Propylene derivatives for oral delivery

Abstract

Provided herein are the use of fatty acid-containing side chains in improving the activity, activity selectivity, and oral availability of polypeptide derivatives, methods for achieving such use, and polypeptide derivatives modified by such side chains.

Description

Technical Field

[0001] Technical Field This application relates to the technical field of biomedicine or biopharmaceuticals, specifically, polypeptides or their derivatives for oral delivery, compositions of polypeptides or their derivatives for oral delivery, and their use for the treatment and / or prevention of metabolic-related diseases.

Background Art

[0002] Background Polypeptides / proteins are not only essential components as the essential cellular components of organisms, but also mainly involved in intercellular or intertissue signal transduction and play an important role in maintaining the normal physiological activities of organisms. Since bovine insulin and porcine insulin were first used in the treatment of diabetes in the early 2000s, with the progress of modern technologies in molecular biology and organic chemistry, more and more polypeptides / proteins have been prepared and applied in the field of human pharmaceuticals. Polypeptide / protein-based pharmaceuticals have clear advantages over other types of pharmaceuticals in the treatment of diseases including cancer, diabetes, cardiovascular diseases, autoimmune diseases, and metabolic disorders due to their high specificity for targets and desirable safety and tolerance. However, most of the therapeutically active polypeptide / protein-based pharmaceuticals have only moderate oral bioavailability due to the limitations imposed by the absorption mechanism of polypeptides / proteins in the human body, so administration by injection or other disease treatment routes is required, which hinders the development of polypeptide / protein-based pharmaceuticals and brings further pain and inconvenience to the patients receiving treatment.

[0003] To compensate for the deficiency of oral polypeptides, numerous attempts have been made to enhance the efficacy of orally administered polypeptide-based drugs. For example, Fiona McCartney et al. (Journal of Controlled Release, Volume 310, 28 September 2019, Pages 115-126) disclosed Labrasol® ALF (Labrasol®), a nonionic surfactant excipient used to enhance the bioavailability of insulin in the gut; P. Uhl et al. (Nanomedicine: Nanotechnology, Biology and Medicine, Volume 24, February 2020, 102132) discovered that oral delivery of liraglutide is possible by coating PLA nanoparticles with a cyclic arginine-rich cell-penetrating peptide; and Vivek Gupta et al. (Journal of Controlled Release, Volume 172, Issue 3, 28 December 2013, Pages 753-762) disclosed the preparation of a mucosal adhesion device by compressing a polymer matrix containing Carbopol, pectin, and sodium carboxymethylcellulose (1:1:2), in We demonstrated the effect of improving salmon calcitonin (sCT) delivery in vivo.

[0004] However, current polypeptide-based drugs still suffer from problems such as low oral efficacy, low bioavailability, uncertainty in the delivery efficacy of the corresponding oral administration regimen for polypeptide molecules, and low versatility. [Overview of the Initiative] [Means for solving the problem]

