KR20230106481A - Long-acting fatty acid-peptide derivatives and uses thereof - Google Patents

Abstract

The present invention relates to a long-acting fatty acid-peptide derivative and its use, and specifically, to a long-acting GLP-1/GLP-2 dual agonist and its use for treating intestinal disorders, intestinal damage, or gastric disorders. The fatty acid-peptide derivative of the present invention is an agonist for both GLP-1 and GLP-2 receptors, effectively stimulating GLP-1 and GLP-2 receptors known as targets of short bowel syndrome, and has excellent sustained efficacy in vivo compared to existing drugs , and exhibits significantly superior therapeutic effects compared to negative and positive controls in short bowel syndrome animal models, and thus can be usefully used for the prevention, improvement, and treatment of intestinal and gastric diseases.

Description

Long-acting fatty acid-peptide derivatives and uses thereof {Long-acting fatty acid-peptide derivatives and uses thereof}

The present invention relates to a long-acting fatty acid-peptide derivative and its use, and specifically, to a long-acting GLP-1/GLP-2 dual agonist and its use for treating intestinal disorders, intestinal damage, or gastric disorders.

Glucagon-like peptide-1 (GLP-1) has various biological activities such as stimulating insulin secretion, inhibiting glucagon secretion, inhibiting gastric emptying, inhibiting gastric or intestinal motility, enhancing glucose utilization and inducing weight loss. induce effect. In addition, GLP-1 prevents pancreatic β cell degeneration caused by the progress of type 2 diabetes, non-insulin dependence diabetes mellitus (NIDDM) and promotes the generation of new β cells It is known that it can act by restoring insulin secretion.

Glucagon-like peptide-2 (GLP-2) is a peptide hormone composed of 33 amino acids produced by L-cells of the small intestine in response to ingested nutrients. GLP-2 induces mucosal growth in the small and large intestine, promotes the growth of enterocytes and crypt cells, and inhibits apoptosis. GLP-2 also increases the absorption of nutrients in the small intestine and reduces intestinal permeability. GLP-2 also inhibits gastric emptying and secretion of gastric acid, increases intestinal blood flow rate, and relaxes intestinal smooth muscle. GLP-2 has shown promising potential as a therapeutic agent in various intestinal diseases and experimental models of intestinal damage due to its characteristics such as energy absorption and protection and activation of intestinal cell functions.

In fact, Gattex (teduglutide), a GLP-2 analogue, was developed at the end of 2012 by NPS Pharmaceuticals, Inc. in the US and received orphan drug designation as a treatment for short bowel syndrome (SBS). Patients are undergoing phase 2 clinical trials.

However, in order to commercialize GLP-2 or an analogue thereof as a drug, there are problems that must first be solved. In general, peptides such as GLP-2 are easily denatured due to their low stability, are degraded by proteolytic enzymes in the body and lose their activity, and are also relatively small in size and easily eliminated through the kidneys. In order to maintain the blood concentration and potency of the drug, it is necessary to frequently administer the peptide drug to the patient. However, most of the peptide drugs are administered to patients in the form of injections, and therefore, frequent injections are required to maintain blood levels of physiologically active peptides, which causes great suffering to patients. Various attempts have been made to overcome these problems. As one of them, there has been an attempt to deliver the peptide drug into the body by inhalation through the oral cavity or nasal cavity by increasing the biomembrane permeability of the peptide drug. However, these methods have lower efficiency of peptide delivery to the body than injections, and therefore, it is still difficult to maintain the activity of the peptide drug under the required conditions. In particular, GLP-2 has a very short physiological activity half-life of less than 7 minutes. This is due to the loss of the potency of GLP-2 by cleavage between amino acids 2 (Ala) and 3 (Asp) of GLP-2 by dipeptidyl peptidase IV (hereinafter referred to as DPPPIV).

Accordingly, the present inventors not only have the activity of GLP-2 and GLP-1, which are the targets of existing drugs, but also exhibit superior efficacy than existing drugs through the synergistic effect between GLP-1 and GLP-2. While developing peptide preparations, an attempt was made to increase the physiological activity half-life of the drug.

Bolette H. et al., The Journal of Clinical Endocrinology & Metabolism. 2000, 85(8): 2884-2888 Jacqueline A. Koehler et al., Cell Metabolism. 2015, 21, 379-391 Daniel J. Drucker, ACS Pharmacol. Transl. Sci. 2019, 2, 134-142 Rasmus Hytting-Andreasen et al., PLoS ONE 13(6):e0198046 K. Bulut. et al., Regulatory Peptides 121 (2004) 137- 143 P. Janssen. et al, Aliment Pharmacol Ther 2013; 37:18-36 Marine Grigoryan. et al, Endocrinology, 2012, 153(7):0000-0000

Therefore, in order to solve the above problems, the present inventors have prepared GLP-1/GLP-2-like peptides that bind to GLP-1 and GLP-2 receptors, which are intestinal cells, and show the effect of treating intestinal and gastric diseases, and to the peptides By combining lysine and palmitic acid, the fatty acid-peptide derivative of the present invention, which exhibits dual action activity on GLP-1 and GLP-2 receptors, has a therapeutic effect on short bowel syndrome and increases half-life in the body.

Therefore, an object of the present invention is a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof in which a fatty acid is linked to a polypeptide represented by the general formula below, wherein the polypeptide represented by the general formula is N-terminal, C-terminal, or It is to provide a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof, characterized in that a fatty acid is linked to the middle chain.

[general meal]

Amino acid sequence-Kn represented by SEQ ID NO: 1

(In the above general formula, K is lysine, and n is an integer from 1 to 10.)

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of intestinal disorders, intestinal damage and gastric diseases, comprising the fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof as an active ingredient. is to do

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention is a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof in which a fatty acid is bound to a polypeptide represented by the following general formula, wherein the polypeptide represented by the general formula is N Provided is a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof, characterized in that a fatty acid is bonded to the terminal, C-terminus, or intermediate chain thereof.

[general meal]

Amino acid sequence-Kn represented by SEQ ID NO: 1

(In the above general formula, K is lysine, and n is an integer from 1 to 10.)

In one embodiment of the present invention, the fatty acid may be a C12 to C22 saturated fatty acid or unsaturated fatty acid, but is not limited thereto.

In another embodiment of the present invention, n may be an integer of 2 to 9, but is not limited thereto.

In another embodiment of the present invention, the fatty acid may be linked to the C-terminus, but is not limited thereto.

In another embodiment of the present invention, the polypeptide represented by the general formula may consist of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 5, but is not limited thereto.

In another embodiment of the present invention, the fatty acid-peptide derivative may have an in vivo half-life (t _1/2 ) of 2 hours or more, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of intestinal disorders, intestinal damage, and gastric diseases, comprising the fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof as an active ingredient. do.

In one embodiment of the present invention, the intestinal disease may be at least one selected from the group consisting of short bowel syndrome, irritable bowel disease, inflammatory bowel disease, Crohn's disease, colitis, colitis, pancreatitis, ileitis, mucositis, and intestinal atrophy, but is limited thereto it is not going to be

In another embodiment of the present invention, the intestinal disease may be short bowel syndrome, but is not limited thereto.

In another embodiment of the present invention, the gastric disease may be at least one selected from the group consisting of gastric spasm, gastritis, gastric ulcer, duodenitis, and duodenal ulcer, but is not limited thereto.

