TW201918259A - Oxyntomodulin analogue GLP-1R / GCGR dual target agonist polypeptide for treating idiopathic pulmonary interstitial fibrosis - Google Patents

Abstract

Disclosed is a use of a polypeptide compound having dual agonistic effects on the glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR). The polypeptide compound has the characteristics of a high enzymolysis stability, a high biological activity and zero adverse reaction, etc., can obviously inhibit the fibrosis transformation and proliferation of human pulmonary epithelial cells induced by TGF-[beta]1, and can obviously improve the degree of pulmonary fibrosis in mice induced by bleomycin. Such a dual target agonist polypeptide can be used for preventing or treating pulmonary diseases indicated by fibrotic symptoms.

Description

Treatment of idiopathic pulmonary interstitial fibrosis based on oxyntomodulin analogue GLP-1R/GCGR dual-target agonist polypeptide

The present invention is in the field of biochemical technology and, in particular, relates to a class of GLP-1R/GCGR dual target agonist polypeptides. The present invention also relates to the use of the above-described dual-target agonist polypeptide for the prophylactic and/or therapeutic use of pulmonary diseases accompanied by fibrotic symptoms such as idiopathic pulmonary interstitial fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease of unknown cause. It is characterized by difficulty in breathing and irreversible decline or even loss of lung function. It is a poor prognosis in chronic non-neoplastic diseases, and the mortality rate is very high. High, glucocorticoid and immunosuppressive therapy results are less than ideal, with a 5-year survival rate of less than 50% (Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, et al. Am. J. Respir. Crit Care Med. 2011 . 183:788–824; Navaratnam V, Fleming KM, West J, Smith CJ, Jenkins RG, et al. Thorax . 2011 . 66:462-467.). IPF is more common in patients between the ages of 40 and 70, and IPF mortality increases as the patient ages. In addition, the occurrence and development of IPF is also related to the patient's gender and body weight, because the incidence of IPF is higher in women than in women, and its development is faster, and the survival rate is lower than that of women (Willis BC, Borok Z Am. J. Physiol. Lung Cell Mol. Physiol. 2012 . 293:525-534.). Most interstitial lung diseases have a common pathological basis. Alveolitis occurs after the initial injury, and as the inflammatory-immune response progresses, inflammation and abnormal repair lead to the proliferation of pulmonary interstitial cells, producing a large amount of collagen and extracellular matrix. Pulmonary interstitial fibrosis ultimately leads to permanent loss of alveolar gas exchange units (Wolters PJ, Collard HR & Jones KD. Annu. Rev. Pathol. Mech. Dis . 2014 . 9: 157-179.).

In normal lung tissue, collagen is its main extracellular matrix (ECM) protein, which forms a three-dimensional network structure with other types of ECM components, and serves as the main skeleton of lung tissue structure. These ECM protein components play an important role in maintaining the integrity of the lung tissue structure and in maintaining the differentiation of the lung epithelium and endothelial cells. After lung injury, various cytokines are produced during the injury-inflammation-repair process, and any one or more of the process disorders can cause abnormalities in ECM metabolism, thereby transforming physiological healing into pathological fibrosis.

At present, the main treatment strategies for idiopathic pulmonary fibrosis are anti-inflammatory, anti-fibrosis and anti-oxidation. However, there is no drug that has proven to be effective against IPF. Glucocorticoid is the traditional drug for the treatment of pulmonary fibrosis. It can inhibit inflammation and reduce alveolitis, thus delaying the progression of pulmonary fibrosis. However, the current study found that it is only effective in 20% of patients with IPF, and does not extend the survival time of patients. Long-term use of glucocorticoids has obvious side effects, often combined with bacterial or fungal infections in the lungs; immunosuppressive agents such as cyclophosphamide, azathioprine, and cyclosporine A can alleviate the body's immune response. However, frequent use not only has potentially serious side effects, but also is essentially ineffective for IPF treatment; pirfenidone (5-methyl 1 phenyl 2 -(1 H)pyridone) is a synthetic molecule and is currently the only approved for IPF. Anti-fibrotic drugs for clinical treatment. Although pirfenidone is an effective drug for the treatment of IPF, as an oral drug, it has many side effects in the clinic, such as gastrointestinal discomfort (nausea, vomiting, indigestion and diarrhea), fatigue and photosensitive rash. In recent years, the exploration of anti-pulmonary fibrosis drugs has attracted more and more attention, and researchers have tried to reduce or regulate the process of pulmonary fibrosis by trying different aspects of synthesis and function. However, at present all treatments have no significant improvement in lung function indicators. Although some cytokine preparations have a certain therapeutic effect on pulmonary fibrosis, none of them have been used for clinical treatment.

Bleomycin (BLM) is a clinically used cancer treatment drug that induces lung damage and pulmonary fibrosis over a long period of time. Thus, the BLM-induced pulmonary fibrosis model is a classic IPF animal model (Chua FJ, Gauldie J, Laurent GJ. Am J Respir Cell Mol Biol, 2005. 33:9-13.).

The inventors obtained a class of GLP-1R/GCGR dual-target agonists as ghrelin analogs in the earlier Chinese patent number: ZL 201510237027.7, which has a long half-life, insulinotropic activity, and no adverse reactions. It can be used for the treatment of diseases such as diabetes. The present invention continues to be intensively conducted and provides novel biological activities and therapeutic and indication uses of such GLP-1R/GCGR dual-target agonist polypeptides.

It is an object of the present invention to provide a prophylactic and/or therapeutic use of such a GLP-1R/GCGR dual-target agonist polypeptide for diseases such as idiopathic pulmonary interstitial fibrosis and associated pulmonary fibrosis.

The present inventors have conducted extensive experimental studies to demonstrate that such GLP-1R/GCGR dual-target agonist polypeptides have a significant effect on TGF-β-induced fibroblast transformation. The inventors have demonstrated through extensive experimental studies that it can effectively delay the treatment of pulmonary fibrosis and significantly reduce the accumulation of cells and fibers in the alveolar space in the treatment of GLP-1R/GCGR dual-target agonist polypeptides. Reduce collagen deposition and expression of pulmonary fibrosis marker protein α-SMA.

