WO2019085773A1

WO2019085773A1 - Oxyntomodulin analogue glp-1r/gcgr dual target agonist polypeptide for treating idiopathic pulmonary interstitial fibrosis

Info

Publication number: WO2019085773A1
Application number: PCT/CN2018/111034
Authority: WO
Inventors: 蒋先兴
Original assignee: 中山大学
Priority date: 2017-11-06
Filing date: 2018-10-19
Publication date: 2019-05-09
Also published as: CN111372945A; CN111372945B; TW201918259A

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-β1, 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

Technical field

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.

Background technique

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. The mortality rate is high, the results of glucocorticoids and immunosuppressive therapy are not satisfactory, and the 5-year survival rate of patients is 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 multiple factors such as gender and weight of patients, because the incidence of IPF in male population is higher than that of female, and its development is faster, and the survival rate is lower than that of female (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 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 processes may 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 potential serious side effects, but also is essentially ineffective for IPF treatment; pirfenidone (5-methyl 1 phenyl 2-(1H) pyridone) is a synthetic molecule and is currently the only approved for IPF clinical use. Treated anti-fibrotic drugs. 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.).

Summary of the invention

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 defect. The reaction 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 this type of GLP-1R/GCGR dual-target agonist polypeptide has 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 cavity 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 a parent peptide represented by the following amino acid sequence:

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 not present;

Xaa29=Gly, Thr or not present;

Xaa30=Gly or does not exist;

Xaa31=Gly or does not exist;

Xaa32=Pro or does not exist;

Xaa33=Ser, Val or does not exist;

Xaa34=Ser or does not exist;

Xaa35=Gly or does not exist;

Xaa36=Ala or does not exist;

Xaa37=Pro or does not exist;

Xaa38=Pro or does not exist;

Xaa39=Pro or does not exist;

Xaa40=Ser or does not exist;

In the amino acid sequence, at least one of 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 in a manner of a lipid substituent having an amide bond with a carboxyl group of an amino group of a bridging group, and a carboxyl group of the amino acid residue of the bridging group forms an amide bond with the N-terminal residue of the Lys of the parent peptide to the parent peptide; The bridging 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 An acyl group of CO-, wherein 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 paired with Glu, and Lys and Asp paired, respectively, can also react to form a lactam 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 solution. , 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 advantages of the invention are:

1) have better anti-pulmonary fibrosis biological activity;

2) It shows stability in drug pharmacokinetic experiments, good stability, easy to scale up production, and low cost;

3) It has lower toxicity than small molecule compounds, and has a larger safety window and a smaller dosage.

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):

Compound 2 (involving SEQ ID NO: 2):

Compound 3 (involving SEQ ID NO: 3):

Compound 4 (involving SEQ ID NO: 4):

Compound 5 (involving SEQ ID NO: 5):

Compound 6 (involving SEQ ID NO: 6):

Compound 7 (involving SEQ ID NO: 7):

Compound 8 (involving SEQ ID NO: 8):

Compound 9 (involving SEQ ID NO: 9):

Compound 10 (involving SEQ ID NO: 10):

Compound 11 (involving SEQ ID NO: 11):

Compound 12 (involving SEQ ID NO: 12):

Compound 13 (involving SEQ ID NO: 13):

Compound 14 (involving SEQ ID NO: 14):

Compound 15 (involving SEQ ID NO: 15):

Compound 16 (involving SEQ ID NO: 16):

Compound 17 (involving SEQ ID NO: 17):

Compound 18 (involving SEQ ID NO: 18):

Compound 19 (involving SEQ ID NO: 19):

Compound 20 (involving SEQ ID NO: 20):

Compound 21 (involving SEQ ID NO: 21):

Compound 22 (involving SEQ ID NO: 22):

Compound 23 (involving SEQ ID NO: 23):

Compound 24 (involving SEQ ID NO: 24):

Compound 25 (involving SEQ ID NO: 25):

Compound 26 (involving SEQ ID NO: 26):

Compound 27 (involving SEQ ID NO: 27):

Compound 28 (involving SEQ ID NO: 28):

Compound 29 (involving SEQ ID NO: 29):

Compound 30 (involving SEQ ID NO: 30):

Compound 31 (involving SEQ ID NO: 31):