In another embodiment of the present invention, the fatty acid-peptide derivative may be administered at 0.1 to 200 mg/kg per week, but is not limited thereto.

In another embodiment of the present invention, the fatty acid-peptide derivative may increase the weight or length of the small intestine, but is not limited thereto.

In another embodiment of the present invention, the fatty acid-peptide derivative may increase villus height, but is not limited thereto.

In another embodiment of the present invention, the fatty acid-peptide derivative may increase the protein or DNA content of the small intestine, but is not limited thereto.

In addition, the present invention provides prevention of at least one disease selected from the group consisting of intestinal disease, intestinal damage, and gastric disease, comprising administering to a subject a composition comprising the fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof. or a method of treatment.

In addition, the present invention provides a use of the fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof for preventing or treating one or more diseases selected from the group consisting of intestinal disorders, intestinal damage, and gastric diseases.

In addition, the present invention provides a use of the fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof for preparing a drug for treating one or more diseases selected from the group consisting of intestinal disorders, intestinal damage, and gastric diseases.

The fatty acid-peptide derivative of the present invention is an agonist for both GLP-1 and GLP-2 receptors, effectively stimulating GLP-1 and GLP-2 receptors known as targets of short bowel syndrome, and has excellent sustained efficacy in vivo compared to existing drugs With, it shows a significantly superior therapeutic effect compared to negative and positive controls in short bowel syndrome animal models, and is expected to be useful for the prevention, improvement, and treatment of intestinal diseases, intestinal damage, and gastric diseases.

Figure 1 shows the Scheme of the cell proliferation efficacy evaluation method in the intestine using the CCD-18co cell line and the Caco-2 cell line.
Figure 2 shows a daily administration test method scheme for confirming the effect on intestinal proliferation of normal mice.
Figure 3 shows the administration test (secondary test) method Scheme for each period to confirm the effect on the intestinal proliferation of normal mice.
Figure 4 shows a test (third test) method scheme for final candidate selection to confirm the effect on intestinal proliferation of normal mice.
Figure 5 shows a test method scheme for confirming the effect on the intestinal growth of the short bowel syndrome rat model.
6 shows the results of comparing the GLP-1 receptor and GLP-2 receptor activity test results of HU003K5FA and HU003K7FA with positive controls (Exendin-4, GLP-2).
Figure 7 shows the results of cell proliferation evaluation of colon-derived serosal cell line CCD-18co cells and colon cancer-derived Caco-2 cells.
8 shows blood concentrations over time after single subcutaneous administration of HU003K2FA and HU003K7FA to rats.
Figure 9 shows the daily weight change according to the daily administration test. Data are presented as mean ± SD.
Figure 10 shows the body weight on the 8th day according to the daily administration test. Data are presented as mean ± SD. *p<0.05 vs. G1, **p<0.01 vs. G1, #p<0.05 vs. G2, ##p<0.01 vs. G2, &&p<0.01 vs. G3.
Figure 11 shows the weight of the small intestine according to the daily administration test. Data are presented as mean ± SD. *p<0.05 vs. G1, **p<0.01 vs. G1.
Figure 12 shows the weight ratio of the small intestine to body weight according to the daily administration test. Data are presented as mean ± SD. **p<0.01 vs. G1.
Figure 13 shows the daily weight change according to the periodic administration test (second test). Data are presented as mean ± SD.
Figure 14 shows the body weight on the 8th day according to the periodic administration test (second test). Data are presented as mean ± SD. *p<0.05 vs. G1, **p<0.01 vs. G1, #p<0.05 vs. G2, ##p<0.01 vs. G2, &p<0.05 vs. G3, &&p<0.01 vs. G3, €€p<0.01 vs. G4.
Figure 15 shows the weight of the small intestine according to the periodic administration test (second test). Data are presented as mean ± SD. **p<0.01 vs. G1, #p<0.05 vs. G2.
Figure 16 shows the weight-to-body ratio of the small intestine according to the periodic administration test (second test). Data are presented as mean ± SD. **p<0.01 vs. G1, ##p<0.01 vs. G2, €€p<0.01 vs. G4.
Figure 17 shows the daily weight change according to the final candidate selection test (third test). Data are presented as mean ± SD.
Figure 18 shows the body weight on the 8th day according to the test for final candidate selection (third test). Data are presented as mean ± SD. **p<0.01 vs. G1, ##p<0.01 vs. G2.
19 shows the weight of the small intestine according to the test for final candidate selection (third test). Data are presented as mean ± SD. **p<0.01 vs. G1, #p<0.05 vs. G2, ##p<0.01 vs. G2, &&p<0.01 vs. G3.
20 shows the weight-to-weight ratio of the small intestine according to the final candidate selection test (third test). Data are presented as mean ± SD. **p<0.01 vs. G1, #p<0.05 vs. G2, ##p<0.01 vs. G2, &p<0.05 vs. G3.
21 shows daily weight changes according to the short bowel syndrome model test. Data are presented as mean ± SD.
22 shows body weight on day 16 according to the short bowel syndrome model test. Data are presented as mean ± SD. **p<0.01 vs. G1, #p<0.05 vs. G2, &p<0.05 vs. G3, &&p<0.01 vs. G3, €p<0.05 vs. G4, €€p<0.01 vs. G4.
23 shows the weight of the small intestine according to the short bowel syndrome model test. Data are presented as mean ± SD. ##p<0.01 vs. G2, &&p<0.01 vs. G3, €€p<0.01 vs. G4.
24 shows the weight ratio of the small intestine to body weight according to the short bowel syndrome model test. Data are presented as mean ± SD. **p<0.01 vs. G1, #p<0.05 vs. G2, ##p<0.01 vs. G2, &p<0.05 vs. G3, &&p<0.01 vs. G3, €p<0.05 vs. G4, €€p<0.01 vs. G4.
25 shows changes in intestinal length for each administration group according to the short bowel syndrome model test. Data are presented as mean ± SD. **p<0.01 vs. G1, #p<0.05 vs. G2.
26 shows the protein content of each administration group according to the short bowel syndrome model test. Data are presented as mean ± SD. *p<0.05 vs. G1, **p<0.01 vs. G1, #p<0.05 vs. G2, ##p<0.01 vs. G2, &p<0.05 vs. G3, €p<0.05 vs. G4, €€p<0.01 vs. G4.
27 shows the total DNA content of each administration group according to the short bowel syndrome model test. Data are presented as mean ± SD. **p<0.01 vs. G1, #p<0.05 vs. G2, ##p<0.01 vs. G2, &p<0.05 vs. G3, &&p<0.01 vs. G3, €€p<0.01 vs. G4.
28 shows the results of H&E staining for each administration group according to the short bowel syndrome model test.
29 shows villus height, crypt depth, and mucosal thickness in the duodenum for each administration group according to the short bowel syndrome model test. Data are presented as mean ± SD. *p<0.05 vs. G1, **p<0.01 vs. G1, #p<0.05 vs. G2, ##p<0.01 vs. G2, &p<0.05 vs. G3, &&p<0.01 vs. G3, €p<0.05 vs. G4, €€p<0.01 vs. G4.
30 shows villus height, crypt depth, and mucosal thickness in the jejunum for each administration group according to the short bowel syndrome model test. Data are presented as mean ± SD. **p<0.01 vs. G1, #p<0.05 vs. G2, ##p<0.01 vs. G2, &p<0.05 vs. G3, €€p<0.01 vs. G4.