[0005] Summary of the Invention Accordingly, this application provides polypeptide derivatives with improved activity and stability, as well as improved oral delivery efficacy and bioavailability, that can effectively treat and / or prevent metabolic diseases. In particular, this application relates to a technical solution including the following embodiments: 1. Use of fatty acid-containing side chains in improving the activity selectivity of polypeptide derivatives, wherein the polypeptide derivative is formed by modification of a polypeptide molecule containing a fatty acid-containing side chain, and the fatty acid-containing side chain is in the form of Z1+Z2+Z3, where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent; and Z3 consists of n AEEA(s), where n≧0. 2. Use of fatty acid-containing side chains in improving the activity of polypeptide derivatives, wherein the polypeptide derivative is formed by modification of a polypeptide molecule containing a fatty acid-containing side chain, and the fatty acid-containing side chain is in the form of Z1+Z2+Z3, where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent; and Z3 consists of n AEEA(s), where n≧0. 3. Use of fatty acid-containing side chains in improving the oral bioavailability of polypeptide derivatives, wherein the polypeptide derivative is formed by modification of a polypeptide molecule containing a fatty acid-containing side chain, and the fatty acid-containing side chain is in the form of Z1+Z2+Z3, where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent; and Z3 consists of n AEEA(s), where n≧0. 4. A method for improving the activity selectivity of a polypeptide derivative, comprising modifying a polypeptide molecule containing a fatty acid-containing side chain to form a polypeptide derivative, wherein the fatty acid-containing side chain is in the form of Z1+Z2+Z3, and where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent; and A method for Z3 to be n AEEA(s) such that n≧0 in the equation. 5. A method for improving the activity of a polypeptide derivative, comprising modifying a polypeptide molecule containing a fatty acid-containing side chain to form a polypeptide derivative, wherein the fatty acid-containing side chain is in the form of Z1+Z2+Z3, and where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent; and A method for Z3 to be n AEEA(s) such that n≧0 in the equation. 6. A method for improving the oral bioavailability of a polypeptide derivative, comprising modifying a polypeptide molecule containing a fatty acid-containing side chain to form a polypeptide derivative, wherein the fatty acid-containing side chain is in the form of Z1+Z2+Z3, and where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent; and A method for Z3 to be n AEEA(s) such that n≧0 in the equation. 7. Use of any one of Embodiments 1 to 3, or method of any one of Embodiments 4 to 6, wherein Z1 is a C16, C17, C18, C19, or C20 fatty acid. 8. A use or method according to any one of Embodiments 1 to 7, wherein Z2 is selected from one of γGlu, βAsp, γGlu, Inp, and Trx, preferably βAsp or γGlu. 9. Any one of Embodiments 1 to 8, wherein n is greater than 0, preferably 1 to 10, more preferably 2 to 8, and even more preferably n = 2. 10. Use or method of any one of Embodiments 1 to 9, wherein Z1 is a C16, C18, or C20 fatty acid. 11. Any one of the uses or methods of Embodiments 1 to 10, wherein Z2 is Inp or Trx, and n is greater than 0. 12. Any one of the uses or methods of Embodiments 1 to 11, wherein Z1 is a C16, C17, C18, C19, or C20 fatty acid; Z2 is γGlu; ​​and n=2. 13. Any one of the embodiments 1 to 12 is used or used in a manner in which Z1, Z2, and Z3 are linked via amide bonds. 14. Any one of Embodiments 1 to 13, wherein the side chain modification is in 1 to 10 amino acids, preferably 1 to 3 amino acids, more preferably 1 amino acid, of the polypeptide. 15. Any one of Embodiments 1 to 14, wherein the side chain modification is located at an amino acid at the N-terminus or any intermediate position of the polypeptide sequence, and this amino acid is preferably selected from glutamic acid, aspartic acid, lysine, glutamine, or asparagine, and more preferably lysine. 16. The polypeptide is preferably a GLP-1 receptor agonist, a GIP receptor antagonist, an insulin receptor agonist, a heparin receptor agonist, somatotrophin, an interferon receptor agonist, an interleukin receptor agonist, a follicle-stimulating hormone receptor agonist, a gonadotropin receptor agonist, an erythropoietin receptor agonist, a peptide YY (PYY) receptor agonist, an oxygenation-modulating receptor agonist, a glucagon-like peptide-2 (GLP-2) receptor agonist, a calcitonin receptor agonist, or a parathyroid hormone (PT) receptor agonist. H) Any one of the uses or methods of Embodiments 1 to 15, which is an agonistic or antagonistic natural or synthetic polypeptide molecule selected from receptor agonists, ghrelin receptor agonists, endocannabinoid receptor agonists, leptin receptor agonists, serotonin receptor agonists, fibroblast growth factor 21 (FGF21) receptor agonists, cholecystokinin (CCK) receptor agonists, oxytomodulin receptor agonists, or glucagon receptor agonists, preferably amylin receptor agonists, and more preferably amylin or calcitonin analogs. 17. The polypeptide contains the amino acid sequence ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH2, KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2, or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2. Preferably, the polypeptide derivative is selected from N1, N2, or one or more of M1 to M30, or Preferably, the polypeptide derivative is selected from one or more of N1, N2, N3, or M1 to M30, in any one of the uses or methods of Embodiments 1 to 16. 18. The use of any one of Embodiment 1 or Embodiments 7 to 17, or any one of Embodiment 4 or Embodiments 7 to 17, wherein the improvement of the activity selectivity of the polypeptide derivative is to preferentially improve the activity of the polypeptide derivative to the amyrin receptor over the calcitonin receptor, preferably by improving the ratio of the activity of the polypeptide derivative to the amyrin receptor to the activity of the polypeptide derivative to the calcitonin receptor, and more preferably by improving the activity of the polypeptide derivative to the amyrin receptor. 19. The improvement in the activity selectivity of the polypeptide derivative is such that the ratio of activity to amyrin receptor activity to activity to calcitonin receptor activity in the cell is improved by more than 1.5 times, preferably more than 2 times, more preferably more than 5 times, and even more preferably more than 10 times, using any one of Embodiments 1 or Embodiments 7 to 18, or using any one of Embodiments 4 or Embodiments 7 to 18. 20. Use or method of Embodiment 18 or 19, wherein the activity is agonist activity. 21. Use of any one of Embodiments 1 or Embodiments 7-20, or method of any one of Embodiments 4 or Embodiments 7-20, wherein the polypeptide derivative is an amyrin receptor agonist, preferably an amyrin or calcitonin derivative, more preferably comprising the amino acid sequence ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH2, or even more preferably comprising the amino acid sequence KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2. 22. Use of any one of Embodiments 2 or Embodiments 7-17, or method of any one of Embodiments 5 or Embodiments 7-17, wherein the activity of the polypeptide derivative is activity in reducing body weight and / or reducing food intake. 23. The improvement of the polypeptide derivative's activity is to improve the polypeptide derivative's activity against intracellular amyrin receptors and calcitonin receptors, using any one of Embodiments 2 or 7-17, or using any one of Embodiments 5 or 7-17. 24. The polypeptide derivative is an amyrin receptor agonist, preferably an amyrin or calcitonin derivative, and more preferably comprises the amino acid sequence KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2, using any one of Embodiments 2 or 7-17 and 22-23, or using any one of Embodiments 5 or 7-17 and 22-23. 25. Use of any one of Embodiments 3 or 7-17, or method of any one of Embodiments 6 or 7-17, wherein a polypeptide derivative having a fatty acid-containing side chain is administered co-administered with one or more oral delivery agents, preferably a salt of N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC), more preferably PNAC. 26. The use of any one of Embodiments 3 or Embodiments 7-17, or the method of any one of Embodiments 6 or Embodiments 7-17, wherein the improvement in the oral bioavailability of the polypeptide derivative achieves higher oral bioavailability than that of the Cagrilintide polypeptide molecule. 27. Use of any one of Embodiments 3 or Embodiments 7-17, or method of any one of Embodiments 6 or Embodiments 7-17, wherein the polypeptide derivative is an amyrin receptor agonist, preferably an amyrin or calcitonin derivative, and more preferably comprises the amino acid sequence KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2 or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2. 28. A polypeptide and a derivative of a polypeptide comprising a side chain attached to the polypeptide in the form of Z1+Z2+Z3, wherein Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent; and A derivative in which Z3 consists of n AEEA(s) such that n ≥ 0. 29. A derivative of Embodiment 28, wherein Z1 is a C16, C17, C18, C19, or C20 fatty acid. 30. A derivative of Embodiment 28 or 29 in which Z2 is selected from one of γGlu, Inp, and Trx, or is absent. 31. A derivative of any one of embodiments 28 to 30, wherein n is greater than 0. 32. A derivative of any one of embodiments 28 to 31, wherein Z2 is Inp or Trx, and n is greater than 0. 33. A derivative of any one of embodiments 28 to 32, wherein Z2 is absent and n is greater than 0. 34. A derivative of any one of embodiments 28 to 33, wherein Z2 is γGlu and n=2. 35. The polypeptide is an amyrin receptor agonist; preferably, the polypeptide derivative is an amyrin or calcitonin derivative; more preferably, the polypeptide comprises the amino acid sequence ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH2, KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH2, or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH2. Preferably, the polypeptide derivative is selected from N1, N2, or one or more of M1 to M30, or Preferably, one derivative of the polypeptide is selected from one or more of N1, N2, N3, or M1 to M30, as a derivative of any one of embodiments 28 to 34. 36. A pharmaceutical composition comprising one derivative of any one of Embodiments 28 to 35 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the composition preferably comprises one or more oral delivery agents, preferably a salt of N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC), more preferably PNAC. Use of any one of the derivatives of Embodiments 28 to 35 or a pharmaceutically acceptable salt thereof or the composition of Embodiment 36 in the manufacture of a polypeptide-based drug, preferably, the polypeptide-based drug is used for the prevention and / or treatment of overweight and / or obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain. 38. The use of Embodiment 37, wherein the polypeptide-based drug is an oral drug; preferably, the oral drug contains one or more oral delivery agents, preferably a salt of N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC), more preferably PNAC. 39. Use of any one of the derivatives of Embodiments 28 to 35 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of Embodiment 36 in reducing food intake. 40. A method for preventing and / or treating overweight and / or obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain, comprising administering a prophylactically or therapeutically effective amount of any one of the derivatives of Embodiments 28 to 35 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of Embodiment 36 to a subject. 41. A method for reducing food intake, comprising administering an effective amount of any one of the derivatives of Embodiments 28 to 35 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of Embodiment 36 to a subject.