A further object of the invention is to provide a novel indication for the therapeutic use of such GLP-1R/GCGR dual target agonist polypeptides.

This kind of GLP-1R/GCGR dual-target agonist polypeptide is expected to be a new generation of preventive or therapeutic drugs for diseases such as idiopathic pulmonary interstitial fibrosis.

The present invention relates to a GLP-1R/GCGR dual-target agonist polypeptide comprising the parent peptide represented by the following amino acid sequence: His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13 -Leu-Asp-Xaa16-Xaa17-Xaa18-Ala-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38 -Xaa39-Xaa40-COR1 wherein R ₁ = -NH ₂ ; Xaa2 = Aib or D-Ser; Xaa10 = Lys or Tyr; Xaa13 = Lys or Tyr; Xaa16 = Ser, Aib, Lys or Glu; Xaa17 = Lys or Arg Xaa18 = Arg or Ala; Xaa20 = His, Gln, or Lys; Xaa21 = Asp or Glu; Xaa23 = Ile, Val; Xaa24 = Glu or Gln; Xaa27 = Met, Leu, Nle; Xaa28 = Asn, Asp, Arg, Ser or absent; Xaa29 = Gly, Thr or absent; Xaa30 = Gly or absent; Xaa31 = Gly or absent; Xaa32 = Pro or absent; Xaa33 = Ser, Val or absent; Xaa34 = Ser or nonexistent Xaa35 = Gly or absent; Xaa36 = Ala or absent; Xaa37 = Pro or absent; Xaa38 = Pro or absent; Xaa39 = Pro or absent; Xaa40 = Ser or absent; Wherein at least one of Xaa10, Xaa16, Xaa17 or Xaa20 is Lys, the at least one Lys or the side chain of the 12th Lys of the sequence is linked to a lipophilic substituent in a manner of the lipophilic substituent Forming a guanamine bond with an amino group of a bridging group, the carboxyl group of the amino acid residue of the bridging group forms a guanamine bond with the N-terminal residue of Lys of the parent peptide to the parent peptide; said bridging The group is Glu, Asp and/or (PEG) _m , wherein m is an integer from 2 to 10; the lipophilic substituent is selected from CH ₃ (CH ₂ ) _n CO- or HOOC(CH ₂ ) _n CO- A thiol group, where n is an integer from 10 to 24. A preferred bridging group can be Glu-(PEG) _m or Asp-(PEG) _m or (PEG) _m in the following manner:

The compounds involved in the present invention can stabilize the helical structure of the molecule based on a theoretical intramolecular bridge, thereby increasing the potency and/or selectivity against GLP-1R or GCGR. The compounds of the invention carry one or more intramolecular bridges in the sequence. Such a bridge is formed between the side chains of two amino acid residues that are typically separated by three amino acids in a linear sequence. For example, the bridge can be formed between residue pairs 12 and 16, 16 and 20, 17 and 21 or 20 and 24 side chains. The two side chains can be linked to each other by ionic interaction or by covalent bonds. Thus, these pairs of residues may comprise oppositely charged side chains to form a salt bridge by ionic interaction. For example, one residue may be Glu or Asp, and the other residue may be Lys or Arg, Lys is paired with Glu, and Lys is paired with Asp, respectively, to react to form an indoleamine ring.

The present invention also provides a pharmaceutical composition comprising the GLP-1R/GCGR dual-target agonist polypeptide of the present invention, wherein the GLP-1R/GCGR dual-target agonist polypeptide is added as a active ingredient to a pharmaceutically acceptable carrier and/or Or excipients are formulated into pharmaceutical compositions.

The polypeptide of the present invention has an improvement and therapeutic effect on pulmonary fibrosis diseases such as idiopathic pulmonary interstitial fibrosis. The polypeptide of the present invention can be used for the direct or indirect treatment of a condition caused by or characterized by pulmonary disease associated with fibrotic symptoms such as idiopathic pulmonary interstitial fibrosis.

Those skilled in the art will appreciate that the pharmaceutical compositions of the present invention are suitable for use in a variety of modes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, and the like. The pharmaceutical composition of the polypeptide of the present invention may be formulated into various suitable dosage forms comprising at least one effective amount of a polypeptide of the present invention and at least one pharmaceutically acceptable pharmaceutically acceptable carrier, depending on the mode of administration employed.

Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for the skin surface, aerosols, nasal sprays, and sterile solutions for injection.

The pharmaceutical composition containing the polypeptide of the present invention may be formulated into a solution or a lyophilized powder for parenteral administration, and the powder may be reconstituted by adding a suitable solvent or other pharmaceutically acceptable carrier before use. The liquid formulation is generally a buffer, Isotonic solution and aqueous solution.

The amount of the polypeptide of the present invention in the pharmaceutical composition can be varied within a wide range, and those skilled in the art can easily according to some objective factors such as the type of the disease, the severity of the disease, the patient's body weight, the dosage form, the administration route and the like. Add to determine.

The invention has the advantages of: 1) having better anti-pulmonary fibrosis biological activity; 2) showing stability in drug pharmacokinetic experiments, good stability, easy scale-up production, low cost; 3) and small molecule compounds Compared to lower toxicity, the safety window is larger and the dosage is smaller.