The present invention relates to a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof in which a fatty acid is bonded to a polypeptide represented by the following general formula, wherein the polypeptide represented by the general formula is at the N-terminus, C-terminus, or intermediate chain thereof. It relates to a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof, characterized in that a fatty acid is bound, and a composition for preventing, improving or treating intestinal diseases, intestinal damage, or gastric diseases comprising the same as an active ingredient.

[general meal]

Amino acid sequence-Kn represented by SEQ ID NO: 1

(In the above general formula, K is lysine, and n is an integer from 1 to 10.)

Hereinafter, the present invention will be described in detail.

In the present invention, amino acids referred to by abbreviations have been described according to the IUPAC-IUB nomenclature:

Alanine Ala, A;

arginine Arg, R;

asparagine Asn, N;

aspartic acid Asp, D;

cysteine Cys, C;

glutamic acid Glu, E;

Glutamine Gln, Q;

glycine Gly, G;

histidine His, H;

isoleucine Ile, I;

Leucine Leu, L;

Lysine Lys, K;

methionine Met, M;

phenylalanine Phe, F;

Proline Pro, P;

serine Ser, S;

threonine Thr, T;

Tryptophan Trp, W;

Tyrosine Tyr, Y;

Balin Val, V.

In the present invention, "peptide" means a linear molecule formed by binding amino acid residues to each other by a peptide bond.

In the present invention, a "peptide" can be easily prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press). , Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989). In addition, the peptides of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, such as using an automated DNA synthesizer (available from Biosearch or Applied Biosystems). The constructed DNA sequence is a vector comprising one or more expression control sequences (eg, promoters, enhancers, etc.) operably linked to the DNA sequence to control the expression of the DNA sequence. and the host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are cultured under appropriate media and conditions to allow expression of the DNA sequence, and the substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery may be performed using a method known in the art (eg, chromatography). As used herein, 'substantially pure peptide' means that the peptide according to the present invention does not substantially contain any other host-derived proteins. For the genetic engineering method for synthesizing the peptide of the present invention, reference may be made to the following documents: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; and Hitzeman et al., J. Biol. Chem., 255:12073-12080, 1990.

The term "glucagon like peptide-1 (GLP-1)" of the present invention is a kind of incretin, a hormone secreted from the digestive tract, and is secreted from L cells in the intestinal tract in an ingestion-dependent manner to increase pancreatic insulin secretion. It is a protein known to play a role in suppressing postprandial blood sugar rise by suppressing the secretion of Glucagon and Glucagon. As such, GLP-1 has been widely known for its use in the treatment of diabetes, and its weight loss effect has also been reported by being involved in the physiological control of appetite. In addition, research reports that GLP-1 induces the division of enterocytes and crypt cells to induce an increase in the number of enterocytes are already known. It is expected that the existing GLP-2 activity and the activity of GLP-1, which induce cell division of small intestine trophoblasts, will be fused, thereby promoting the proliferation and growth of small intestine trophoblastic cells through two active mechanisms, which will be more effective in treating short bowel syndrome. expected

The term "glucagon like peptide-2 (GLP-2)" of the present invention refers to 33 short peptides produced in the form of proglucagon precursors in enteroendocrine L cells of the intestine and specific regions of the brain and produced through enzymatic cleavage by intestinal enzymes. As a peptide hormone with amino acids, it is co-secreted with glucagon-like peptide-1 (GLP-1), oxyntomodulin and glycentin in response to food intake and nutrient digestion. GLP-2 is widely known to be effective not only in inflammatory bowel disease but also in short bowel syndrome (SBS). In fact, Gattex (teduglutide), a GLP-2 analog, was developed at the end of 2012 by NPS Pharmaceuticals, Inc. in the United States and was designated as an orphan drug for the treatment of short bowel syndrome. In progress.

The polypeptide represented by the following general formula of the present invention is SEQ ID NO: 1 (His-Gly-Asp-Gly-Ser-Phe-Thr-Ser-Glu-Leu-Ser-Thr-Tyr-Leu-Asp-Ala-Leu- Ala-Thr-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp) may contain 1 to 10 lysines, but is not limited thereto. .

[general meal]

Amino acid sequence-Kn represented by SEQ ID NO: 1

(In the above general formula, K is lysine, and n is an integer from 1 to 10.)

The amino acid sequence represented by SEQ ID NO: 1 of the present invention may be obtained by changing N (Asn), which is the 16th amino acid of native GLP-2, to A (Ala), but is not limited thereto. This may be for reducing the overall peptide size, but is not limited thereto.

The amino acid sequence represented by SEQ ID NO: 1 of the present invention may be obtained by changing A (Ala), the second amino acid of native GLP-2, to G (Gly) of Exendin-4 and gattex, but is not limited thereto. This may be for increasing the activity of GLP-1 and GLP-2, but is not limited thereto.

In addition, the amino acid sequence represented by SEQ ID NO: 1 of the present invention is one in which the amino acid at position 13 is substituted with Tyr (Y) and/or the amino acid at position 19 is replaced with Thr (T) based on the native GLP-2 amino acid. It may be, but is not limited thereto. The substitution may be for increasing the biological activity of GLP-1 and GLP-2, but is not limited thereto.

In the present invention, the amino acid sequence represented by SEQ ID NO: 1 may be a variant having 80%, 90%, 95%, 97%, 98% or 99% homology thereto, and some of the sequences in the sequence of SEQ ID NO: 1 Deletions, substitutions or additions may also be included within the scope of the present invention.

In the present invention, the fatty acid-peptide derivative may have dual agonist activity for GLP-1 receptor and GLP-2 receptor, but is not limited thereto.

In the present invention, the fatty acid-peptide derivative may have greater agonist activity on the GLP-2 receptor than agonist activity on the GLP-1 receptor, but is not limited thereto.

In the present invention, the fatty acid-peptide derivative has a relative agonist activity for the GLP-1 receptor of 5% to 30%, 8% to 30%, 10% to 30%, 10% to 25%, 10% to 23%, 13% to 25%, 13% to 23%, 14% to 23%, 14% to 21%, 15% to 25%, 17% to 23%, or 18% to 22%. It is not.

In the present invention, the fatty acid-peptide derivative has a relative agonist activity for the GLP-2 receptor of 30% to 300%, 30% to 250%, 30% to 200%, 30% to 180%, 30% to 150%, 30% to 120%, 40% to 300%, 40% to 250%, 40% to 200%, 40% to 180%, 40% to 150%, 40% to 120%, 50% to 300%, 50% to 250%, 50% to 200%, 50% to 180%, 50% to 150%, 50% to 130%, 70% to 300%, 70% to 250%, 70% to 200%, 70% to 180 %, it may be 70% to 150%, 70% to 130%, or 80% to 100%, but is not limited thereto.

The fatty acid-peptide derivative of the present invention may include one or more amino acid changes within the range of showing effects of preventing, treating, or improving intestinal disorders, intestinal damage, or gastric disorders.

In the fatty acid-peptide derivative of the present invention, modification of the amino terminal or carboxy terminal may be induced to select a part of the amino acid sequence and increase its activity. Through this modification, the peptides of the present invention can have an increased half-life upon in vivo administration.