[0006] Technical effects The polypeptide derivative of the present application has high activity in cells, strong efficacy or effectiveness against amylin receptor and calcitonin receptor, and little change even after long-term storage, and the properties and qualities including concentration and purity are stable.

[0007] The polypeptide derivative of the present application brings a significant effect on weight loss and reduction of food intake, and achieves improvement in effectiveness and bioavailability as an oral drug.

[0008] Brief description of the drawings The drawings are not intended to unduly limit this application, but rather to facilitate a better understanding. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 is a trend chart showing the effect of polypeptide derivatives on body weight changes in rats. [Figure 2] Figure 2 is a trend chart showing the effect of polypeptide derivatives on the cumulative food intake of rats. [Figure 3] Figure 3 is a trend chart showing the effects of several polypeptide derivatives on body weight change in rats. [Figure 4] Figure 4 is a trend chart showing the effects of several polypeptide derivatives on the cumulative food intake of rats. [Figure 5] Figure 5 shows the plasma levels of polypeptide derivatives D1 and N2. [Figure 6] Figure 6 shows the plasma levels of several polypeptide derivatives as oral tablets. [Modes for carrying out the invention]

[0010] Detailed explanation Exemplary embodiments of this application are described below, but various details of the embodiments in this application are included for the sake of clarity and should be considered as illustrative only. Accordingly, those skilled in the art will recognize that numerous changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. The following descriptions also omit well-known descriptions of functions and structures for clarity and brevity, and the scientific and technical terms used herein have the same meaning as generally understood by those skilled in the art unless otherwise defined.

[0011] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to amino acid polymers of any length. The polymers may be linear or branched and may contain naturally occurring amino acids, as well as non-naturally occurring amino acids.

[0012] The term "N-terminus" refers to the N-terminus or amino-terminus of a linear polypeptide consisting of multiple amino acids linked by peptide bonds and containing free α-amino groups (-NH2) and free α-carboxyl groups (-COOH). When unmodified, the free α-amino groups (-NH2) and free α-carboxyl groups (-COOH) are not bound to the respective amino acids at either end; that is, the N-terminus has unbound free α-NH2 of the polypeptide. N-terminal modification refers to modifying the amino acids at the N-terminus of a polypeptide.

[0013] In this specification, the term “derivative” refers to a product obtained by modifying a functional group (e.g., an amino acid residue) of a biomolecule (e.g., a polypeptide or protein) with a specific compound or molecule. Modifications include, but are not limited to, acylation, amidation, esterification, and thioesterification.

[0014] In this specification, the term “activity selectivity” of a polypeptide or polypeptide derivative refers to the characteristic of a polypeptide or polypeptide derivative that has a stronger effect on one of the receptors it acts on than on other receptors. In some embodiments of this application, activity refers to agonist activity.

[0015] In some embodiments of this application, activity selectivity may further refer to a preferential improvement in the activity of the polypeptide derivative toward the amyrin receptor compared to the calcitonin receptor. Preferably, activity selectivity refers to an improvement in the ratio of the activity of the polypeptide derivative toward the amyrin receptor to the activity of the polypeptide derivative toward the calcitonin receptor.

[0016] The pharmaceutical compositions described herein may be used for the treatment of diseases or in in vitro cell culture assays. When used for the treatment of diseases, the term “pharmaceutical composition” generally refers to a unit dosage form, which can be prepared by any method well known in the field of pharmacy. The method used involves the step of mixing an active ingredient with one or more excipients, which are auxiliary components. Typically, compositions are prepared by homogeneously and appropriately mixing an active compound with a liquid excipient, a fine solid excipient, or both.

[0017] In this specification, “pharmaceutically acceptable” means the properties of a substance or composition that must be chemically and / or toxicologically compatible with the other components of the formulation and / or with the mammal being treated by the formulation.

[0018] The term “pharmaceutically acceptable excipients” as used herein may include any solvent, solid excipient, diluent, or other liquid excipient suitable for a particular target dosage form. The use of any conventional excipients is also within the scope contemplated herein, unless they are incompatible with the compounds of this application, for example, resulting in any undesirable biological effect or interacting hazardously with any other components of the pharmaceutically acceptable composition.

[0019] As used herein, “treatment” means the achievement of a desired pharmacological and / or physiological effect. This effect may be preventive in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it completely or partially cures a disease and / or side effects caused by the disease. As used herein, “treatment” with respect to diseases of mammals, in particular humans, includes (a) preventing the development of a disease or condition in an individual susceptible to the disease but not diagnosed with the disease; (b) suppressing the disease, e.g., preventing the onset of the disease; or (c) alleviating the disease, e.g., reducing the symptoms associated with the disease. As used herein, “treatment” includes, but is not limited to, administering a drug or compound to an individual to treat, cure, alleviate, improve, reduce, or suppress an individual’s disease, including administering a drug containing a compound described herein to an individual in need of it.

[0020] In this specification, the term “prevention” means reducing the likelihood of the onset (or recurrence) of a disease, condition, or disorder or related symptoms (e.g., cancer).

[0021] Abbreviation The corresponding names or structures of some of the abbreviations used in the embodiments of this application are as follows: [ka]

[0022] AA: amino acid; Boc: t-butyloxycarbonyl; DCM: dichloromethane; DMF: N,N-dimethylformamide; DIEA: N,N-diisopropylethylamine; EDT: 1,2-ethanedithiol; Fmoc: 9-fluorenylmethyloxycarbonyl; OtBu: t-butyl ester; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl chloride; Pip: piperidine; TBTU: O-(benzotriazol-1-yl)-N,N,N',N',-tetramethyluronium tetrafluoroborate; tBu: 3-butyl; TFA: trifluoroacetic acid; TIS: triisopropylsilane; Trt: triphenylmethyl; αGlu: α-glutamic acid; γGlu: γ-glutamic acid; αAsp: α-aspartic acid; and βAsp: β-aspartic acid. [Examples]

[0023] Examples Example 1 Preparation of polypeptide derivatives Table 2 shows the polypeptide derivatives prepared in this application.

[0024] The target products in Table 2 were prepared by solid-phase organic synthesis, specifically by solid-phase peptide synthesis (SPPS) using Fmoc-protected amino acids, followed by cleavage, oxidation, and purification.

[0025] Taking compound D1 (Cagrilintide) as an example, it has the following structure [ka] The synthesis process is as follows:

[0026] 1.1 Solid phase synthesis Using Fmoc-Linker MBHA Resin S=0.32 mmol / g and Fmoc / tBu processing, amino acids were linked from the C-terminus to the N-terminus (right to left) according to the peptide sequence, as shown in Table 1.