In a specific embodiment, the invention relates to a GLP-1R/GCGR dual target agonist polypeptide having the sequence: Compound 1 (involving SEQ ID NO: 1 ): His-(D-Ser)-Gln-Gly-Thr -Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp -Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYS -K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 2 (involving SEQ ID NO: 2 ): His-(D-Ser)-Gln- Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S) -QGTFTSDYS-K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 3 (involving SEQ ID NO: 3 ): His-(D-Ser)-Gln -Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CH ₃ )-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln -Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-A la-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYS-K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CH ₃ )-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 4 (directed to SEQ ID NO: 4): His- (D-Ser) -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG 2 -PEG 2 -γGlu-CO (CH 2) 14 CH 3) -Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly -Ala-Pro-Pro-Pro- Ser-NH 2 H- (d -S) -QGTFTSD-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 14 CH 3) -SKYLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS compound 5 (directed to SEQ ID NO: 5): His- (D-Ser) -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CO 2 H )-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser- gly-Ala-Pro-Pro- Pro-Ser-NH 2 H- (d -S) -QGTFTSD-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CO 2 H) -SKYLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS -NH ₂ compound 6 (involving SEQ ID NO: 6 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys ( PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met- Asn-Thr-Gly-Gly- Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 H- (d -S) -QGTFTSDYSKYLD-K (PEG 2 -PEG 2 -γGlu-CO ( CH ₂ ) ₁₄ CH ₃ )-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 7 (involving SEQ ID NO: 7 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys -Tyr-Leu-Asp-Aib-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met- Asn-Thr-Gly-Gly- Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 H- (d -S) -QGTFTSDYSKYLD-Aib-K (PEG 2 -PEG 2 -γGlu- CO(CH ₂ ) ₁₆ CO ₂ H)-RAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 8 (involving SEQ ID NO: 8 ): His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG ₂ -PEG _2- γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp- Gly-Gly-Pro-Ser- Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 H-Aib-QGTFTSD-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 14 CH 3) - SKYLD-Aib-RRAQDFVQWLLDGGPSSGAPPPS-NH ₂ Compound 9 (involving SEQ ID NO: 9 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys (PEG ₂ - PEG ₂ -CO(CH ₂ ) ₁₄ CH ₃ )-Lys-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-V al-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYS-K( PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₄ CH ₃ )-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 10 (involving SEQ ID NO: 10 ): His-(D-Ser)-Gln-Gly-Thr-Phe -Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Lys(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-Arg-Ala-Gln-Asp-Phe- Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYSKYLD-Aib- K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-RAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 11 (involving SEQ ID NO: 11 ): His-Aib-Gln-Gly-Thr-Phe-Thr-Ser- Asp-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CO ₂ H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln -Trp-Leu-Leu-Asp- Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 H-Aib-QGTFTSD-K (PEG 2 -PEG 2 -γGlu-CO (CH ₂ ) ₁₄ CO ₂ H)-SKYLD-Aib-RRAQDFVQWLLDGGPSSGAPPPS-NH ₂ Compound 12 (involving SEQ ID NO: 12 ): His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CH ₃ )-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Al a-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-Aib-QGTFTSD- K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CH ₃ )-SKYLD-Aib-RRAQDFVQWLLDGGPSSGAPPPS-NH ₂ Compound 13 (involving SEQ ID NO: 13 ): His-Aib-Gln-Gly-Thr-Phe -Thr-Ser-Asp-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CO ₂ H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp- Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-Aib-QGTFTSD-K (PEG ₂ -PEG _2- γGlu-CO(CH ₂ ) ₁₄ CO ₂ H)-SKYLDERRAQDFVQWLLDGGPSSGAPPPS-NH ₂ Compound 14 (involving SEQ ID NO: 14 ): His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys ( PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CH ₃ )-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu- Leu-Asp-Gly-Gly- Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 H-Aib-QGTFTSD-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CH ₃₎ -SKYLDERRAQDFVQWLLDGGPSSGAPPPS-NH ₂ compound 15 (relates to SEQ ID NO: 15): His -Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG 2 -PEG 2 -γGlu-CO (CH ₂ ) ₁₆ CO ₂ H)-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg -Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-Aib-QGTFTSD -K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-SKYLDERRAQDFVQWLLDGGPSSGAPPPS-NH ₂ Compound 16 (involving SEQ ID NO: 16 ): His-Aib-Gln-Gly-Thr-Phe-Thr -Ser-Asp-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val -Gln-Trp-Leu-Leu- Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 H-Aib-QGTFTSD-K (PEG 2 -PEG 2 -γGlu -CO(CH ₂ ) ₁₄ CH ₃ )-SKYLDERRAQDFVQWLLDGGPSSGAPPPS-NH ₂ Compound 17 (involving SEQ ID NO: 17 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu _{-Leu-Arg-Ala-NH 2} H- (d -S) -QGTFTSDYSKYLDS-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CO 2 H) -AAHDFVEWLLRA-NH 2 compound 18 (SEQ ID involving NO:18 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys (PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Arg -Ala-NH ₂ H-( d -S)-QGTFTSDYSKYLDEKAA-K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-EFIEWLLRA-NH ₂ Compound 19 (involving SEQ ID NO: 19 ) : His-Aib-Gln-Gly -Thr-Phe-Thr-Ser-Asp-Lys (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CO 2 H) -Ser-Lys-Tyr-Leu-Asp- Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-Aib-QGTFTSD-K (PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-SKYLD-Aib-RRAQDFVQWLLDGGPSSGAPPPS-NH ₂ Compound 20 (involving SEQ ID NO: 20 ): His-(D -Ser) -Gln-Gly-Thr- Phe-Thr-Ser-Asp-Tyr-Ser-Lys (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CO 2 H) -Lys-Leu-Asp-Aib -Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYS-K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-KLD-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 21 (involving SEQ ID NO: 21 ) : His- (D-Ser) -Gln -Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys (PEG 2 -PEG 2 -gGlu-CO (CH ₂ ) ₁₆ CO ₂ H)-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser -Gly-Ala-Pro-Pro- Pro-Ser-NH 2 H- (d -S) -QGTFTSDYSKYLDS-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CO 2 H) -AAHDFVEWLLNGGPSSGAPPPS-NH 2 Compound 22 (involving SEQ ID NO: 22 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys (PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CH ₃ )-Ala-Ala-His-Asp-Phe-Val-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly -Ala-Pro-Pro-Pro- Ser-NH 2 H- (d -S) -QGTFTSDYSKYLDS-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CH 3) -AAHDFVEWLLNGGPSSGAPPPS-NH 2 compound 23 ( References to SEQ ID NO: 23 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala- Lys(PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Arg-Ala-NH ₂ H-( d -S)-QGTFTSDYSKYLDEKAA- K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-EFIEWLLRA-NH ₂ Compound 24 (involving SEQ ID NO: 24 ): His-(D-Ser)-Gln-Gly-Thr-Phe -Thr-Ser-Asp-Tyr- Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys (PEG 2 -PEG 2 -gGlu-CO (CH 2) 16 CH 3) -Glu-Phe -Ile-Glu-Trp-Leu-Leu-Arg-Ala-NH ₂ H-( d -S)-QGTFTSDYSKYLDEKAA-K(PEG ₂ -PEG ₂ - γGlu-CO(CH ₂ ) ₁₆ CH ₃ )-EFIEWLLRA-NH ₂ Compound 25 (involving SEQ ID NO: 25 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp- Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Lys(PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CH ₃ )-Ala-Ala-His-Asp-Phe-Val-Glu-Trp- Leu-Leu-Arg-Ala- NH 2 H- (d -S) -QGTFTSDYSKYLDS-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CH 3) -AAHDFVEWLLRA-NH 2 compound 26 (SEQ ID involving NO:26 ) : His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys (PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYSKYLDEKAA-K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-EFIEWLLNGGPSSGAPPPS-NH ₂ Compound 27 (involving SEQ ID NO :27 ) : His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Lys (PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₄ CO ₂ H)-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro _{-Pro-Ser-NH 2 H- (} d -S) -QGTFTSDYSKYLDEKAA-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 14 CO 2 H) -EFIEWLLNG GPSSGAPPPS-NH ₂ Compound 28 (involving SEQ ID NO: 28 ) : His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu -Lys-Ala-Ala-Lys(PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CH ₃ )-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser -Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH 2 H- (d -S) -QGTFTSDYSKYLDEKAA-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CH 3) -EFIEWLLNGGPSSGAPPPS-NH ₂ Compound 29 (involving SEQ ID NO: 29 ) : His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Lys- Ala-Ala-Lys(PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Glu-Phe-Ile-Glu-Trp-Leu-Leu-Asn-Gly-Gly-Pro-Ser-Ser- gly-Ala-Pro-Pro- Pro-Ser-NH 2 H- (d -S) -QGTFTSDYSKYLDEKAA-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 14 CH 3) -EFIEWLLNGGPSSGAPPPS-NH 2 compound 30 (Involving SEQ ID NO: 30 ) : His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)- Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly- Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSD-K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-SKYL D-Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 31 (involving SEQ ID NO: 31 ) : His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG ₂ -PEG ₂ -CO (CH ₂ ) ₁₄ CH ₃ )-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly -Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSD-K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₄ CH ₃ )-SKYLD -Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 32 (involving SEQ ID NO: 32 ) : His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG ₂ -PEG ₂ -CO( CH ₂ ) ₁₆ CH ₃ )-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly- Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSD-K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CH ₃ )-SKYLD- Aib-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 33 (involving SEQ ID NO: 33 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp -Aib-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly -Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYSKYLD-Aib-K(PEG ₂ -PEG _2- γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-RAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 34 (involving SEQ ID NO: 34 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp -Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln- Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYSKYLD-K (PEG ₂ -PEG _2- γGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 35 (involving SEQ ID NO: 35 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp -Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYSKYLD-K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 36 (involving SEQ ID NO: 36 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr -Ser-Lys-Tyr-Leu-Asp-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CH ₃ )-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu -Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-Q GTFTSDYSKYLD-K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CH ₃ )-RRAQDFVQWLMNTGGPSSGAPPPS-NH ₂ Compound 37 (involving SEQ ID NO: 37 ): His-(D-Ser)-Gln-Gly-Thr -Phe-Thr-Ser-Asp- Tyr-Ser-Lys-Tyr-Leu-Asp-Lys (PEG 2 -PEG 2 -γGlu-CO (CH 2) 14 CH 3) -Arg-Arg-Ala-Gln-Asp -Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYSKYLD -K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-RRAQDFVQWL-Nle-NTGGPSSGAPPPS-NH ₂ Compound 38 (involving SEQ ID NO: 38 ): His-(D-Ser)-Gln- gly-Thr-Phe-Thr- Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys (PEG 2 -PEG 2 -gGlu-CO (CH 2) 16 CO 2 H) -Arg-Arg-Ala -Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d - S)-QGTFTSDYSKYLD-K(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-RRAQDFVQWL-Nle-NTGGPSSGAPPPS-NH ₂ Compound 39 (involving SEQ ID NO: 39 ): His-(D- Ser) -Gln-Gly-Thr- Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys (PEG 2 -PEG 2 -gGlu-CO (CH 2) 16 CO 2 H) - Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser- Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH 2 H- (d -S) -QGTFTSDYSKYLD-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 16 CO 2 H) -RRAQDFVQWLLNTGGPSSGAPPPS-NH ₂ Compound 40 (involving SEQ ID NO: 40 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys (PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser- Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH 2 H- (d -S) -QGTFTSDYSKYLD-K (PEG 2 -PEG 2 -γGlu-CO (CH 2) 14 CH 3) -RRAQDFVQWLLNTGGPSSGAPPPS-NH 2 Compound 41 (involving SEQ ID NO: 41 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys (PEG ₂ - PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser- Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYSKYLD-K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-RRAQDFVQWL-Nle-NTGGPSSGAPPPS-NH ₂ Compound 42 (involving SEQ ID NO: 42 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys (PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₄ CH ₃ )-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu- Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSDYSKYLD-K(PEG ₂ -PEG ₂ -CO(CH _{2 14} CH ₃ )-RRAQDFVQWLLNTGGPSSGAPPPS-NH ₂ Compound 43 (involving SEQ ID NO: 43 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr -Gly-Gly-Pro-Ser- Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 H- (d -S) -QGTFTSD-K (PEG 2 -PEG 2 -gGlu-CO (CH 2) ₁₄ CH ₃ )-SKYLD-Aib-RRAQDFVQWLLNTGGPSSGAPPPS Compound 44 (involving SEQ ID NO: 44 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₄ CH ₃ )-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly -Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSD-K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₄ CH ₃ ) - SKYLD-Aib-RRAQDFVQWLLNTGGPSSGAPPPS Compound 45 (involving SEQ ID NO: 45 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (PEG ₂ -PEG ₂ -CO(CH) ₂ ) ₁₄ CH ₃ )-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-L eu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSD-K(PEG ₂ -PEG ₂ - CO(CH ₂ ) ₁₄ CH ₃ )-SKYLD-Aib-RRAQDFVQWL-Nle-NTGGPSSGAPPPS Compound 46 (involving SEQ ID NO: 46 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser- Asp-Lys(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp -Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSD-K(PEG ₂ -PEG ₂ -CO(CH ₂ ) ₁₆ CO ₂ H)-SKYLD-Aib-RRAQDFVQWL-Nle-NTGGPSSGAPPPS Compound 47 (involving SEQ ID NO: 47 ): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr- Ser-Asp-Lys(PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val -Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S)-QGTFTSD-K (PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-SKYLD-Aib-RRAQDFVQWLLNTGGPSSGAPPPS Compound 48 (involving SEQ ID NO: 48 ): His-(D-Ser)-Gln-Gly-Thr-Phe- Thr-Ser-Asp-Lys(PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-G ln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ H-( d -S )-QGTFTSD-K(PEG ₂ -PEG ₂ -gGlu-CO(CH ₂ ) ₁₆ CO ₂ H)-SKYLD-Aib-RRAQDFVQWL-Nle-NTGGPSSGAPPPS