In the present invention, the fatty acid may be a C12 to C22 saturated fatty acid or unsaturated fatty acid. The fatty acid may be bonded to a polypeptide represented by the general formula in the form of a fatty acid group, but is not limited thereto.

The saturated fatty acid group mentioned in the present invention includes a lauryl group (C12:0, lauryl), a tridecyl group (C13:0, tridecyl), a myristyl group (C14:0, myristil), and a pentadecyl group (C15:0, pentadecyl). ), palmitoyl group (C16:0, palmitoyl), margaryl group (C17:0, margaryl), stearyl group (C18:0, stearyl), nonadecyl group (C19:0, nonadecyl), arachidyl group (C20 :0, arachidyl), heneicosyl group (C21:0, heneicosyl), or behenyl group (C22:0, behenyl).

The C12 to C22 unsaturated fatty acid groups referred to in the present invention are palmitoleyl (C16: 1, palmitoleyl), oleyl (C18: 1, oleyl), myristoleyl (C14: 1 myristoleyl), linoleyl (C18) :2 linoleyl) or arachidonyl group (C20:3, arachidonyl).

In the present invention, n may be an integer of 1 to 10, 1 to 9, 1 to 8, 2 to 9, 2 to 8, 2 to 7, 3 to 7, 4 to 7, 5 to 7, or 6 to 8 However, it is not limited thereto.

In the present invention, the fatty acid may be bonded to the C-terminus of the polypeptide represented by the general formula, but is not limited thereto.

In the present invention, the polypeptide represented by the general formula may consist of the amino acid sequence represented by SEQ ID NO: 2 (HGDGSFTSELSTYLDALATRDFIAWLIQTKITDKKKKKKK), but is not limited thereto.

In the present invention, the fatty acid-peptide derivative has an in vivo half-life (t _1/2 ) of 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more. or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, or 19 hours or more, but is not limited thereto. . In one embodiment of the present invention, the fatty acid-peptide derivative of the present invention showed a half-life in blood of about 19 hours, compared to the blood half-life of 1.8 hours of the positive control teduglutide.

In the present invention, the fatty acid-peptide derivative has a mean retention time (MRT) in blood of 3 to 100 hours, 3 to 90 hours, 3 to 80 hours, 3 to 70 hours, 3 to 60 hours, 3 to 50 hours, and 3 to 50 hours. 40 hours, 5 to 100 hours, 5 to 90 hours, 5 to 80 hours, 5 to 70 hours, 5 to 60 hours, 5 to 50 hours, 5 to 40 hours, 10 to 100 hours, 10 to 90 hours, 10 to 80 hours, 10 to 70 hours, 10 to 60 hours, 10 to 50 hours, 10 to 40 hours, 20 to 100 hours, 20 to 90 hours, 20 to 80 hours, 20 to 70 hours, 20 to 60 hours, 20 to 60 hours It may be 50 hours, 20 to 40 hours, 25 to 35 hours, or about 30 hours, but is not limited thereto. In one embodiment of the present invention, the fatty acid-peptide derivative of the present invention showed an average retention time in blood of about 31.8 hours, compared to the average retention time in blood of 2.1 hours of the positive control teduglutide.

In the present invention, the fatty acid-peptide derivative may be administered at 0.1 to 200 mg/kg per week, but is not limited thereto.

In the present invention, the fatty acid-peptide derivative may increase the weight or length of the small intestine, but is not limited thereto.

In the present invention, the fatty acid-peptide derivative may increase villus height, but is not limited thereto.

A protecting group such as acetyl group, fluorenyl methoxy carbonyl group, formyl group, myristyl group, stearyl group or polyethylene glycol (PEG) may be bound to the N-terminus or C-terminus of the fatty acid-peptide derivative of the present invention. The carboxy terminal of may be modified with a hydroxyl group (-OH), an amino group (-NH ₂ ), an azide (-NHNH ₂ ), and the like. In addition, oligosaccharides chains, all nanoparticles (gold particles, liposomes, heparin, hydrogel, etc.), amino acids, carrier proteins, etc. can be combined. Modification of the amino acids described above serves to improve the potency and stability of the peptides of the present invention.

In the present invention, "bowel disease" may be short bowel syndrome, irritable bowel disease, inflammatory bowel disease, Crohn's disease, colitis, colitis, pancreatitis, ileitis, mucositis or intestinal atrophy, but is not limited thereto.

In the present invention, "gastric disease" may be gastric spasm, gastritis, gastric ulcer, duodenitis, or duodenal ulcer, but is not limited thereto.

In the present invention, "short bowel syndrome" refers to various metabolic abnormalities caused by a decrease in digestion and absorption of nutrients due to a short small intestine, and is classified into congenital and acquired according to the type of onset. Congenital means that the fetus is born with a short small intestine, and acquired means that the length of the small intestine remaining after cutting out due to other diseases or trauma is too short, causing disability.

Acquired short bowel syndrome refers to a situation in which the intestine is extensively resected for various reasons, resulting in a decrease in intestinal absorption capacity, resulting in diarrhea, dehydration, and malnutrition. Although many cases are cured with medical treatment, including TPN, in some cases, an intestinal lengthening procedure or an intestinal transplantation may be required. It is a disease that requires complex treatment.

The present invention may also include a pharmaceutically acceptable salt of the fatty acid-peptide derivative as an active ingredient. As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.

Examples of suitable acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid and the like. Acid addition salts can be prepared by conventional methods, for example, by dissolving a compound in an aqueous solution of excess acid and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can also be prepared by heating equimolar amounts of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or suction filtering the precipitated salt.

Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable for pharmaceutical purposes to prepare a sodium, potassium or calcium salt, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

The content of the fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the progress of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9 based on the weight of the total composition. % by weight, or 0.001 to 50% by weight, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.

The pharmaceutical compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, and limonadese, respectively, according to conventional methods. , tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta agents, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external agents are creams, gels, patches, sprays, ointments, and warning agents. , lotion, liniment, pasta, or cataplasma may have formulations such as the like.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin Binders such as hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch arc, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, Hydroxypropyl Methyl Cellulose, Corn Starch, Agar Powder, Methyl Cellulose, Bentonite, Hydroxypropyl Starch, Sodium Carboxymethyl Cellulose, Sodium Alginate, Calcium Carboxymethyl Cellulose, Calcium Citrate, Sodium Lauryl Sulfate, Silicic Anhydride, 1-Hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopod, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid; may be used.

Additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, and the like may be used.

In the syrup according to the present invention, a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.

Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

Acacia, tragacantha, methylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, HPMC 2910, etc. and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV injection, dextrose injection, dextrose + sodium chloride injection, polyethylene glycol (PEG), lactated IV injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; tonicity agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid; Sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; Suspending agents such as Siemesis sodium, sodium alginate, Tween 80, aluminum monostearate may be included.

The suppository according to the present invention includes cacao butter, lanolin, witapsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lannet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolen (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Buytyrum Tego-G -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hyde Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), testosterone triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. ) or by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. Since the pharmaceutical composition of the present invention has excellent in vivo persistence, the number and frequency of administration of the pharmaceutical composition of the present invention can be significantly reduced.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.

In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. It may be a mammal of, but is not limited thereto.

In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method.