[0027] [Table 1]

[0028] The following amino acids were linked together in order: A-01 Fmoc-Pro-OH, A-02 Fmoc-Thr(tBu)-OH, A-03 Fmoc-Asn(Trt)-OH, A-04 Fmoc-Ser(tBu)-OH, A-05 Fmoc-Gly-OH, A-06 Fmoc-Val-OH, A-07 Fmoc-Asn(Trt)-OH, A-08 Fmoc-Thr(tBu)-OH, A-09 Fmoc-Pro-OH, A-10 Fmoc-Pro-OH, A-11 Fmoc-Leu-OH, A-12 Fmoc-Ile-OH, A-13 Fmoc-Pro-OH, A-14 Fmoc-Gly-OH, A-15 Fmoc-Phe-OH, A-16 Fmoc-Asn(Trt)-OH, A-17 Fmoc-Asn(Trt)-OH, A-18 Fmoc-Ser(tBu)-OH, A-19 Fmoc-Ser(tBu)-OH, A-20 Fmoc-His(Trt)-OH, A-21 Fmoc-Arg(Pbf)-OH, A-22 Fmoc-Leu-OH, A-23 Fmoc-Phe-OH, A-24 Fmoc-Glu(OtBu)-OH, A-25 Fmoc-Ala-OH, A-26 Fmoc-Leu-OH, A-27 Fmoc-Arg(Pbf)-OH, A-28 Fmoc-Gln(Trt)-OH, A-29 Fmoc-Thr(tBu)-OH, A-30 Fmoc-Ala-OH, A-31 Fmoc-Cys(Trt)-OH, A-32 Fmoc-Thr(tBu)-OH, A-33 Fmoc-Ala-OH, A-34 Fmoc-Thr(tBu)-OH, A-35 Fmoc-Asn(Trt)-OH, A-36 Fmoc-Cys(Trt)-OH, A-37 Fmoc-Lys(Boc)-OH, A-38 Fmoc-Glu-otbu, and A-39 C20 diacid.

[0029] Finally, polypeptide derivatives were formed on the resin.

[0030] The polypeptide derivatives on the resin were washed, transferred, and dried to a certain weight for cutting.

[0031] 1.2 Cutting The cleavage reagent was used in an amount of 10 mL ± 2 mL per gram of polypeptide derivative on the resin. For cleavage, H2O, TFA, EDT, and TIS, which constitute the necessary cleavage reagent, were sequentially added to the cleavage reaction flask in the ratio TFA:H2O:EDT:TIS = 95:1:2:2, and the temperature was controlled at 0 to 10°C. The cleavage reagent was added to the resin while stirring, and after the system temperature stabilized, the reactants were stirred for a further 2.5 hours at a controlled temperature of 25 to 30°C. The cleavage solution was filtered and precipitated using 5 times the volume of ice diethyl ether. The precipitate was filtered, washed three times with 3 times the volume of ice diethyl ether, and then dried at room temperature under reduced pressure to obtain a crude solid.

[0032] 1.3 Oxidation The crude solid was finely ground and slowly added to purified water while stirring, and an aqueous solution of acetonitrile was added dropwise. After the crude solid was added and completely dissolved, a methanol solution of iodine was added and the mixture was stirred for 30 minutes.

[0033] 1.4 Purification and freeze-drying The oxidizing solution described above was filtered through a 0.45 μm microporous filtration membrane. The crude solid was separated and purified using a column packed with C-18 column material at room temperature with an appropriate gradient, and then the target product was recovered, detected, analyzed, and sorted. A purity of ≥90% was required. To achieve a qualified liquid peak, substandard target products were collected and separated and purified again with an appropriate gradient. The qualified liquid samples described above were freeze-dried under reduced pressure to obtain freeze-dried powders of the purified polypeptide derivatives.

[0034] Table 2 shows polypeptide derivatives prepared using a method similar to that described in Example 1 of this application, and they are distinguished from each other by the amino acid sequence and / or fatty acid-containing side chain used in solid-phase synthesis.

[0035] [Table 2-1]

[0036] [Table 2-2]

[0037] N1-N3 or M1-M30 represent derivatives resulting from modifications of the amino acid sequences shown in SEQ ID NOs: 1-5, which have corresponding fatty acid-containing side chains in Table 2, or other polypeptides (in the absence of fatty acid modification). The fatty acid-containing side chain is in the form of Z1+Z2+Z3, where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is absent; Z3 is 0-8, e.g., 0, 1, 2, 3, 4, 5, or 6 AEEA(s); and Z1, Z2, and Z3 are linked by an amide bond. The C16-C22 fatty acid moiety is located outside the fatty acid-containing side chain for modification of the amino acid sequence and is therefore located at the distal end of the linkage between the side chain and the amino acid sequence. For example, M1 represents a derivative obtained by modifying the amino acid sequence shown in SEQ ID NO: 1 with a fatty acid-containing side chain "C18 diacid + γGlu". The γGlu terminus of the side chain is linked to the amino acid sequence by a peptide bond, and the C18 fatty acid terminus is located at the distal end of the link between the side chain and the amino acid sequence.

[0038] Example 2: In vitro activity testing of polypeptide derivatives The purpose of this assay is to test the efficacy of the polypeptide derivatives of this application against amyrin receptors and calcitonin receptors in vitro using a luciferase assay.

[0039] 2.1 Amylin receptor agonism test Using a standard protocol for constructing amyrin receptor / CRE-luc cell lines, CHO-K1 / Ga15 / AMY3 cells (purchased from GenScript, with calcitonin receptor and receptor activity regulatory peptide (RAMP) already constructed) were transfected with a plasmid containing a luciferase expression cassette driven by multiple copies of the cAMP response element (CRE). Cells were cultured in F12 medium containing 200 ug / mL zeosin, 2 ug / mL puromycin, 100 ug / mL hygromycin, and 400 ug / mL G418 to obtain stably transfected amyrin receptor / CRE-luc cell lines.

[0040] In the amyrin luciferase assay, the lyophilized powder obtained according to the method of Example 1 was dissolved in 20 mM phosphate buffer at pH 7.0 and diluted with growth medium (F12 medium containing 10% FBS) to obtain a derivative sample with an initial concentration of 10 nM. This was then serially diluted fivefold with growth medium to obtain seven samples at concentrations including 10 nM, 2 nM, and 0.4 nM. 50 μL of the test sample solution at each concentration was added to each well of a white 96-well plate.

[0041] Stable transfected amyrin receptor / CRE-luc CHO cells were resuspended in growth medium at a constant density, and 50 μL of the cell suspension was added to a white 96-well plate containing the test sample solution at a density of approximately 20,000 cells per well. After incubation at 37°C and 5% CO2 for 24 hours, 100 μL of luciferase substrate was added to each well and incubated for 3 minutes. Finally, luminescence was tested using SpectraMax L (Molecular Devices) with SoftMax Pro 7.0.3 GxP software. A standard curve was plotted using fluorescence intensity for EC50 calculation.