The abbreviations used in the present invention have the following specific meanings : Boc is a tert-butoxycarbonyl group, Fmoc is a fluorenylmethoxycarbonyl group, t-Bu is a tert-butyl group, and ivDDe is 1-(4,4-dimethyl-2,6-dioxo. Resin and lipophilic substituents of resinocyclohexyl)-3-methyl-butyl, resin is resin, TFA is trifluoroacetic acid, EDT is 1,2-ethanedithiol, Phenol is phenol, FBS is tire Bovine serum, BSA is bovine serum albumin, HPLC is high-performance liquid phase, GLP-1R is glucagon-like peptide 1 receptor, GCGR is glucagon receptor, and GLP-1 is glucagon-like peptide. mPEG is monomethoxypolyethylene glycol, OXM is oxyntomodulin, His is histidine, Ser is serine, D-Ser is D-serine, Gln is glutamine, Gly is glycine, Glu Is glutamic acid, Ala is alanine, Thr is threonine, Lys is lysine, Arg is arginine, Tyr is tyrosine, Asp is aspartic acid, Trp is tryptophan, Phe is Phenylalanine, Ile is isoleucine, Leu is leucine, Cys is cysteine, Pro is valine, Val is valine, Met is methionine, Asn is aspartame, HomoLys is High lysine, Orn is ornithine, Dap is diaminopimelic acid, Dab is 2,4-diaminobutyric acid, Nle is norleucine, Aib is 2-aminoisobutyric acid, Palmitoyl is a palmitoyl group, Cholesteryl is a cholesterol group, and AEEA is [2-[2-(amino) Ethoxy]ethoxy]acetic acid, CA is 4-imidazolylethane.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, however, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, the reagents or instruments used are commercially available. : Example 1 Synthesis of the compound of polypeptide material