In the present invention, "prevention" refers to any action that suppresses or delays the onset of a desired disease, and "treatment" means that a desired disease and its associated metabolic abnormalities are improved or treated by administration of the pharmaceutical composition according to the present invention. Any action that is beneficially altered, and "improvement" means any action that reduces a parameter related to a desired disease, for example, the severity of a symptom, by administration of the composition according to the present invention.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Experimental Example 1. Synthesis and purification of long-acting GLP-1/GLP-2 dual agonist

1-1. Synthesis of HU003 peptide

A peptidyl resin for 9-fluorenylmethyloxycarbonyl (Fmoc) SPPS (stationary phase peptide synthesis) was prepared. First, Rink Amide MBHA resin was resin swelled in a DMF solvent for 60 minutes. Then, Fmoc-amino acid was sequentially coupled to the resin by solid phase synthesis together with a coupling reagent. The resin was then washed three times with DMF. Fmoc-Gln(Trt)-Thr(Psi,Me,Me,Pro)-OH was used at amino acids 5 and 6. Deprotection of Fmoc was performed on the resin using 20% Piperidine/DMF, and then washed 5 times using DMF. Then, all amino acids were bound to the peptidyl resin in a fully protected state. Then, the completely protected peptidyl resin was separated using E solution (TFA:Thioanisole:EDT:Phenol:H ₂ O=87.5:5:2.5:2.5:2.5) for 3 hours and then filtered. Then, the filtrate was precipitated with cold ether and dried under vacuum to obtain crude peptide. The crude peptide was purified to 70% by reverse phase HPLC (Reverse Phase Preparative HPLC) using a luna C18 5 μm column and a fraction collector with Hanbon equipment, and A buffer (4 Purification was performed using g/L ammonium acetate, aq.) and buffer B (4 g/L ammonium acetate, 70% ACN, aq.). Afterwards, the obtained samples were analyzed using HPLC and MS and lyophilized. The obtained material (13.7 mg) was prepared in Buffer A (0.1% TFA, aq.) and Buffer B (0.1% TFA, 80% ACN, aq.) under gradient conditions using HPLC and MS to obtain a purity of 95%. The second purification was performed using , and finally, 10.8 mg of HU003 peptide sample was obtained.

HU003 (SEQ ID NO: 1): His-Gly-Asp-Gly-Ser-Phe-Thr-Ser-Glu-Leu-Ser-Thr-Tyr-Leu-Asp-Ala-Leu-Ala-Thr-Arg-Asp-Phe -Ile-Ala-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp

[Formula 1]

Peptide of SEQ ID NO: 1

1-2. Conjugation of HU003 peptide and palmitic acid

In order to conjugate palmitic acid, amino acids protected by Boc and/or Fmoc (Fmoc-Lys(Palm-Glu-OtBu)-OH, Fmoc-Lys(Trt)-OH) were additionally used for synthesis. Palmitic acid was conjugated to lysine through a peptide bond, and Fmoc-Lys(Pal-Glu-OtBu)-OH was purchased from Polypeptide in the form of synthesized chemicals and synthesized (Cas no. 1491158-62-3, PPL item no: 500495). The binding of lysine-palmitic acid to the HU003 peptide was performed in the same manner as in Experimental Example 1-1.

The sequences of the prepared lysine (K) and peptides are shown in Table 1 below. K marked in red represents the site where fatty acids are conjugated.

In the present specification, when a fatty acid is attached to each of the following peptide sequences, it is indicated as HU003K2FA, HU003K3FA, HU003K5FA, HU003K7FA, and HU003K9FA.

HU003K2 (SEQ ID NO: 2): His-Gly-Asp-Gly-Ser-Phe-Thr-Ser-Glu-Leu-Ser-Thr-Tyr-Leu-Asp-Ala-Leu-Ala-Thr-Arg-Asp-Phe -Ile-Ala-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-Lys-Lys

HU003K3 (SEQ ID NO: 3): His-Gly-Asp-Gly-Ser-Phe-Thr-Ser-Glu-Leu-Ser-Thr-Tyr-Leu-Asp-Ala-Leu-Ala-Thr-Arg-Asp-Phe -Ile-Ala-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys

HU003K5 (SEQ ID NO: 4): His-Gly-Asp-Gly-Ser-Phe-Thr-Ser-Glu-Leu-Ser-Thr-Tyr-Leu-Asp-Ala-Leu-Ala-Thr-Arg-Asp-Phe -Ile-Ala-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys

HU003K7 (SEQ ID NO: 5): His-Gly-Asp-Gly-Ser-Phe-Thr-Ser-Glu-Leu-Ser-Thr-Tyr-Leu-Asp-Ala-Leu-Ala-Thr-Arg-Asp-Phe -Ile-Ala-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys-Lys

HU003K9 (SEQ ID NO: 6): His-Gly-Asp-Gly-Ser-Phe-Thr-Ser-Glu-Leu-Ser-Thr-Tyr-Leu-Asp-Ala-Leu-Ala-Thr-Arg-Asp-Phe -Ile-Ala-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys-Lys

Experimental Example 2. cAMP assay (GLP-1 and GLP-2 receptor efficacy analysis)

2-1. Cell lines expressing human GLP-1 and GLP-2 receptors

To verify the activity of the novel GLP-1/GLP-2 peptide, a cell-based assay test was performed. The cell lines used were GLP-1R/CHO and GLP-2/CHO cells, and information on the cells is shown in Table 2 below.

cell Manufacturing number (Code) manufacturing company GLP-1R/CHO 95-0062C2 DiscoverX GLP-2R/CHO 95-0112C2 DiscoverX

2-2. Human GLP-1/GLP-2 Biological Activity Assay

The synthetic peptides were treated with GLP-1R/CHO and GLP-2R/CHO cells, respectively, to confirm intracellular cAMP concentration. cAMP concentration was measured using a cAMP assay kit (Discover X, 90-0075-LM10). EC50 values were compared using exendin-4 (DiscoverX, 92-1115) for GLP-1 and GLP-2 (DiscoverX, 92-1079) for GLP-2 as standards. Information on standard products is shown in Table 3 below.

GLP-1 activity measurement GLP-2 activity measurement substance name Exendin-4 GLP-2 (synthetic) manufacturing company Discover X Discover X water >95% >95%

Experimental Example 3. Cell proliferation assay

CCD-18co cells are colon-derived serosal cells (fibroblasts) that have GLP-2 receptors and express cell growth factors such as IGF-1, VEGF, and FGF when combined with GLP-2 agonists. Increased cell growth factor induces proliferation of colon cancer-derived cell line Caco-2 cells. In this test, the long-acting GLP-1/GLP-2 peptide developed by the company and the positive control group, GLP-2 agent teduglutide and GLP-1 agent liraglutide, were mixed to increase the The proliferation effect of Caco-2 cells was confirmed by the cell growth factor. The evaluation scheme using CCD-18co and Caco-2 cells is shown in FIG. 1 .

First, CCD-18co cells cultured in a 75T flask were subcultured in a 6-well plate at a concentration of 2x10 ⁵ cells/well. When the CCD-18co cells grew to the maximum density, serum starvation conditions were created by reducing the concentration of FBS to 0.1% FBS in 10% FBS/DMEM medium, and further cultured at 4 °C for 24 hours. Subsequently, CCD-18co cells were treated with 100 nM, 250 nM, or 1000 nM of GLP-1 and GLP-2 agonists (Liraglutide, Teduglutide), or synthetic peptides (HU003K5FA, HU003K7FA) diluted in 0.1% FBS/DMEM medium. did Treated cells were incubated at 37 °C for 24 hours. After obtaining the supernatant of the synthetic peptide-treated CCD-18co cell medium (conditioned medium, CM), centrifugation was performed at 700 xg for 3 minutes, and suspended matter was removed with a 0.22 μm filter.