[0042] Samples of compounds N2 and N3 were tested in the same manner as described above, except that the test sample solution and CHO cells were incubated at 37°C and 5% CO2 for 4 hours, followed by the addition of 100 μL of luciferase substrate to each well.

[0043] The results are shown in Table 3.

[0044] [Table 3]

[0045] The data in Table 3 demonstrate that the polypeptide derivative of this application exhibits high intracellular activity and shows excellent agonist activity against the amyrin receptor.

[0046] 2.2 Calcitonin receptor agonism 2.2.1 Construction of a calcitonin receptor / CRE-luc cell line Using a standard protocol, CHO-K1 / CALCR / Gα15 cells (purchased from GenScript and pre-constructed with calcitonin receptors) were transfected with a plasmid containing a luciferase expression cassette driven by multiple copies of cAMP response elements (CRE). The cells were cultured in F12 medium containing 200 ug / mL zeosin, 2 ug / mL puromycin, and 100 ug / mL hygromycin to obtain a stably transfected calcitonin receptor / CRE-luc cell line.

[0047] 2.2.2 Calcitonin luciferase assay The lyophilized powder from Example 1 was dissolved in 20 mM phosphate buffer at pH 7.0 and diluted with growth medium (F12 medium containing 10% FBS) to obtain a derivative sample with an initial concentration of 10 nM. This sample was then sequentially diluted fivefold with growth medium to obtain seven samples at concentrations including 10 nM, 2 nM, and 0.4 nM. 50 μL of the test sample solution at each concentration was added to each well of a white 96-well plate.

[0048] Stable transfected calcitonin receptor / CRE-luc CHO cells were resuspended in growth medium at a constant density, and 50 μL of the cell suspension was added to a white 96-well plate containing the test sample solution at a density of approximately 20,000 cells per well. After incubation at 37°C and 5% CO2 for 24 hours, 100 μL of luciferase substrate was added to each well and incubated for 3 minutes. Finally, luminescence was tested using SpectraMax L (Molecular Devices) with the software SoftMax Pro 7.0.3 GxP. A standard curve was plotted using fluorescence intensity to calculate EC50. The results are shown in the following table.

[0049] [Table 4]

[0050] The data in Table 4 demonstrate that the polypeptide derivative of this application exhibits high intracellular activity and agonist activity against the calcitonin receptor.

[0051] Table 5 shows an integrated analysis of data for the activity of several polypeptide derivatives of this application against intracellular amyrin receptor (hAMY3R) and calcitonin receptor (hCTR). Using the activity of molecule D1 against intracellular hAMY3R and hCTR as a benchmark, polypeptide derivatives of this application containing fatty acid-containing side chains in the form of Z1+Z2 (i.e., fatty acid + amino acid) showed a surprisingly significant difference in activity against intracellular amyrin receptor and calcitonin receptor compared to polypeptide derivatives with fatty acid-containing side chains in the form of Z1+Z2+Z3 (i.e., fatty acid + amino acid + AEEA), exhibiting greater agonist / relative activity against hAMY3R than against hCTR, and this improvement in activity was particularly pronounced when Z2 was Trx or Inp. For example, M30 (SEQ ID NO: 4 modified with C18 dioxide + Inp + 2AEEAs) showed a nearly 70-fold increase in agonist activity against the amyrin receptor compared to M17 (SEQ ID NO: 4 modified with C18 dioxide + Inp), and in contrast to the 1.7-fold increase achieved by M17 compared to the benchmark, it showed an approximately 42.5-fold increase in the ratio of agonist activity against the amyrin receptor to that against the calcitonin receptor, indicating a stronger tendency toward amyrin receptor agonism.

[0052] Similarly, another derivative N3, containing a different amino acid sequence (SEQ ID NO: 2) modified by a fatty acid-containing side chain in the form of Z1+Z2+Z3 (i.e., fatty diacid + amino acid + AEEA), also exhibited high agonist activity against the amyrin receptor.

[0053] [Table 5]

[0054] Example 3: Stability test of polypeptide derivatives 3.1 Materials and equipment Stability testing chamber (BINDER GmbH), 0.01 mg precision balance (METTLER TOLEDO), pH meter (METTLER TOLEDO), biosafety cabinet (ESCO), T2G-II small motor-driven capping device (Changsha zhongya pharmaceutical equipment co. LTD), high-speed refrigerated centrifuge (Eppendorf), sterilization cabinet (Shinva Medical), Agilent 1260 high-performance liquid chromatography, and Sepax Bio-C18 4.6 * A 250 mm 3 μm 200 Å reversed-phase column was used.

[0055] Reagents and consumables include: ultrapure water (18.2 MΩ, homemade), acetonitrile (HPLC grade), trifluoroacetic acid (HPLC grade), sodium dihydrogen phosphate (pharmaceutical grade, Hunan Jiudian Hongyang Pharmaceutical Co., Ltd), hydrochloric acid (pharmaceutical grade, Hunan Er-Kang Pharmaceutical Co., Ltd), sodium hydroxide (analytical reagent grade, Hushi®, Sinopharm Chemical Reagent Co., Ltd), vials, rubber stoppers, disposable syringes, and sterile filters.

[0056] 3.2 Assay Method Lyophilized polypeptide derivative powder (1 mg / mL) prepared according to the method of Example 1 was mixed with sodium dihydrogen phosphate (1.42 mg / mL) and dissolved in ultrapure water in the indicated mass-volume ratio. After adjusting the pH to approximately 7.4 with hydrochloric acid / sodium hydroxide, the solution was filtered into a sterile vial in a clean bench using a 0.22 μm sterile filter. The vial was then capped and placed in a stability test chamber at 40°C. Detection was performed on day 7 (7d) and day 26 (26d), and changes in the characteristics and properties of the polypeptide during the assay were observed and recorded. The sample was then centrifuged at 4°C and 10,000 rpm for 3 minutes, and the supernatant was transferred to a liquid-phase sample vial. The concentration and purity of the polypeptide were detected using liquid-phase chromatography as described below. Polypeptide concentration = peak area / injection volume / extinction coefficient / 60 × flow rate, and polypeptide purity = target peak area / total peak area × 100%.

[0057] The conditions for reversed-phase chromatography were: flow rate: 1.0 ml / min; autosampler temperature: 15°C; column temperature: 25°C; detection wavelengths: 280 nm and 214 nm; mobile phase A: 100% H2O + 0.05% TFA; mobile phase B: 100% ACN; and the elution gradient is shown in Table 6.

[0058] [Table 6]

[0059] 3.3 Assay Results As observed in the heat-accelerated stability assay, the solution of the polypeptide derivative of this application remained clear on day 7 of the heat-accelerated assay, showing significantly higher clarity than the solution of molecule D1, in which foreign matter was easily observed on day 26 of the assay.