Compound 32 (involving SEQ ID NO: 32):

Compound 33 (involving SEQ ID NO: 33):

Compound 34 (involving SEQ ID NO: 34):

Compound 35 (involving SEQ ID NO: 35):

Compound 36 (involving SEQ ID NO: 36):

Compound 37 (involving SEQ ID NO: 37):

Compound 38 (involving SEQ ID NO: 38):

Compound 39 (involving SEQ ID NO: 39):

Compound 40 (involving SEQ ID NO: 40):

Compound 41 (involving SEQ ID NO: 41):

Compound 42 (involving SEQ ID NO: 42):

Compound 43 (involving SEQ ID NO: 43):

Compound 44 (involving SEQ ID NO: 44):

Compound 45 (involving SEQ ID NO: 45):

Compound 46 (involving SEQ ID NO: 46):

Compound 47 (involving SEQ ID NO: 47):

Compound 48 (involving SEQ ID NO: 48):

The abbreviations used in the present invention have the following specific meanings:

Boc is tert-butoxycarbonyl, Fmoc is fluorenylmethoxycarbonyl, t-Bu is tert-butyl, and ivDDe is 1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3- Base-butyl removal and lipophilic substituent, resin is resin, TFA is trifluoroacetic acid, EDT is 1,2-ethanedithiol, Phenol is phenol, FBS is fetal bovine serum, BSA is bovine serum albumin HPLC is a high-performance liquid phase, GLP-1R is a glucagon-like peptide 1 receptor, GCGR is a glucagon receptor, GLP-1 is a glucagon-like peptide, and mPEG is a monomethoxy polyethylene. Alcohol, OXM is oxyntomodulin, His is histidine, Ser is serine, D-Ser is D-serine, Gln is glutamine, Gly is glycine, Glu is glutamic acid, and 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, and Ile is isoleucin Acid, Leu is leucine, Cys is cysteine, Pro is proline, Val is valine, Met is methionine, Asn is asparagine, HomoLys is high lysine, Orn is ornithine, Dap is diaminopimelic acid, Dab is 2,4-diaminobutyric acid, and Nle is positively bright ammonia. Acid, Aib is 2-aminoisobutyric acid, Palmitoyl is palmitoyl group, Cholesteryl is cholesterol group, AEEA is [2-[2-(amino)ethoxy]ethoxy]acetic acid, CA is 4-imidazolyl Acetyl.

DRAWINGS

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.

Detailed ways

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 polypeptide compounds

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 polypeptide. 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:

Structure sequence:

a) Master peptide chain assembly:

The following polypeptides of 0.25 mmol scale were synthesized on a CS336X peptide synthesizer (CS Bio, USA) according to the Fmoc/t-Bu strategy:

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-dimethylformamide (DMF) ) Wash the resin sufficiently 3 times;

(2) The second step: using Rink amide resin as carrier and coupling agent from 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), organic base N,N-diisopropylethylamine (DIEPA) is a 1:1 molar ratio of N,N-dimethylformamide (DMF) as a solvent, and the condensation reaction is carried out in sequence.

Fmoc-Ser(t-Bu)-OH, Fmoc-Pro-OH(3x), Fmoc-Ala-OH, Fmoc-Gly-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 gets:

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, the resin is thoroughly washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N,N-dimethylformamide (DMF). 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 to 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 to 8 times, and the reaction time is 1 to 5 hours.

b) Removal of 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl (ivDde) and introduction of lipophilic substituents:

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 hydrate N, N was added. a solution of dimethylformamide (DMF), the reaction mixture was shaken at room temperature for 10 to 30 minutes for a well treatment step, and then filtered. Repeat the 肼 processing step 5 times to get:

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, the resin is thoroughly washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N,N-dimethylformamide (DMF). 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, the resin is thoroughly washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N,N-dimethylformamide (DMF). Get 3 times each:

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, the resin is thoroughly washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N,N-dimethylformamide (DMF). 3 times each.