Meanwhile, Caco-2 cells cultured in a 5T flask were subcultured in a 96-well plate at a concentration of 0.5x10 ⁴ cells/well. Caco-2 cells were replaced with 100 μl of fresh 10% FBS/MEM medium, and CCD-18co CM was treated with FBS/MEM medium in a ratio of 1:1. After treatment with CCD-18co CM, incubation was performed at 37 °C for 3 days. Caco-2 cells were replaced with 100 μl of fresh 10% FBS/MEM medium, and then 10 μl of EZ-cytox assay solution was added so that the medium became 10%. After the EZ-cytox assay solution was added, cell proliferation was confirmed by measuring absorbance at a wavelength of 450 nM using a spectrophotometer every hour for a total of 4 hours.

Experimental Example 4. Pharmacokinetic test

SD rats (Male, 7 weeks old at the time of administration, Coretech Co., Ltd.) were used as test animals, and the designed test groups are shown in Table 4 below.

Group administration substance dose Administration route/ blood collection time (hr) One Teduglutide 1 mg/kg - Route of administration: IV administration (tail vein administration)
- Blood sampling time: 10m, 20m, 30m, 45m, 1h, 2h, 4h, 6h 8h
Blood collection method: Jugular vein or tail vein blood collection 2 HU003K2FA 3 HU003K7FA

About 300 μL of blood was collected from the jugular vein or tail vein using a disposable syringe (1 mL, 26G, Korean vaccine) and a heparin tube at regular intervals after administration for all subjects in each test group. The collected blood was immediately placed in a plasma tube containing an anticoagulant, and plasma was separated by centrifugation at 13,000 rpm for 5 minutes within 30 minutes after blood collection. A maximum of 200 μL of each separated plasma was dispensed into one tube, stored in a cryogenic freezer, and delivered after all blood collection was completed. The concentration of peptides in blood was measured by performing ELISA analysis using antibodies using plasma obtained from blood collection. The pharmacokinetic test of the drug in the blood used a model-independent method (Non-compartmental analysis), and the pharmacokinetic parameters, the maximum concentration in blood (C _max ), the time to reach the maximum concentration in blood (T _max ), and the drug concentration-time curve in blood Analysis of lower area (AUClast) and elimination half-life (T _1/2 ) was performed using Phoenix Winnonlin.

Experimental Example 5. Evaluation of drug efficacy on intestinal proliferation of normal mice

SD rats (Male, 7 weeks old at the time of administration, Orient Bio Co., Ltd.) were used as test animals. Test substances and positive control substances (Liraglutide, Teduglutide) were each converted to individual doses based on body weight, and were administered subcutaneously (s.c.) at a dose of 5 ml per kg body weight. The designed test groups are shown in Table 5 below.

At the time of acquisition, at the time of group separation, and during the test substance administration period, the body weight of each individual was measured once a day. Measurements were made using the same scale in order to minimize errors in each measurement.

In addition, after the end of the test, the animals were anesthetized, laparotomy, and blood were exsanguinated, and intestinal tissues (from the stomach to the large intestine) were extracted for each individual. The extracted intestinal tissue was photographed, and the intestinal tissue after the photograph was incised only in the small intestine (from the duodenum to the cecum), and the contents were removed with a cotton swab and then weighed.

For the experimental results, a commercial statistical processing program (IBM SPSS statistics version 25.0) owned by NCEED (Inha University Digestive Disease Efficacy Evaluation Support Center) was used. Data were statistically analyzed by Mann-Whitney analysis.

Experimental Example 6. Evaluation of drug efficacy in short bowel syndrome (SBS) rat model

6-1. Construction of short bowel syndrome model

SD rats (Male, 7 weeks old at the time of administration, Orient Bio Co., Ltd.) were used as test animals. Rats were fasted for 16 hours before surgery, anesthetized by intraperitoneal injection of ketamine and xylene (3:2), and an incision was made along the central abdomen. The small intestine, cecum, and large intestine were removed with tweezers and sterile swabs. After resection from the jejunum part at a distance of 40 cm from the Ligament of Treitz to the proximal colon at a distance of 1 cm from the cecum (including 60% small intestine and cecum), End-to-end anastomosis was performed. The small intestine was placed in place, and 5 ml of sterile saline was put into the abdominal cavity for body fluid resuscitation and sutured with 2-0 silk suture. In addition, 200 mg/kg ampicillin was subcutaneously injected to prevent infection after surgery.

6-2. administration method

Test substances and positive controls (Liraglutide, Teduglutide) were each converted to individual doses based on body weight, and administered subcutaneously (s.c.) at a dose of 1 ml per kg body weight. The designed test groups are shown in Table 8 below.

6-3. Histopathological analysis

Villus height, crypt depth, and mucosal thickness were measured through histopathology and H&E staining (Hematoxylin & Eosin staining). First, the duodenum, jejunum, and colon tissues were fixed in 10% buffered neutral formalin. The fixed tissue was cut to a certain thickness, and then paraffin-embedded through a general tissue treatment process to produce a tissue section of 4-5 μm. H&E staining was performed on the tissue sections to observe the histopathological findings of each tissue, and the height, depth, and mucosal thickness of the villi of the jejunum and duodenum, and the depth of the colon were measured.

The formula for calculating villi height is as follows: villi height (μm) = mucosal thickness - villi depth

6-4. DNA and protein content determination

DNA and protein contents of duodenal and jejunal mucosa were measured. Intestinal mucosal DNA was extracted from frozen tissue using QIAZEN's Dneasy Blood & Tissue Kit and measured. Intestinal mucosal proteins were extracted and measured using Thermo Scientific's Pierce BCA protein assay kit. Each method was performed according to the manufacturer's kit instructions.

Example 1. Activity analysis of long-acting GLP-1/GLP-2 peptides according to palmitic acid binding sites

The long-acting peptide synthesized by the method of Experimental Example 1-2 was reacted with GLP-1R/CHO cells and GLP-2R/CHO cells, respectively, to confirm whether cAMP increase was induced. Cells in which an increase in cAMP was induced were disrupted, and the increased amount of intracellular cAMP was quantified by an ELISA method. The test for each test group and the control group was performed together to confirm the contrast activity. In order to ensure the accuracy of the activity results, repeated tests were performed three or more times for each peptide. On the other hand, since the maximum number of lysines to which palmitic acid can be bound was 9, the activity of the long-acting GLP-1/GLP-2 peptide bound to palmitic acid was measured by adding and synthesizing up to 9 lysines.

As a result of activity evaluation according to the number of lysines in Table 9, it was confirmed that 7 lysines were added to the HU003 peptide to have the highest activity.

As a result, HU003K7FA showed the highest activity with about 20.4% of GLP-1 activity and 90.3% of GLP-2 activity in GLP-1, and HU003K5FA showed the next highest activity. Accordingly, the comparison results of HU003K7FA and HU003K5FA with the positive control group are shown in FIG. 6 .