[0060] Table 7 shows that there was no significant change in the concentration of the polypeptide derivatives described in this application during the heat-accelerated stability assay under neutral conditions. In contrast to molecule D2, which showed a concentration change of -40% on day 7 of the accelerated test, the polypeptide derivatives showed minimal concentration changes, with some (e.g., M6, M19, M22, M23, M24, M27, M29, and M30) showing a concentration change of less than 1% (indicated as "no change"). The concentration changes of the polypeptide derivatives in this application remained below 10% even on day 26 of the heat-accelerated assay, and some were even lower, below 3%.

[0061] [Table 7]

[0062] Table 8 shows that, as determined by reverse-phase chromatography, there was no significant change in the purity of the polypeptide derivatives shown in this application during the heat-accelerated stability assay under neutral conditions. On day 7 of the accelerated test, the purity change of most polypeptide solutions was less than 1% as determined above, or no decrease in purity was observed at all. The polypeptide derivatives of this application maintained high purity, while the prior art molecule D1 showed a 12.73% decrease in purity on day 7 of the accelerated test. Molecules such as M7, M18, M19, and M23 were further found to show almost no significant change in purity even on day 26 of the accelerated test, demonstrating excellent purity stability.

[0063] [Table 8]

[0064] Example 4: Results of animal experiments SD (Sprague Dawley) rats weighing 200-250g were used in this experiment. The rats were received at least 10-14 days prior to the start of the experiment to allow time for adaptation to the experimental environment. Upon arrival, the rats were exposed to a reverse light-dark cycle (i.e., lighting off during the day and on at night) for two weeks, and were housed individually for the first week to ensure high data accuracy and test sensitivity. During adaptation and the experimental period, the rats had free access to food and water. Five to eight rats were assigned to each derivative test group, and a dose of 10 nmol / kg of the derivative or vehicle (20 mM phosphate buffer, PB, pH 7.0) was administered subcutaneously, with the administration time recorded for each group. After administration, the rats were returned to cages where they were housed and had access to food and water. Rats' food intake and body weight changes were recorded online or manually every 24 hours. Body weight change = (BWT) n -BWT0) / BWT0 * It is 100%, and in the formula, BW represents the value of body weight, and T0 and T n The terms represent the time of administration and n hours after administration, respectively. The dots in the figure represent the mean ± standard error (SEM).

[0065] As shown in Figures 1 to 4, the polypeptide derivatives of this application demonstrated a clearly superior effect in reducing body weight and cumulative food intake in SD rats compared to the control (vehicle or D1). More importantly, for example, the addition of AEEA to the linker in polypeptide derivatives M18, M23, M25, M27, and M29 clearly enabled a further reduction in body weight and cumulative food intake in SD rats compared to a single amino acid linker in molecules with the same polypeptide structure.

[0066] Example 5: Efficacy assay-dose-dependent study in SD rats SPF male SD rats (7-8 weeks old, 220-230g) were used in this study after health checks and quarantine. The experimental animals were exposed to room temperature of 20°C-23°C and relative humidity of 40%-50%, and were provided with Co60 mouse breeding feed 1035 and purified water supplied from a water bottle, which were freely available throughout the health checks, quarantine, and study.

[0067] In the efficacy comparison assay between compounds D1 and N3, animals were assigned to five groups (n=5) based on mean body weight: a vehicle group (20 mM phosphate buffer, PB, pH 7.0), a group receiving compound D1 at various doses (30 nmol / kg and 100 nmol / kg), and a group receiving compound N3 at various doses (30 nmol / kg and 100 nmol / kg).

[0068] In the efficacy comparison assay between compounds N2 and N3, animals were assigned to seven groups (n=5) based on mean body weight: a vehicle group (20 mM phosphate buffer, PB, pH 7.0), compound N2 groups with varying doses (5 nmol / kg, 25 nmol / kg, and 125 nmol / kg), and compound N3 groups with varying doses (5 nmol / kg, 25 nmol / kg, and 125 nmol / kg).

[0069] In this study, animals were given a single subcutaneous injection, with the administration day designated as day 1 / hour 0 (T0). Initial body weight was recorded, the first meal was given on day 0, and the body weight and remaining food were recorded daily to calculate the percentage change in body weight and food intake. Any abnormalities during this period were also recorded and reported. During the study, the rats were in good condition, and no abnormalities were observed. Percentage change in body weight = (BWT) n -BWT0) / BWT0 * It is 100%, and in the formula, BW represents the value of body weight, and T0 and T n The values ​​represent the time of administration and n hours after administration, respectively. The dots in the figure represent the mean ± standard error (SEM). Food intake is the amount of food each animal consumes per day.

[0070] [Table 9]

[0071] [Table 10]

[0072] Compound N2 showed a significantly greater effect on weight loss than D1 (see Example 3 in CN 202310614769.1). Table 9 shows that N3 also showed a clearly stronger dose-dependent effect on reducing body weight and cumulative food intake in SD rats compared to D1. N2 and N3 are additional derivatives containing different amino acid sequences (SEQ ID NO: 2) modified by fatty acid-containing side chains in the form of Z1+Z2+Z3 (i.e., fatty diacid + amino acid + AEEA), and in comparison, N3 showed an unexpectedly strong effect on reducing animal body weight and food intake (Table 10).

[0073] Example 6: Study on the PK profile of amylin derivative tablets The amylin derivative is formulated as an oral tablet and further contains an equal amount of an oral delivery agent PNAC having the following structure: [ka]

[0074] N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC) was prepared using the method described in Example 1 of International Publication No. 2008 / 028859. Isopropanol (22070.0 ml, 4.0 vol) was added to a 50 L reactor and stirred, and then NAC (5518 g, 1.0 equivalent) was added. After warming the system to 50°C, 50% potassium hydroxide solution (1304.0 g, 1.0 equivalent) was added dropwise until a clear yellow solution was obtained, and the mixture was reacted at 50°C for 1 hour. The reaction solution was then concentrated little by little at 40°C to obtain a pale orange crude solid.

[0075] The combined crude solid was added to isopropanol (19310.0 ml, 3.5 vol), ground for 1 hour, and then filtered by suction. The filter cake was rinsed with isopropanol (2760.0 ml, 0.5 vol), transferred to a vacuum dryer, pressure equalized with nitrogen, dried at 60°C for 16 hours, then transferred back to the vacuum dryer and dried at 100°C for 24 hours. After drying, a total of 4.52 kg of PNAC solid product was obtained as an off-white powder, with a yield of 72.8%.

[0076] Table 11 shows the amounts of essential components contained in tablets of polypeptide derivatives containing fatty acid-containing side chains.

