):

[0577] Table 7. Comparative Example 2 (CE-2, N-Ac-Epithalon-Arg-NHj) (SEQ. ID 2):

[0578] For all the comparative examples, it can be noticed that the moisture content rises during storage, reaching after 6 weeks of storage at room temperature as much as 10%. Moisture content is an important indicator of product stability, and the higher moisture content the lower resultant stability.

[0579] 2. Comparison of visual inspection for Examples 1 and 2 of the inventions as well as for Comparative Examples 3 and 4 (CE-3, CE-4) after long-term of storage (6 months) at room temperature (20°C). Table 8.

[0580] Blood test

[0581] Blood test for EXAMPLE 50 and COMPARATIVE EXAMPLE CE-3 (table 2) and Comparative Example 2.1 from the EP 22156902.3.

[0582] Preparation of the dosage form of Example 50 from the table 1.

[0583] The single dosage form of the invention containing Epithalon peptide (prepared by the manual technology. The peptide agent (20 mg) and microcrystalline cellulose (1000 mg) were weighed out and blended.

[0584] Each gelatine capsule of size 4 (inner capsule) was filled manually with 102 mg of the above blend so that the capsule contains 2 mg of the peptide agent and 100 mg of microcrystalline cellulose.

[0585] 275 mg of 80% pellets citric acid (220mg API) and 145mg of 50% lauroyl L-carnitine pellets (72,5mg API) was introduced into the enteric capsule size 00 and (outer capsule). Inner capsule was then placed in the outer capsule filled previously with pellets and the outer capsule was closed.

[0586] Comparative example CE-3 of table 2 is the same as Comparative example 2 from EP22156902.3. It was prepared as shown in table 2. Comparative example 2.1 from EP22156902.3

[0587] Comparative capsule was prepared by intermixing powdered 2 mg of Epithalon peptide, 100 mg of acyl L- carnitine and 400 mg of citric acid and placing them in the enteric capsule size 00.

[0588] The single dosage form of the invention of EXAMPLE 50 (invention) as well as the single dosage form from COMPARATIVE EXAMPLE CE-3 of table 2, and Comparative Example 2.1 from the EP 22156902.3 were administered p.o. (orally) to a patient.

[0589] The plasma concentration of the peptide was studied in the range time from 0 min to 360 min after the swallowing of a capsule, wherein the 0 min time point was taken shortly after taking the capsule.

[0590] The peptide agent in the blood samples was quantified by high-performance liquid chromatography (HPLC) with the use of post-column ninhydrin derivatization and UV detection at the 220 nm, using standard curve method.

[0591] Liquid chromatograph Perkin-Elmer LC-200 with autosampler LC-200 and DIODE ARRAY LC-235 detector, TOTALCHROM ver. 6.3.2 software were used.

[0592] Column: Acclaim Mixed-Mode WAX-1 5 um (2,1 x 150mm), precolumn Acclaim Mixed-Mode WAX- 1. Conditions of the HPLC run:

[0593] - Mobile phase flow: 0.2 mL / min

[0594] - Temperature: 35 °C

[0595] - Mobile phase: A: 50 mM potassium phosphate; B: methanol

[0596] - Dosing loop - 10 pl

[0597] - Detector UV : 220 nm

[0598] - Gradient program: For comparative purposes, the peaks from HPLC analysis of: Example 50 (solid circle), COMPARATIVE EXAMPLE CE-3 of table 2 (triangle), and Comparative Example 2.1 from the EP 22156902.3 and Comp. Ex. 2.1 (square) for the samples tested after 120 minutes were superimposed on one plot shown on Fig. 1.

[0599] Table 9 below shows the concentrations of the peptide agent calculated for Example 50, COMPARATIVE EXAMPLE CE-3 and Comparative Example 2.1 from the EP 22156902.3 shown at Fig.l (after 120 min).

[0600] Table 9

[0601] Corresponding data were collected for all samples taken in the time range from 0 min to 360 min. Fig 1. shows plot of the concentrations of the peptide agent in the blood for Example 50 (solid circle), Comp. Ex. CE-3 from Table 2 (triangle) and Comp. Ex. 2.1 from the EP 22156902.3 (square). Three time points were chosen for detailed analysis: after 60, 90 and 120 min and concentrations in plasma are listed in the table 10 below:

[0602] Table 10

[0603] Fig. 1 shows graphically the pharmacokinetic profiles for the peptides agent Epithalon quantified in the blood in the specific time range for Example 50, Comp. Ex. CE-3 of table 2 and Comp. Ex. 2.1 from the EP 22156902.3.

[0604] After 60 minutes, the concentration of Epithalon for Example 50 is 430 pg / ml, whereas for the same composition as of the invention, but intermixed in one simple enteric capsule (Comp. Example 2.1 from the EP 22156902.3) is 310 pq / ml, and for capsule -in-capsule dosage form where lauroyl L-carnitine is powdered and located in the inner capsule - 360 pg / ml.