Removal of the Fmoc group by 20% piperidine/N,N-dimethylformamide (DMF) solution (30 minutes, repeated removal twice), adding Fmoc-PEG ₂ -OH, 2-(7 -azozotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), diisopropylethylamine (DIEPA) N,N-dimethyl Formamide (DMF) mixed coupling solution (5 times excess), coupled reaction

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, the resin is thoroughly washed with N,N-dimethylformamide (DMF), dichloromethane (DCM), methanol (Methanol), dichloromethane (DCM), and N,N-dimethylformamide (DMF). 3 times each.

The Fmoc group was removed by 20% piperidine/N,N-dimethylformamide (DMF) solution (30 minutes, repeated twice), and then Fmoc-γGlu-OtBu was sequentially coupled according to the conventional conditions. Add hexadecanoic acid (palmitic acid) to get:

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 (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 is added to the cutting solution TFA/Phenol/thioanisole/EDT/H ₂ O ( 82.5:5:5:2.5:5, volume ratio), the temperature is raised, the temperature of the lysate is controlled at 25 ° C, and the reaction is carried out 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:

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 column of &lt Starting with 30% acetonitrile (containing 0.05% trifluoroacetic acid) / H ₂ O (containing 0.05% trifluoroacetic acid), the gradient (increased ratio of acetonitrile at a rate of 1.33% / min), the flow rate is 15 mL / min The column was eluted for 30 minutes, and the fraction containing the peptide was collected and lyophilized to obtain a pure product having an HPLC purity of more than 95%. The separated product was analyzed by liquid chromatography.

Based on the above synthetic procedures, the following polypeptide compounds of the invention were synthesized (Table 1):

Table 1. Structure of the polypeptide compound synthesized in the examples of the present invention:

Example 2. Inhibitory effect of GLP-1R/GCGR dual-target agonistic polypeptide on proliferation of human lung type II epithelial cells A549 in vitro:

Inflammatory cells produce chemokines and cytokines to repair and remodel lung structures. TGF-β1 is a key pro-fibrotic 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 constant temperature. In the incubator, culture for 24 hours; (2) aspirate the original medium and replace it with serum-free medium. Grouped: 1 normal control group without treatment; TGF-β control group of 25.0 ng/mL; 3 candidate peptide drug (15.0 nM) group; 4 candidate polypeptide drug (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 of DMSO, and mix for 15 min. 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 cell proliferation, and the difference was extremely significant. The double-target agonist polypeptide 1-48 had 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 effect of dual-target agonistic polypeptide on pulmonary fibrosis

1. Materials and methods

1.1 Animals

Eight-week-old SPF female C57BL/6 mice, 20-25 g, were provided by the Experimental Animal Center of Guangdong Province and were tested in the SPF laboratory of the Experimental Animal Center of Sun Yat-sen University School of Pharmacy. 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 treatment of pulmonary fibrosis with GLP-1R/GCGR dual-target agonistic polypeptide

144 SPF female C57BL/6 mice were randomly divided into 18 groups (n=8), a total of 18 groups: blank control group, bleomycin group and peptide drug administration group. Based on the above results of 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).

After all groups of C57BL/6 mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (40 mg/kg), pulmonary fibrosis was induced by intratracheal infusion of 5 mg/kg bleomycin in the bleomycin group. A control group of mice was intratracheally infused with an equal volume of sterile saline.

Groups

4,6,7,12,15,21,24,27,30,37,38,39,40,44,48, and the liraglutide group were given a subcutaneous injection of 200 μg/kg of the corresponding polypeptide drug every other day. , blank control group (control), 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 for histogram analysis to observe collagen deposition.

As shown in Figure 2: HE staining results in mouse lung:

Blank control group: the lung tissue structure is clear, no inflammatory cell infiltration;

Bleomycin fibrosis group: see fibroblasts in the alveolar septum, subepithelial muscle fibroblasts increased significantly, capillary congestion and lymphocyte, macrophage infiltration, fibrous tissue hyperplasia, alveolar septal destruction, fibrous tissue formation Patchy distribution

The double-target agonistic peptide administration group compared with the liraglutide-administered group: the candidate polypeptide compounds alleviated the alveolar structural disorder, inflammatory cell infiltration, reduced cell and fiber accumulation in the alveolar cavity, and reduced collagen deposition. It is effective enough to delay the progression of pulmonary fibrosis and has a therapeutic effect on idiopathic pulmonary fibrosis.