[0077] [Table 11]

[0078] Tablets of polypeptide derivatives containing fatty acid-containing side chains were prepared by sieving the polypeptide derivative and PNAC, homogeneously mixing with excipients, and directly compressing the mixture. Male beagle dogs (9-12 kg) aged 10-15 months were orally administered amylin derivatives (D1 and N2) at a dose of 7 mg once daily for 5 consecutive days (n=5), with the first day of oral administration of the protein molecule-based drug designated as day 1 and the last day as day 5. Whole blood from the animals was collected on day 1 before administration (-10 minutes) and 2, 4, and 8 hours after administration; on days 2 through 4 before administration (-10 minutes) and 2 and 4 hours after administration; and on day 5 before administration (-10 minutes) and 2, 4, 8, 24, 48, and 72 hours after administration. The blood was then treated with heparin sodium as an anticoagulant. Plasma was stored at -80°C, and then the plasma levels of APIs were analyzed using LC-MS / MS (Waters ACQUITY I Class Premier UPLC tandem with Sciex 6500+QQQ). The charge-to-mass ratio of the D1 ion pair was 1102.9 / 1074.5, and the charge-to-mass ratio of N2 was 945.6 / 919.8. The experimental data were plotted using GraphPad Prism 9.3.1.

[0079] The results are shown in Figure 5, demonstrating that, compared to tablets containing compound D1, the molecule of compound N2 exhibits a special synergistic effect with the delivery agent PNAC in the tablet, resulting in very high oral bioavailability.

[0080] In a comparison of oral bioavailability between D1 and N3, the combination of N3 in the tablet and the delivery agent PNAC also produced a synergistic effect that significantly improved bioavailability.

[0081] Example 7: Study on the effect of side-chain modification on the PK profile of oral tablets of polypeptide derivatives. The effect of side-chain modification on the oral bioavailability of polypeptide derivatives was further investigated using additional polypeptide analogs of amyrin containing different sequences. Oral tablets of polypeptide derivatives D1, M2, and M3, which differ only in their side chains as shown in Table 1, were tested for their PK profiles using a method similar to that described in Example 6.

[0082] The results are shown in Figure 6, where polypeptide derivatives M2 and M3 were able to achieve significantly higher absorption than molecule D1 after oral administration, similar to the results in Example 6. This further demonstrates that the modification by the side chain structure shown in this application effectively contributes to improving the oral bioavailability of polypeptide derivatives, and that M3, which contains C18 fatty acid, has higher oral bioavailability than M2, which contains C20 fatty acid.

[0083] While embodiments of this application have been described above, this application is not limited to the above-described, illustrative, and non-limiting embodiments, nor to the field of application. Those skilled in the art will be able to draw inspiration from this specification and arrive at numerous variations without departing from the claims of this application.

Claims

1. The use of fatty acid-containing side chains in improving the activity selectivity of polypeptide derivatives, wherein the polypeptide derivative is formed by modification of a polypeptide molecule containing the fatty acid-containing side chain, and the fatty acid-containing side chain is in the form of Z1 + Z2 + Z3, where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is not present; and Z3 consists of n AEEA(s), where n ≥ 0.

2. The use of fatty acid-containing side chains in improving the activity of polypeptide derivatives, wherein the polypeptide derivative is formed by modification of a polypeptide molecule containing the fatty acid-containing side chain, and the fatty acid-containing side chain is in the form of Z1 + Z2 + Z3, where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is not present; and Z3 consists of n AEEA(s), where n ≥ 0.

3. The use of fatty acid-containing side chains in improving the oral bioavailability of polypeptide derivatives, wherein the polypeptide derivative is formed by modification of a polypeptide molecule containing the fatty acid-containing side chain, and the fatty acid-containing side chain is in the form of Z1 + Z2 + Z3, where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is not present; and Z3 consists of n AEEA(s), where n ≥ 0.

4. A method for improving the activity selectivity of a polypeptide derivative, comprising modifying a polypeptide molecule containing a fatty acid-containing side chain to form the polypeptide derivative, wherein the fatty acid-containing side chain is in the form of Z1 + Z2 + Z3, and where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is not present; and A method in which Z3 consists of n AEEA(s) such that n ≥ 0.

5. A method for improving the activity of a polypeptide derivative, comprising modifying a polypeptide molecule containing a fatty acid-containing side chain to form the polypeptide derivative, wherein the fatty acid-containing side chain is in the form of Z1 + Z2 + Z3, where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is not present; and A method in which Z3 consists of n AEEA(s) such that n ≥ 0.

6. A method for improving the oral bioavailability of a polypeptide derivative, comprising modifying a polypeptide molecule containing a fatty acid-containing side chain to form the polypeptide derivative, wherein the fatty acid-containing side chain is in the form of Z1 + Z2 + Z3, where Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is not present; and A method in which Z3 consists of n AEEA(s) such that n ≥ 0.

7. The use according to any one of claims 1 to 3, or the method according to any one of claims 4 to 6, wherein Z1 is C16, C17, C18, C19, or C20 fatty acid.

8. The use or method according to any one of claims 1 to 7, wherein Z2 is selected from one of γGlu, βAsp, γGlu, Inp, and Trx, preferably βAsp or γGlu.

9. The use or method according to any one of claims 1 to 8, wherein n is greater than 0, preferably 1 to 10, more preferably 2 to 8, and even more preferably n = 2.

10. The use or method according to any one of claims 1 to 9, wherein Z1 is C16, C18, or C20 fatty acid.

11. The use or method according to any one of claims 1 to 10, wherein Z2 is Inp or Trx, and n is greater than 0.

12. The use or method according to any one of claims 1 to 11, wherein Z1 is a C16, C17, C18, C19, or C20 fatty acid; Z2 is γGlu; ​​and n = 2.

13. The use or method according to any one of claims 1 to 12, wherein Z1, Z2, and Z3 are linked via amide bonds.

14. The use or method according to any one of claims 1 to 13, wherein the modification of the polypeptide molecule including the side chain is in 1 to 10 amino acids, preferably 1 to 3 amino acids, more preferably 1 amino acid of the polypeptide.

15. The use or method according to any one of claims 1 to 14, wherein the modification of the polypeptide molecule including the side chain is located at an amino acid at the N-terminus or any intermediate position of the polypeptide, and the amino acid is preferably selected from glutamic acid, aspartic acid, lysine, glutamine, or asparagine, and more preferably lysine.

16. The polypeptide is a naturally occurring or artificially synthesized polypeptide molecule and is an agonist or antagonist, preferably a GLP-1 receptor agonist, a GIP receptor antagonist, an insulin receptor agonist, a heparin receptor agonist, a somatotrophin, an interferon receptor agonist, an interleukin receptor agonist, a follicle-stimulating hormone receptor agonist, a gonadotropin receptor agonist, an erythropoietin receptor agonist, a peptide YY (PYY) receptor agonist, an oxygenation-modulating receptor agonist, or a glucagon-like peptide-2 (GLP-2). Use or method according to any one of claims 1 to 15, wherein a receptor agonist, calcitonin receptor agonist, parathyroid hormone (PTH) receptor agonist, ghrelin receptor agonist, endocannabinoid receptor agonist, leptin receptor agonist, serotonin receptor agonist, fibroblast growth factor 21 (FGF21) receptor agonist, cholecystokinin (CCK) receptor agonist, oxytomodulin receptor agonist, or glucagon receptor agonist, preferably an amyrin receptor agonist, and more preferably amyrin or a calcitonin analog.