Example 2. Confirmation of cell proliferation assay results

Two long-acting GLP-1/GLP-2 peptide candidates (HU003K5FA, HU003K7FA) and GLP-2 agent Teduglutide, GLP-1 agent Liraglutide, and Teduglutide + Liraglutide as positive control substances were tested in CCD-18co cells and Caco- 2 cells were used to confirm the effect of inducing intestinal epithelial cell proliferation. For the growth of Caco-2 cells, the cell growth of the drug-treated group was calculated as an increase in the number of cells compared to the control group, with the cell growth of the untreated CM-treated control group as 100%.

As shown in Table 10 and FIG. 7, when the test result was treated at a drug concentration of 100 nM, Liraglutide was 99.9 ± 7.6% compared to the control group, and Teduglutide was 106.0 ± 3.7% compared to the control group. Contrast was 107.6 ± 1.8%. 103.6 ± 5.4% in the group treated with the long-acting peptide HU003K5FA of the present invention, and 113.0 ± 1.0% in the group treated with HU003K7FA.

In addition, when treated at a drug concentration of 250 nM, Liraglutide was 109.18 ± 5.6 %, Teduglutide was 115.7 ± 6.2 %, Teduglutide + Liraglutide combined treatment group was 112.4 ± 3.6 %, and HU003K5FA treated group was 122.2 ± 9.9 % , It was confirmed that the cell proliferation of the group treated with HU003K7FA increased by 125.1 ± 11.7% compared to the control group.

When treated at a drug concentration of 1000 nM, Liraglutide was 109.5 ± 6.4 %, Teduglutide was 120.9 ± 3.8 %, Teduglutide + Liraglutide combined treatment group was 117.8 ± 3.3 %, HU003K5FA treated group was 125.5 ± 9.9 %, HU003K7FA It was confirmed that the treated group increased cell proliferation compared to the control group by 133.0 ± 8.0%.

Therefore, it was confirmed that the long-acting peptides HU003K5FA and HU003K7FA of the present invention showed a similar effect to that of the positive control at 100 nm and a significantly increased effect than the positive control at a concentration of 250 nM or more. In particular, HU003K7FA It was confirmed that the effect of inducing proliferation of intestinal epithelial cells was excellent.

Example 3. Confirmation of pharmacokinetic test results of HU003K2FA and HU003K7FA

A single intravenous administration of Gattex (teduglutide) or candidate substances HU003K2FA and HU003K7FA to normal rats was used to measure their ability to persist in the blood.

As a result, as shown in FIG. 8 and Table 11, compared to the positive control group teduglutide, the half-life (T _1/2 ) in blood was increased by 10.6 times in the HU003K2FA-administered group and 2.7-fold in the HU003K7FA-administered group, and the mean retention time (MRT) in blood was also It increased by 12.6 times in the HU003K2FA-administered group and 2.8-fold in the HU003K7FA-administered group.

PK Parameter T _1/2 T _max C _max C ₀ AUC _last MRT(min) CL (mL/min/kg) min min ng/mL ng/mL ng/mL*min min mL/min/kg Teduglutide 13.8 5 7797 12014 175037 16.2 5.7 HU003K2FA 145.9 10 9190 10875 1563897 203.6 0.5 HU003K7FA 37.7 10 10642 11994 677550 46.0 1.5

Example 4. Confirmation of effect on normal mice: 1st test

4-1. check weight

As shown in FIGS. 9 and 10 , the body weight decreased the most in the G4 and G5 Liraglutide-administered groups, and showed a similar decrease in the HU003K5FA and HU003K7FA-administered groups. This was predicted to be due to the GLP-1 receptor activating effect of the GLP-1/GLP-2 dual agonist.

4-2. small intestine weight check

It was confirmed whether the long-acting peptide of the present invention can improve the function of the small intestine by increasing the weight of the small intestine in normal mice.

As shown in FIG. 11, it was confirmed that the weight of the small intestine increased compared to vehicle in both groups administered with HU003K5FA or HU003K7FA.

As shown in FIG. 12, in the group administered with 1 mg/kg of HU003K5FA or HU003K7FA, the intestinal weight-to-body weight increase was significantly increased compared to teduglutide 10 mg/kg, which is considered to be due to the high persistence in blood. In the liraglutide-administered group, the rate of increase in intestinal weight due to weight loss was similar to that of teduglutide.

According to the results of Example 4, the second test of Example 5 was conducted by designing a test group corresponding to a formulation suitable for administration once or twice a week.

Example 5. Confirmation of effect on normal mice: 2nd test

According to the results of Example 4, it was confirmed whether small intestine function could be improved only by administering the long-acting peptide of the present invention once to three times a week instead of daily administration.

5-1. check weight

As shown in FIGS. 13 and 14, the Teduglutide + Liraglutide and HU003K7FA-administered group lost weight at the first administration, but then recovered and lost weight only in the Teduglutide + Liraglutide group and the high-concentration administration group of HU003K7FA compared to the vehicle on the 8th day before sacrifice. confirmed that It was predicted that weight loss was due to the GLP-1 receptor activating effect of the GLP-1/GLP-2 dual agonist.

5-2. small intestine weight check

As shown in FIGS. 15 and 16, it was confirmed that the weight of the small intestine increased significantly in all administration groups compared to Vehicle, and in particular, HU003K7FA showed a dose-dependent difference. In the case of Liraglutide, it was measured that the small intestine weight-to-body weight increased more as the body weight decreased.

In the results of G6, G8, and G10, in which HU003K7FA was administered at a total dose of 21 mg/kg during the administration period, it was confirmed that the effect of increasing the weight of the small intestine was significantly increased compared to Tedulgutide 10 mg/kg (daily, total dose of 70 mg/kg).

Example 6. Confirmation of effect on normal mice: 3rd test

According to the results of Example 5, it was confirmed whether small intestine function could be improved by administering the long-acting peptide of the present invention with only 1/3 the dose of Teduglutide once or twice a week.

6-1. check weight

As shown in FIGS. 17 and 18, the Teduglutide + Liraglutide and HU003K7FA-administered groups lost weight at the first administration, but then recovered and on the 8th day before sacrifice, it was confirmed that only the Teduglutide + Liraglutide-administered group compared to the Vehicle group lost weight.

6-2. small intestine weight check

As shown in FIG. 19, it was confirmed that the weight of the small intestine increased significantly in all administration groups compared to Vehicle, and in particular, a dose-dependent effect difference was confirmed when HU003K5FA or HU003K7FA was administered.

As shown in FIG. 20, it was confirmed that the small intestine weight-to-body weight showed a high effect in the results of G7 administered with HU003K5FA twice a week and G8 administered with HU003K7FA once a week. In the case of small intestine weight change, it was confirmed that HU003K7FA exhibited a 12% higher efficacy than HU003K5FA compared to the same dose.

Based on the above results, HU003K7FA, which had the highest effect, was finally selected as a candidate substance for the treatment of short bowel syndrome, and an efficacy test was conducted in a short bowel syndrome model.

Example 7. Confirmation of treatment effect on short bowel syndrome model

7-1. check weight

Changes in body weight of the short bowel syndrome model according to the administration of the long-acting peptide of the present invention were evaluated.

As shown in FIGS. 21 and 22 , the short bowel syndrome model (G2) lost 50-60% of the duodenum of the small intestine and removed the entire ileum and cecum, resulting in overall weight loss. On the other hand, it was confirmed that G5 to G7 administered the long-acting peptide HU003K7FA of the present invention for 2 weeks significantly increased their body weight compared to the existing drug Teduglutide or Teduglutide + Liraglutide-administered group.