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 horseshoe staining results: Image J software was used to calculate the blue areas in the same area image. 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 result shows:

The blank control group: Mazong staining of mouse lung tissue showed 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: Masonian staining of mouse lung tissue showed a large amount of bundled blue-stained collagen tissue in lung tissue;

In the 15th administration group, the lung tissue of the mice was stained with a small amount of collagen and other markers of pulmonary fibrosis; in the 37th 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. There was no obvious collagen deposition in the lung tissue; there was a small amount of structural collagen in the bronchial wall and blood vessel wall in the lung tissue of the mice in the 38th administration group, and the cell exudate in the alveolar cavity did not secrete collagen, so No obvious collagen deposition was observed in the lung tissue; in the 40-administered group, the horseshoe staining of the lung tissue showed only a small amount of collagen deposition in the alveolar wall except for a small amount of structural collagen observed in the bronchial wall and the vessel wall; Masonian staining of mouse lung tissue showed that although there was no obvious deposition in the alveolar cavity of the lung tissue, there was a small amount of collagen deposition in the alveolar wall. The horseshoe staining of the lung tissue of the 48-administered group showed only a small amount of deposition and collagen in the alveolar wall. Deposition phenomenon. Polypeptide compounds 15,37,38,40,44 and 48 alleviated alveolar structural disorders, inflammatory cell infiltration, significantly inhibited hydroxyproline content, reduced cell and fiber accumulation in alveolar spaces, and reduced collagen precipitation. Effectively delay the progression of pulmonary fibrosis and have a therapeutic effect on idiopathic pulmonary fibrosis.

Indirect immunofluorescence 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 intended to fall within the scope of the appended claims.

Claims

Use of a oxyntomodulin analog GLP-1R/GCGR double agonist polypeptide for the preparation of a medicament for the prevention or treatment of pulmonary disease associated with fibrotic symptoms such as idiopathic pulmonary interstitial fibrosis.
The use according to claim 1, wherein the polypeptide has a 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 R1=-NH2;

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 not present;

Xaa29=Gly, Thr or not present;

Xaa30=Gly or does not exist;

Xaa31=Gly or does not exist;

Xaa32=Pro or does not exist;

Xaa33=Ser, Val or does not exist;

Xaa34=Ser or does not exist;

Xaa35=Gly or does not exist;

Xaa36=Ala or does not exist;

Xaa37=Pro or does not exist;

Xaa38=Pro or does not exist;

Xaa39=Pro or does not exist;

Xaa40=Ser or does not exist.
The use according to claim 2, wherein at least one of Xaa10, Xaa16, Xaa17 or Xaa20 is Lys, and the at least one Lys or the 12th Lys side chain of the sequence is linked to a lipophilic substituent, and is linked. The manner is that the lipophilic substituent forms an amide bond with the amino group of a bridging group at a carboxyl group thereof, and the carboxyl group of the amino acid residue of the bridging group forms an amide bond with the N-terminal residue of the Lys of the parent peptide to On the parent peptide, the bridging group is Glu, Asp and/or (PEG)m, wherein m is an integer from 2 to 10; the lipophilic substituent is selected from CH3(CH2)nCO- or HOOC(CH2) An acyl group of nCO-, wherein n is an integer from 10 to 24.
The use according to claim 2, characterized in that the bridging group is Glu-(PEG)m or Asp-(PEG)m or (PEG)m.
The use according to claim 2, characterized in that 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, characterized in that 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, characterized in that lipophilic substitution to the Lys side chain when the 10th, 12th, 16th, 17th or 20th position of the amino acid sequence is Lys The base 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 amino acid sequence of the parent peptide.
The use of the oxyntomodulin analog GLP-1R/GCGR double agonist polypeptide for the prevention or direct or indirect treatment of pulmonary fibrosis associated with idiopathic pulmonary interstitial fibrosis, according to claim 1. A condition caused or characterized by it.
The use according to any of the preceding claims, wherein the polypeptide of any one of claims 2-8 and at least one pharmaceutically acceptable pharmaceutically acceptable carrier are contained in an effective amount to produce an oxyntomodulin. The GLP-1R/GCGR dual agonist polypeptide pharmaceutical composition is prepared in a variety of suitable dosage forms.