[0605] After 90 minutes, the concentration of Epithalon for the Example 50 is much higher than for comparative examples. After 120 minutes, the differences are more pronounces, and the concentration of Epithalon of Example 50 is 22300 pq / ml, for Comparative Example CE-3 of table 2 is 18600 pq / ml, whereas for Comp. Example 2.1 from the EP 22156902.3 is 3000 pg / ml.

[0606] The highest values as shown on Fig. 1 were obtained after 120 minutes for all tested single dosage forms, but for Example 50 the concentration of the peptide agent in the blood was over 7 times higher than for Comp. Example 2.1 from the EP 22156902.3, where citric acid, the absorption enhancer and the peptide agent were in the same compartment intermixed in one capsule. Epithalon concentration in blood was over 20% higher than for Comparative Example CE-3 of table 2.

[0607] For all tested peptides included in the dosage form of the invention, an increased bioavailability was observed relative to a dosage form where all ingredients are powdered and contained in one capsule. The blood levels for inventive dosage forms with various of peptides were similar or better than for the dosage forms where lauroyl L-carnitine is not in the form of pellets. Probably this is due to the fact, that dosage form of the invention is more stable and peptide is not degraded when stored.

Claims

Claims1. A pharmaceutical single dosage form for oral delivery consisting of two capsules, an outer enteric capsule, and an inner capsule, wherein said inner capsule comprises at least one physiologically active peptide agent and wherein said outer capsule contains inner capsule and an organic acid, characterized in that said outer capsule contains an absorption enhancer which is a carnitine compound in the form of optionally coated pellets.

2. The pharmaceutical single dosage form according to claim 1, wherein the carnitine compound is an acyl L-carnitine, preferably lauroyl L-carnitine.

3. The pharmaceutical single dosage form according to claim 1 or 2, wherein the organic acid is in the form of optionally coated pellets.

4. The pharmaceutical single dosage form according to claim 1 to 3, wherein the peptide agent is composed of 3-20 amino acids, preferably 3-16 amino acids.

5. The pharmaceutical single dosage form according to claim 4, wherein the peptide agent is selected from GHK-Cu, Epithalon, ARG* BPC-157, Selank, Semax, Cortagen, Ac-BPC-157- Arg-NHz having a sequence of Ac-Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly- Leu-Val-Arg-NHz (SEQ. ID 1); Ac-Epithalon-Arg-NJL having a sequence of Ac-Ala-Glu-Asp- Gly-Arg-NH2 (SEQ.. ID 2); Ac-GHK-Arg-NJL having a sequence of Ac-Gly-His-Lys-Arg-NPE (SEQ. ID 3); Ac-Semax-Arg-NJL having a sequence of Ac-Met-Glu-His-Phe-Pro-Gly-Pro-Arg- NH2 (SEQ. ID 4), Ac-Selank-Arg-NH2 having a sequence of Ac-Thr-Lys-Pro-Arg-Pro-Gly-Pro- Arg-NH2 (SEQ. ID 5), Ac-Cortagcn-Arg-NIL having a sequence of Ac-Ala-Glu-Asp-pro-ARG- NH2(SEQ. ID 6).

6. The pharmaceutical single dosage form according to claim 5 , wherein the peptide agent is selected from Ac-BPC-157-Arg-NH2 having a sequence of Ac-Gly-Glu-Pro-Pro-Pro-Gly-Lys- Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val-Arg-NH2 (SEQ. ID 1); Ac-Epithalon-Arg-NJL having a sequence of Ac-Ala-Glu-Asp-Gly-Arg-NIL (SEQ. ID 2); Ac-GHK-Arg-NJL having a sequence of Ac-Gly-His-Lys-Arg-NH2 (SEQ. ID 3); Ac-Semax-Arg-NJL having a sequence of Ac-Met- Glu-His-Phe-Pro-Gly-Pro-Arg-NH2 (SEQ. ID 4), Ac-Sclank-Arg-NEL having a sequence of Ac- Thr-Lys-Pro-Arg-Pro-Gly-Pro-Arg-NH2 (SEQ. ID 5), Ac-Cortagcn-Arg-NIL having a sequence of Ac-Ala-Glu-Asp-pro-ARG-NH2 (SEQ. ID 6).

7. The pharmaceutical single dosage form according to any proceeding claim wherein the inner capsule is enteric capsule or non-enteric capsule, preferably non-enteric capsule.

8. The pharmaceutical single dosage form according to any proceeding claim wherein organic acid is selected from the group of carboxylic acids such as acetylsalicylic, acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glyoxylic, isocitric, isovaleric, lactic, maleic, oxaloacetic, propionic, pyruvic, succinic, tartaric, valeric acid, or a mixture thereof.

9. The pharmaceutical single dosage form according to claim 8 wherein organic acid is citric acid or ascorbic acid or a mixture thereof.

10. The pharmaceutical single dosage form according to any proceeding claim wherein the amount of peptide agent is 0.2mg - 20mg, the amount of absorption enhancer is 50mg - 150mg and the amount of organic acid is lOOmg - 500mg per single dosage form.

11. Use of the pharmaceutical single dosage form according to claim 1 to 6 as a dietary supplement.

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