All amino acids were purchased from NovaBiochem. All other reagents were of analytical grade and were purchased from Sigma unless otherwise stated. A Protein Technologies PRELUDE 6-channel peptide synthesizer was used. A Phenomenex Luna C18 preparative column (46 mm x 250 mm) was used to purify the peptide. The high performance liquid chromatograph is a product of Waters Corporation. Mass spectrometry was performed using an Agilent mass spectrometer. The synthesis method of the polypeptide compound of the present invention is illustrated by taking the polypeptide compound 6 as an example: structural sequence: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu- Asp-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly- Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ a) Master peptide chain assembly:

According to the Fmoc/ t- Bu strategy, a 0.25 mmol scale of the following polypeptide was synthesized on a CS336X peptide synthesizer (CS Bio, USA): Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly- Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu )-Leu-Asp(OtBu)-Lys(ivDde)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)- Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t- Bu)-rink amide resin (1) First step: 0.75 g of Rink amide MBHA-LL resin (Novabiochem, loading 0.34 mmol/g) was swollen in dichloromethane (DCM) for one hour with N , N - II Methylformamide (DMF) thoroughly washes the resin 3 times; (2) Step 2: Rink amide resin as carrier and coupling agent from 6-chlorobenzotriazole-1,1,3,3- Tetramethylurea hexafluorophosphate (HCTU), organic base N , N -diisopropylethylamine (DIEPA) in a ratio of 1:1 by mass to N , N -dimethylformamide ( DMF) is a solvent, the reaction is carried out, and the condensation reaction is carried out in sequence to link Fmoc-Ser(t-Bu)-OH, Fmoc-Pro-OH (3x), Fmoc-Ala-OH, Fmoc-G. ly-OH, Fmoc-Ser(t-Bu)-OH (2x), Fmoc-Pro-OH, Fmoc-Gly-OH (2x), Fmoc-Thr(t-Bu)-OH, Fmoc-Asn(Trt) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu )-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH (2x), Fmoc-Lys(ivDde)-OH, Fmoc-Asp(OtBu)-OH, Fmoc -Leu-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Phe-OH, Thr(t-Bu)-OH, Fmoc-Gly-OH, Fmoc -Gln(Trt)-OH, Fmoc-D-Ser(t-Bu)-OH, Boc-His(Boc)-OH gives: Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu )-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr (t-Bu)-Leu-Asp(OtBu)-Lys(ivDde)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp (Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro- Ser(t-Bu)-rink amide resin. Thereafter, N , N -dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N , N -dimethylformamide (DMF) were used in this order. The resin was washed 3 times each.

In the reaction, 1) the ratio of the amount of the first amino acid Fmoc-Ser(t-Bu)-OH to the amount of the resin is 1:1~6:1; 2) the next condensation reaction in Fmoc Protected amino acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), organic base N , N -diisopropylethylamine (DIEPA) The excess is 2~8 times, and the reaction time is 1~5 hours. b) 1- (4,4- dimethyl-2,6-dioxo-cyclohexylidene) -3-methyl - butyl (ivDde) removal of the lipophilic substituent is introduced:

The resin was washed twice with a solution of N , N -dimethylformamide (DMF) / dichloromethane (DCM) = 1:1 (volume ratio), and freshly prepared 3.0% hydrazine N was added . A solution of N -dimethylformamide (DMF) was shaken at room temperature for 10 to 30 minutes for the hydrazine treatment step, followed by filtration. The hydrazine treatment step was repeated 5 times to obtain: Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser( tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys-Arg(Pbf)-Arg( Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro -Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Thereafter, N , N -dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N , N -dimethylformamide (DMF) were used in this order. The resin was washed 3 times each.