17. The polypeptide is ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH ２ , KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH ２ , or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH ２ The use or method according to any one of claims 1 to 16, comprising the amino acid sequence, preferably wherein the polypeptide derivative is selected from one or more of N1, N2, N3, or M1 to M30.

18. The use according to any one of claims 1 and 7 to 17, or the method according to any one of claims 4 and 7 to 17, wherein the improvement of the activity selectivity of the polypeptide derivative is to improve the activity of the polypeptide derivative to the amyrin receptor preferentially over the calcitonin receptor, preferably to improve the ratio of the activity of the polypeptide derivative to the amyrin receptor to the activity of the polypeptide derivative to the calcitonin receptor, and more preferably to improve the activity of the polypeptide derivative to the amyrin receptor.

19. The use according to any one of claims 1 and 7 to 18, or the method according to any one of claims 4 and 7 to 18, wherein the improvement in the activity selectivity of the polypeptide derivative is such that the ratio of activity to amyrin receptor activity to activity to calcitonin receptor activity in cells is improved by more than 1.5 times, preferably more than 2 times, more preferably more than 5 times, and even more preferably more than 10 times.

20. The use or method according to claim 18 or 19, wherein the activity is agonist activity.

21. The polypeptide derivative is an amyrin receptor agonist, preferably an amyrin or calcitonin derivative, and more preferably ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH ２ The amino acid sequence includes KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH ２ The use according to any one of claims 1 and 7 to 20, or the method according to any one of claims 4 and 7 to 20, comprising the amino acid sequence.

22. The use according to claim 2, or the method according to claim 5, wherein the activity of the polypeptide derivative is an activity in reducing body weight and / or reducing food intake.

23. The use according to any one of claims 2 and 7 to 17, or the method according to any one of claims 5 and 7 to 17, wherein the improvement of the activity of the polypeptide derivative is an improvement of the activity of the polypeptide derivative with respect to intracellular amyrin receptors and calcitonin receptors.

24. The polypeptide derivative is an amyrin receptor agonist, preferably an amyrin or calcitonin derivative, and more preferably KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH ２ Or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH ２ A use according to any one of claims 2, 7-17, and 22-23, comprising the amino acid sequence, or a method according to any one of claims 5, 7-17, and 22-23.

25. The use according to any one of claims 3 and 7 to 17, or the method according to any one of claims 6 and 7 to 17, wherein the polypeptide derivative comprising the fatty acid-containing side chain is administered simultaneously with one or more oral delivery agents, preferably a salt of N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC), more preferably PNAC.

26. The use according to any one of claims 3 and 7-17, or the method according to any one of claims 6 and 7-17, wherein the improvement in the oral bioavailability of the polypeptide derivative achieves a higher oral bioavailability than that of the Cagrilintide polypeptide molecule.

27. The polypeptide derivative is an amylin receptor agonist, preferably an amylin or calcitonin derivative, more preferably, KCNTATCATQ RLADFLRHS SNNLKPI LPPT NVGSNT-trans-Hyp-NH ２ or KCNTATCATQ RLAEFLRHS SNNFGPIL PPT NVGSNTP-NH ２ Use according to any one of claims 3 and 7 to 17, or method according to any one of claims 6 and 7 to 17, comprising the amino acid sequence of

28. A polypeptide derivative comprising the polypeptide and a side chain attached to the polypeptide in the form of Z1 + Z2 + Z3, wherein Z1 is a C16-C22 fatty acid; Z2 is selected from one of γGlu, αGlu, βAsp, αAsp, Inp, and Trx, or is not present; and A derivative in which Z3 consists of n AEEA(s) such that n ≥ 0.

29. The derivative according to claim 28, wherein Z1 is C16, C17, C18, C19, or C20 fatty diacid.

30. The derivative according to claim 28 or 29, wherein Z2 is selected from one of γGlu, Inp, and Trx, or is absent.

31. A derivative according to any one of claims 28 to 30, wherein n is greater than 0.

32. The derivative according to any one of claims 28 to 31, wherein Z2 is Inp or Trx, and n is greater than 0.

33. The derivative according to any one of claims 28 to 32, wherein Z2 is absent and n is greater than 0.

34. The derivative according to any one of claims 28 to 33, wherein Z2 is γGlu and n = 2.

35. The polypeptide is an amyrin receptor agonist; preferably, a derivative of the polypeptide is an amyrin or calcitonin derivative; more preferably, the polypeptide is ASQLS TAVLG RLSDE LHRLQ DYPRT DVGSG SP-NH ２ , KCNTATCATQ RLADFLRHSS NNLKPILPPT NVGSNT-trans-Hyp-NH ２ , or KCNTATCATQ RLAEFLRHSS NNFGPILPPT NVGSNTP-NH ２ The derivative according to any one of claims 28 to 34, comprising the amino acid sequence, preferably the derivative of the polypeptide being selected from one or more of N1, N2, N3, or M1 to M30.

36. A pharmaceutical composition comprising a derivative or a pharmaceutically acceptable salt thereof according to any one of claims 28 to 35, and a pharmaceutically acceptable excipient, preferably comprising one or more oral delivery agents, more preferably a salt of N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC), and even more preferably PNAC.

37. Use of a derivative according to any one of claims 28 to 35 or a pharmaceutically acceptable salt thereof or a composition according to claim 36 in the manufacture of a polypeptide-based agent, preferably the polypeptide-based agent being used for the prevention and / or treatment of overweight and / or obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain.

38. The use according to claim 37, wherein the polypeptide-based agent is an oral agent; more preferably, the oral agent comprises one or more oral delivery agents, preferably a salt of N-[8-(2-hydroxybenzoyl)amino]caprylic acid (NAC), and more preferably PNAC.

39. Use of a derivative according to any one of claims 28 to 35 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 36, in reducing food intake.

40. A method for preventing and / or treating overweight and / or obesity and / or type I or type II diabetes and / or osteoporosis and / or neuropathic pain, comprising administering a prophylactic or therapeutically effective amount of a derivative or pharmaceutically acceptable salt thereof according to any one of claims 28 to 35 or the pharmaceutical composition according to claim 36 to a target.

41. A method for reducing food intake, comprising administering an effective amount of a derivative or a pharmaceutically acceptable salt thereof according to any one of claims 28 to 35 or the pharmaceutical composition according to claim 36.

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