7-2. small intestine weight check

It was confirmed that the increase in body weight due to the administration of the long-acting peptide was due to the increase in the weight of the small intestine.

23 and 24, there was no significant change in the weight of the small intestine in the group administered with Teduglutide. In contrast, when the long-acting peptide of the present invention, HU003K7FA, was administered once a week, the weight of the small intestine increased by 14.6% or 28.0% compared to the vehicle, and when administered twice a week, it increased by 33.9%.

7-3. Check for changes in the length of the small intestine

Changes in the length of the small intestine following administration of the long-acting peptide of the present invention were measured.

As shown in Table 12 and FIG. 25, the length and weight of the small intestine hardly increased in the Teduglutide-administered group (G3), but in the HU003K7FA-administered group (G5 to G7), the length of the small intestine increased significantly by about 10% compared to the vehicle. seemed On the other hand, the Teduglutide + Liraglutide administration group (G4) increased the length of the small intestine, but rather decreased the weight of the small intestine, confirming that it was not suitable for the treatment of short bowel syndrome.

7-4. Confirmation of protein and DNA content comparison results in the small intestine

The increase in DNA and protein content in the small intestine following administration of the long-acting peptide of the present invention was evaluated.

As shown in FIGS. 26 and 27, in the case of Duodenum (duodenum), it was confirmed that the amount of DNA and protein increased in the group administered with HU003K7FA at 3.5 mg/kg twice a week compared to Vehicle.

On the other hand, in the case of Jejunum (factory), protein and DNA were rather decreased in the group administered with Teduglutide compared to the vehicle, but the amount of DNA and protein significantly increased in the group administered with HU003K7FA compared to the vehicle-administered group.

7-5. Confirmation of histopathological analysis of the small intestine

In order to confirm the intestinal tissue formation effect according to the administration of the long-acting peptide of the present invention, the histopathological changes of the small intestine were evaluated.

First, as shown in FIG. 28, tissues including duodenum, jejunum, and large intestine were stained by H&E staining. Based on the results of FIG. 28, the histopathological analysis of FIGS. 29 and 30 was performed.

As shown in FIG. 29, in the case of Duodenum (duodenum), the villus height increased by 6.2% for Teduglutide compared to Vehicle, and when the total dose of HU003K7FA was administered at 14 mg/week, the twice-weekly formulation (G7 ) increased by 16.1%, and one-time formulation (G6) increased by 14.5%.

As shown in FIG. 30, in the case of Jejunum (factory), the villus height increased by 18.3% in Teduglutide compared to Vehicle, and when the total dose of HU003K7FA was administered at 14 mg/week, the twice-weekly formulation (G7) increased by 31.4% , the one-time formulation (G6) increased significantly by 42.6%.

This is a result indicating that the HU003K7FA of the present invention has a strong intestinal tissue formation promoting effect compared to the positive control group.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

<110> HuonsLab Co., Ltd. <120> Long-acting fatty acid-peptide derivatives and uses thereof <130> MP21-173P1 <150> KR 10-2022-0001657 <151> 2022-01-05 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 33 <212> PRT <213> artificial sequence <220> <223> HU003 <400> 1 His Gly Asp Gly Ser Phe Thr Ser Glu Leu Ser Thr Tyr Leu Asp Ala 1 5 10 15 Leu Ala Thr Arg Asp Phe Ile Ala Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 2 <211> 35 <212> PRT <213> artificial sequence <220> <223> HU003K2 <400> 2 His Gly Asp Gly Ser Phe Thr Ser Glu Leu Ser Thr Tyr Leu Asp Ala 1 5 10 15 Leu Ala Thr Arg Asp Phe Ile Ala Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Lys Lys 35 <210> 3 <211> 36 <212> PRT <213> artificial sequence <220> <223> HU003K3 <400> 3 His Gly Asp Gly Ser Phe Thr Ser Glu Leu Ser Thr Tyr Leu Asp Ala 1 5 10 15 Leu Ala Thr Arg Asp Phe Ile Ala Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Lys Lys Lys 35 <210> 4 <211> 38 <212> PRT <213> artificial sequence <220> <223> HU003K5 <400> 4 His Gly Asp Gly Ser Phe Thr Ser Glu Leu Ser Thr Tyr Leu Asp Ala 1 5 10 15 Leu Ala Thr Arg Asp Phe Ile Ala Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Lys Lys Lys Lys Lys 35 <210> 5 <211> 40 <212> PRT <213> artificial sequence <220> <223> HU003K7 <400> 5 His Gly Asp Gly Ser Phe Thr Ser Glu Leu Ser Thr Tyr Leu Asp Ala 1 5 10 15 Leu Ala Thr Arg Asp Phe Ile Ala Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Lys Lys Lys Lys Lys Lys Lys 35 40 <210> 6 <211> 42 <212> PRT <213> artificial sequence <220> <223> HU003K9 <400> 6 His Gly Asp Gly Ser Phe Thr Ser Glu Leu Ser Thr Tyr Leu Asp Ala 1 5 10 15 Leu Ala Thr Arg Asp Phe Ile Ala Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp Lys Lys Lys Lys Lys Lys Lys Lys Lys 35 40

Claims (13)

A fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof in which a fatty acid is bound to a polypeptide represented by the following general formula,
The polypeptide represented by the general formula is characterized in that a fatty acid is bonded to the N-terminus, C-terminus, or a middle chain thereof, a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof.
[general meal]
Amino acid sequence-Kn represented by SEQ ID NO: 1
(In the above general formula, K is lysine, and n is an integer from 1 to 10.)
According to claim 1,
Characterized in that the fatty acid is a C12 to C22 saturated fatty acid or unsaturated fatty acid, a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof.
According to claim 1,
Characterized in that n is an integer of 2 to 9, a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof.
According to claim 1,
Characterized in that the fatty acid is bonded to the C-terminus, a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof.
According to claim 1,
Characterized in that the polypeptide represented by the general formula consists of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 5, a fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof.
According to claim 1,
The fatty acid-peptide derivative is characterized in that the in vivo half-life (t _1/2 ) is 2 hours or more, the fatty acid-peptide derivative or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of intestinal disorders, intestinal damage, and gastric diseases, comprising the fatty acid-peptide derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
According to claim 7,
The intestinal disease is characterized in that at least one selected from the group consisting of short bowel syndrome, irritable bowel disease, inflammatory bowel disease, Crohn's disease, colitis, colitis, pancreatitis, ileitis, mucositis and intestinal atrophy, pharmaceutical composition.
According to claim 8,
Characterized in that the intestinal disease is short bowel syndrome, a pharmaceutical composition.
According to claim 7,
The gastric disease is characterized in that at least one selected from the group consisting of gastric cramps, gastritis, gastric ulcer, duodenitis, and duodenal ulcer, a pharmaceutical composition.
According to claim 7,
The fatty acid-peptide derivative is characterized in that the administration of 0.1 to 200 mg / kg per week, pharmaceutical composition.
According to claim 7,
Characterized in that the fatty acid-peptide derivative increases the weight or length of the small intestine, the pharmaceutical composition.
According to claim 7,
Characterized in that the fatty acid-peptide derivative increases villus height, a pharmaceutical composition.

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