Add FmocNH-PEG ₂ -OH (Quanta BioDesign), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), two different A mixed solution of propylethylamine (DIEPA) in N , N -dimethylformamide (DMF) (5 times excess) was shaken for 2 hours and then filtered. Thereafter, N , N -dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N , N -dimethylformamide (DMF) were used in this order. Washing the resin three times each time: Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(tBu) -Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys(Fmoc-PEG ₂ )-Arg(Pbf )-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly -Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Thereafter, N , N -dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N , N -dimethylformamide (DMF) were used in this order. The resin was washed 3 times each.

20% of Piperidine / N , N - dimethylformamide (DMF) solution to remove Fmoc group (30 minutes, repeated removal twice), added Fmoc-PEG ₂ -OH, 2- ( 7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), diisopropylethylamine (DIEPA) N , N -dimethyl The mercaptoamine (DMF) mixed coupling solution (5 times excess) was subjected to a coupling reaction to obtain Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t- Bu)-Phe-Thr(t-Bu)-Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu- Asp(OtBu)-Lys(Fmoc-PEG ₂ -PEG ₂ )-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc )-Leu-Met-Asn(Trt)-Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser( t-Bu)-rink amide resin. Thereafter, N , N -dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N , N -dimethylformamide (DMF) were used in this order. The resin was washed 3 times each.

20% of Piperidine / N , N - dimethylformamide (DMF) solution to remove Fmoc group (30 minutes, repeated removal twice), and then coupled Fmoc-gGlu- according to normal conditions OtBu, adding hexadecanoic acid (palmitic acid) to give: Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu) -Ser(tBu)-Asp(OtBu)-Tyr(t-Bu)-Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(OtBu)-Lys(PEG ₂ -PEG _2- C16)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)- Thr(t-Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin. Thereafter, the resin was sufficiently washed three times with N , N -dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol) and dichloromethane (DCM), and then evaporated to dryness.

c ) Removal of the complete protection of the polypeptide: Boc-His(Boc)-D-Ser(t-Bu)-Gln(OtBu)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser( tBu) -Asp (OtBu) -Tyr ( tBu) -Ser (tBu) -Lys (Boc) -Tyr (tBu) -Leu-Asp (OtBu) -Lys (PEG 2 -PEG 2 -C16 )-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(OtBu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t -Bu)-Gly-Gly-Pro-Ser(t-Bu)-Ser(t-Bu)-Gly-Ala-Pro-Pro-Pro-Ser(t-Bu)-rink amide resin added to cutting solution TFA/Phenol In /thioanisole/EDT/H ₂ O (82.5:5:5:2.5:5, volume ratio), the temperature was raised, and the temperature of the lysate was controlled at 25 ° C for 2.5 hours. After filtration, the filter cake was washed 3 times with a small amount of lysate, and the filtrate was combined. The filtrate was slowly poured into ice diethyl ether with stirring. After standing for more than 2 hours, the precipitate was completely precipitated, centrifuged, and washed with ice diethyl ether for 3 times to obtain a crude compound: His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser- Lys-Tyr-Leu-Asp-Lys(PEG ₂ -PEG ₂ -γGlu-CO(CH ₂ ) ₁₄ CH ₃ )-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met- Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ d ) Purification and purification of peptide compounds:

The obtained crude compound was dissolved in a solution of acetonitrile (ACN) / H ₂ O = 1:2 (volume ratio) and purified by preparative HPLC on a &_lt;RTIgt;&_lt;/RTI&gt_; Start with 30% acetonitrile (containing 0.05% trifluoroacetic acid) / H ₂ O (containing 0.05% trifluoroacetic acid), gradient (inclination rate of acetonitrile at 1.33% / min), flow rate of 15 mL / min The column was eluted for 30 minutes, and the fraction containing the peptide was collected and lyophilized to give a pure product having an HPLC purity of more than 95%. The separated product was analyzed by liquid chromatography.

Based on the above synthetic steps, the following polypeptide compounds of the present invention were synthesized (Table 1): Table 1 , the structure of the polypeptide compound synthesized in the examples of the present invention: Example 2 , GLP-1R/GCGR dual-target agonistic polypeptide inhibits proliferation of human lung type II epithelial cells A549 in vitro:

Inflammatory cells produce chemokines and cytokines to repair and reconstruct lung structures. TGF-β1 is a key profibrotic factor secreted by macrophages and is the backbone of fibroblast repair responses. TGF-β1 was significantly increased in animal models and lung tissue of IPF patients. TGF-β1 regulates the transfer, proliferation and differentiation of fibroblasts, which are characterized by the expression of α-SMA and the deposition of extracellular matrix (ECM).

Experimental methods: (1) A549 cells were digested, mixed and counted, diluted according to the concentration of 10000 cells per well, and then added to a 96-well plate at 100 μL per well, and the plate was placed at 37 ° C, 5% CO ₂ . In a constant temperature incubator, culture for 24 hours; (2) aspirate the original medium and replace it with serum-free medium. Grouped: 1 normal control group without treatment; 2 TGF-β control group of 5.0 ng/mL; 3 candidate peptide drug (15.0 nM) group; 4 candidate polypeptide drugs (15.0 nM) + 5.0 ng/mL TGF-β group ; 5 liraglutide (15.0 nM) group; 6 liraglutide (15.0 nM) + 5.0 ng / mL TGF-β group. Set 10 parallel holes in each group and continue to culture in the incubator for 24 hours. (3) Add 10.0 μL of MTT to each well after 24 hours, continue to culture for 4 hours, aspirate the supernatant, add 150.0 μL DMSO, and mix for 15 min. After that, the absorbance A value of each well was measured at 490 nm with a microplate reader. Liraglutide standard (purchased from Jill Biochemical (Shanghai) Co., Ltd., purity >98%, liraglutide acetate Cas No.: 204656-20-2.)

Experimental results: Through human lung type II epithelial cells A549, we first induced proliferation of A549 cells with exogenous TGF-β1, and then introduced GLP-1R/GCGR dual-target agonistic polypeptide. Compared with the normal control group, TGF-β induced a large number of cell proliferation, and the difference was extremely significant. The double-target agonist polypeptide 1-48 had a strong inhibition of TGF-β1-induced A549 proliferation at 15.0 nM (Fig. 1). The results also show that the overall dual-target agonist polypeptide, liraglutide, has a more pronounced effect on TGF-β-induced fibroblast transformation at 15.0 nM. Example 3 : Therapeutic efficacy of dual-target agonistic polypeptides on pulmonary fibrosis 1. Materials and methods 1.1 Animals

Eight-week-old SPF female C57 BL/6 mice, 20-25 g, were provided by the Guangdong Experimental Animal Center and were tested in the SPF laboratory of the Experimental Animal Center of the School of Medicine, Sun Yat-sen University. Adaptive feeding for 1 week. Breeding environment: temperature 20 ~ 25 ° C, humidity 70%, 12 hours of light and dark cycle controllable room, free access to food and water. 1.2 drugs and reagents

Bleomycin (BLM, purchased by TCI); sodium pentobarbital, bleomycin hydrochloride for injection, hydroxyproline (HYP) standard (purchased from Sigma, USA). 1.3 Establishment of an animal model of bleomycin-induced pulmonary fibrosis and the therapeutic effect of GLP-1R/GCGR dual-target agonistic polypeptide on pulmonary fibrosis

144 SPF female C57 BL/6 mice were randomly divided into 18 groups (n=8), a total of 18 groups: control group, bleomycin group and peptide drug administration group. Based on the above studies on the inhibition of TGF-β1-induced proliferation of A549, we selected peptide compounds: 4, 6, 7, 12, 15, 21, 24, 27, 30, 37, 38, 39, 40, 44, 48 And liraglutide for the study of the effect of intervention on pulmonary fibrosis. The drug administration components are: No. 4 drug group, No. 6 drug group, No. 7 drug group, No. 12 drug group, No. 15 drug group, No. 21 drug group, No. 24 drug group, No. 27 drug group, No. 30 drug group , No. 37 drug group, No. 38 drug group, No. 39 drug group, No. 40 drug group, No. 44 drug group, No. 48 drug group and Liraglutide drug group (18 groups).

All groups of C57 BL/6 mice were anesthetized with intraperitoneal injection of 2% sodium pentobarbital (40 mg/kg). The bleomycin group was intratracheally infused with 5 mg/kg bleomycin to induce pulmonary fibers. Chemical. A control group of mice was intratracheally infused with an equal volume of sterile saline. 4, 6, 7, 12, 15, 21, 24, 27, 30, 37, 38, 39, 40, 44, 48 groups, and the liraglutide group was given a subcutaneous injection of 200 μg/kg of the corresponding peptide once every other day. Drug, blank control group, bleomycin group (Bleomycin) subcutaneous injection of the same amount of sterile saline, once every other day, continuous administration for 21 days, taken. 1.4 histopathological examination

The right lower lobe of the mouse was cut and fixed in neutral formaldehyde with a volume fraction of 10%, hematoxylin-eosin staining (HE staining) and horseshoe collagen staining (Masson staining). The collagen staining procedure was strictly in accordance with the Masson staining kit. Product manuals are carried out. Image J software calculates the area of the blue area in the same area image, and then uses the graphpad Prismversion6 software to perform histogram analysis to observe collagen deposition.

As shown in Figure 2: HE staining results of mouse lung: blank control group: clear lung tissue structure, no inflammatory cell infiltration; bleomycin fibrosis group: see alveolar septal fibroblasts, subepithelial muscle fibroblasts Increased, capillary congestion and lymphocyte, macrophage infiltration, fibrous tissue hyperplasia, alveolar septal destruction, fibrous tissue patchy distribution; dual-target agonistic peptide administration group compared with liraglutide administration group : Candidate peptide compounds alleviate the alveolar structural disorder, inflammatory cell infiltration, reduce the accumulation of cells and fibers in the alveolar cavity, reduce collagen deposition, effectively delay the progression of pulmonary fibrosis, and treat idiopathic pulmonary fibrosis effect.

Further, we selected Masson staining in the lung tissues of the 15, 37, 38, 40, 44 and 48 administration groups (Fig. 3); semi-quantitative analysis of the results of the horseshoe staining: Calculating the blue areas in the same area image using Image J software Area, and then the area value was analyzed by histogram using graphpad software (Fig. 4), t-test was performed between each group; hydroxyproline was used to detect collagen deposition in the lung (Fig. 5). The results showed: blank control group: horse lung tissue was found to have a small amount of structural collagen in the bronchial wall and blood vessel wall, and no obvious collagen deposition in the lung tissue; bleomycin group: mouse lung tissue horse pine Staining showed a large amount of bundled blue-stained collagen tissue in the lung tissue; the lung tissue of the 15th administration group showed only a small amount of collagen and other markers of pulmonary fibrosis; There was a small amount of structural collagen in the bronchial wall and blood vessel wall, and no obvious collagen deposition in the lung tissue. In the 38th administration group, the lung tissue of the mice was stained with a small amount of structural collagen in the bronchial wall and the blood vessel wall, and the alveolar cavity was found. The cell exudate did not secrete collagen, so no obvious collagen deposition was observed in the lung tissue; the horseshoe staining of the lung tissue of the 40-administered group was observed in the alveolar except for a small amount of structural collagen observed in the bronchial wall and vessel wall. There was only a small amount of collagen deposition on the wall; the horseshoe staining of the lung tissue of the mice in the 44th administration group showed that although there was no obvious deposition in the alveolar space of the lung tissue, there was a small amount of collagen deposition in the alveolar wall. 48 lung of mice administered group Masson staining revealed the presence of only a small amount of collagen deposition and deposition phenomena in the alveolar wall. Polypeptide compounds 15, 37, 38, 40, 44 and 48 all attenuated alveolar structural disorders, inflammatory cell infiltration; significantly inhibited hydroxyproline content, reduced cell and fiber accumulation in the alveolar cavity, reduced collagen deposition, enough Effectively delay the progression of pulmonary fibrosis and have a therapeutic effect on idiopathic pulmonary fibrosis.

Indirect immunofluorescence assay of α-SMA, Figure 6 shows that dual-target candidate drugs 15, 37, 38, 40, 44 and 48 significantly reduced the expression of pulmonary fibrosis marker protein α-SMA during the treatment of pulmonary fibrosis.

Summary: The results indicate that the GLP-1R/GCGR dual-target agonist polypeptide of the present invention can effectively delay the progression of pulmonary fibrosis, and can significantly reduce the accumulation of cells and fibers in the alveolar cavity and reduce collagen precipitation during therapeutic administration. And pulmonary fibrosis marker protein α-SMA expression.

The present invention has been described by way of example, and although not shown, all the polypeptides within the scope of the present invention can achieve the technical effects of the present invention, and those skilled in the art can make modifications and variations of the present invention without departing from the present invention. The spirit of the invention is within the scope of the appended claims.

no

Figure 1 is a graph showing the inhibitory effect of a dual-target agonistic polypeptide on TGF-β1-induced proliferation of A549 (#: indicates a significant decrease in 95% confidence (p < 0.05) compared with the control group; ##: indicates The control group was within 99% confidence (p < 0.01 was significantly reduced; **: indicated a significant increase in 99% confidence (p < 0.01) compared to the normal diet group).

Figure 2 shows the dual-target agonistic polypeptides 4, 6, 7, 12, 15, 21, 24, 27, 30, 37, 38, 39, 40, 44, 48 and liraglutide for pulmonary fibrosis in mice Effect of HE staining slices.

Figure 3 is a graph showing Masson stained sections of dual-target agonistic polypeptides 15, 37, 38, 40, 44 and 48 for treatment of pulmonary fibrosis in mice.

Figure 4 is a graph showing semi-quantitative analysis of the results of Marsson staining (*: indicates 95% confidence in comparison with the control (p < 0.05); **: indicates 99% confidence in comparison with the control (p < 0.01)).

Figure 5 is a graph showing the amount of collagen deposition in the lungs detected by hydroxyproline (*: expressed within 95% confidence level compared to the control (p < 0.05); **: expressed within 99% confidence level compared to the control (p<0.01)).

Figure 6 is a graph showing indirect immunofluorescence of α-SMA - green is α-SMA.

Claims (10)

Use of an oxyntomodulin analog GLP-1R/GCGR double agonist polypeptide for the preparation of a medicament for preventing or treating pulmonary disease associated with fibrotic symptoms such as idiopathic pulmonary interstitial fibrosis. The use according to claim 1, wherein the polypeptide has the parent peptide represented by the following amino acid sequence: His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Leu- Asp-Xaa16-Xaa17-Xaa18-Ala-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39- Xaa40-CO R ₁ wherein R ₁ = -NH ₂ ; Xaa2 = Aib or D-Ser; Xaa10 = Lys or Tyr; Xaa13 = Lys or Tyr; Xaa16 = Ser, Aib, Glu or Lys; Xaa17 = Lys or Arg; Xaa18=Arg or Ala; Xaa20=His, Gln or Lys; Xaa21=Asp or Glu; Xaa23=Ile,Val; Xaa24=Glu or Gln; Xaa27=Met, Leu, or Nle; Xaa28=Asn, Asp, Arg, Ser Or absent; Xaa29=Gly, Thr or absent; Xaa30=Gly or absent; Xaa31=Gly or absent; Xaa32=Pro or absent; Xaa33=Ser, Val or absent; Xaa34=Ser or absent; Xaa35=Gly or absent; Xaa36=Ala or absent; Xaa37=Pro or absent; Xaa38=Pro or absent; Xaa39=Pro or absent; Xaa40=Ser or absent. The use according to claim 2, wherein at least one of the Xaa10, Xaa16, Xaa17 or Xaa20 is Lys, and the side chain of the at least one Lys or the 12th Lys of the sequence is linked to a lipophilic substituent, The linkage is such that the lipophilic substituent forms a guanamine bond with a carboxy group of the amino group of a bridging group, and the carboxy group of the amino acid residue of the bridging group forms a guanamine with the N-terminal residue of the Lys of the parent peptide. a bond to the parent peptide, which is Glu, Asp and/or (PEG) _m , wherein m is an integer from 2 to 10; the lipophilic substituent is selected from CH ₃ (CH ₂ ) _n CO - or a thiol group of HOOC(CH ₂ ) _n CO-, wherein n is an integer from 10 to 24. The use according to claim 2, wherein the bridging group is Glu-(PEG) _m or Asp-(PEG) _m or (PEG) _m . The use according to claim 2, wherein the bridging group forms a molecular bridge between the residue pairs 12 and 16, 16 and 20, 17 and 21 or 20 and 24 side chains of the amino acid sequence. The use according to claim 2, wherein the Lys linked to the lipophilic substituent is replaced by HomoLys, Orn, Dap or Dab. The use according to any one of claims 2 to 5, wherein when the 10th, 12th, 16th, 17th or 20th position of the amino acid sequence is Lys, the lipophilic substituent attached to the Lys side chain is One of the following structures: . The use according to claim 2, wherein the amino acid sequence of the parent peptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO :14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22. SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48 The amino acid sequence of the parent peptide. According to the application of claim 1, the oxyntomodulin analog GLP-1R/GCGR double agonist polypeptide is prepared for prevention or direct or indirect treatment of pulmonary fibrosis accompanied by idiopathic pulmonary interstitial fibrosis A condition caused or characterized by it. The use according to claim 1, comprising at least one effective amount of the polypeptide of any one of claims 2 to 8 and at least one pharmaceutically acceptable pharmaceutically acceptable carrier to prepare an oxyntomodulin analog GLP -1R/GCGR dual agonist polypeptide pharmaceutical composition and formulated into a variety of suitable dosage forms.