Application of GLP-1 (Glucagon Like Peptide-1) similar peptide dimer in preparation of medicine for preventing and / or treating diabetic complications

Abstract

The invention provides an application of a GLP-1 (Glucagon Like Peptide-1) similar peptide dimer in preparation of a medicine for preventing and / or treating diabetic complications, and belongs to the technical field of biological medicines. It is found in repairing cell damage of diabetic retinopathy that the GLP-1 similar peptide dimer has the effect of preventing oxidative stress of diabetic retinopathy, the dimer can further remarkably reduce the active oxygen level after the retinopathy is damaged, and the GLP-1 similar peptide dimer has the remarkable effect of preventing and repairing the damage of the diabetic retinopathy. The research result of treating a mouse with diabetic nephropathy by using the GLP-1 similar peptide dimer shows that the GLP-1 similar peptide dimer can effectively treat diabetic nephropathy. Therefore, more choices are provided for clinical medicines for preventing and / or treating diabetic complications.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a GLP-1 analog dimer in the preparation of a drug for preventing and / or treating diabetic complications. Background Art

[0002] Diabetes is a group of lifelong metabolic diseases caused by multiple etiologies and characterized by persistent chronic hyperglycemia. Diabetes has become an increasingly serious health problem. Diabetes is a disease with the most known complications, and most diabetic patients die of diabetic complications. Long-term elevated blood glucose damages large and small blood vessels and endangers the heart, kidneys, peripheral nerves, eyes, feet, etc. Among them, diabetic nephropathy is the main cause of death in diabetic patients, and diabetic retinopathy is one of the most common complications of diabetes. The clinical manifestations are mainly the formation of retinal neovascularization and microaneurysms, and hard exudates in the vitreous, which ultimately lead to a continuous decline in the patient's vision and even blindness.

[0003] Stress injury is related to multiple diseases, such as diabetic nephropathy and diabetic retinopathy complicated by diabetes. Under normal physiological conditions, the free radicals generated can be completely degraded by in vivo reducing agents such as glutathione, and the normal body will strictly regulate the level of reactive oxygen species (ROS) to maintain cell homeostasis. However, when diabetic nephropathy or diabetic retinopathy occurs, the tissues of diabetic nephropathy or retinal cells are more likely to be damaged. Disease stress leads to the accumulation of ROS levels, and the retina, pancreas, and kidneys are damaged. Diabetic nephropathy or diabetic retinopathy is a diabetic complication caused by diabetes affecting the kidneys or retina, which seriously affects the health of patients. Although there are currently various treatment methods, there are still many challenges in the treatment of the disease. Therefore, finding a drug that is non-toxic and side-effect-free and can be safely and effectively used for diabetic complications is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the application of a GLP-1 analog dimer in the preparation of a drug for treating diabetic complications.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides the application of a GLP-1 analog dimer in the preparation of a drug for preventing and / or treating diabetic complications, and the amino acid sequence of the GLP-1 analog dimer is as follows:

[0007]

[0008] In the amino acid sequence, the three-letter amino acids are L-α-amino acids. "|" indicates that Cys-Cys is linked by a disulfide bond. β-Aib is β-aminoisobutyric acid. Lys[ε-N(γE-palmitic acid)] is lysine modified with γ-glutamic acid-palmitic acid on the ε-amino group of the lysine side chain. γE represents γ-glutamic acid.

[0009] Preferably, the diabetic complication is diabetic retinopathy and / or diabetic nephropathy.

[0010] Preferably, the lesions of the diabetic nephropathy include diabetes and kidney injury.

[0011] Preferably, the diabetic retinopathy includes retinal oxidative damage.

[0012] Preferably, the dosage form of the drug is an ointment, an emulsion or a solution.

[0013] The present invention provides a drug for treating diabetic complications, and the drug comprises the above-mentioned GLP-1 analogue dimer and a pharmaceutically acceptable excipient.

[0014] The present invention provides an eye drops for treating diabetic retinopathy, and the eye drops comprise the above-mentioned GLP-1 analogue dimer and a pharmaceutically acceptable excipient.

[0015] Preferably, the final concentration of the GLP-1 analogue dimer is 0.1-0.6 nM and pH = 8.0.

[0016] Preferably, the excipient comprises an osmotic pressure regulator, a pH regulator, an antibacterial agent, a thickening agent or a solubilizer.

[0017] Preferably, the mass percentage of the GLP-1 analogue dimer in the eye drops is 20%-80%.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides an application of a GLP-1 analogue dimer in the preparation of a drug for treating diabetic complications. In the treatment of diabetic retinopathy, it is found that the GLP-1 analogue dimer has a stress injury treatment effect on diabetic retinopathy, significantly down-regulates the reactive oxygen species level in diabetic retinopathy, and has an obvious preventive, protective and reparative effect on diabetic retinopathy. The research results of treating diabetic nephropathy mice with the GLP-1 analogue dimer show that the GLP-1 analogue dimer can effectively treat diabetic nephropathy. Therefore, the present invention provides more choices for clinically treating drugs for diabetic complications. Description of the Drawings

[0020] Figure 1 To study the effects of GLP-1 analog dimer at different concentrations on the repair of diabetic retinopathy;

[0021] Figure 2 To study the results of using GLP-1 analog dimer for the treatment of diabetic nephropathy. Specific Embodiments

[0022] The present invention provides an application of a GLP-1 analog dimer in the preparation of a medicament for treating diabetic complications. The amino acid sequence of the GLP-1 analog dimer is as follows:

[0023]

[0024] In the amino acid sequence, the three-letter amino acids are L-α-amino acids. "|" indicates that Cys-Cys is linked by a disulfide bond. β-Aib is β-aminoisobutyric acid, and Lys[ε-N(γE-palmitic acid)] is lysine modified with γ-glutamic acid-palmitic acid on the ε-amino group of the lysine side chain, where γE represents γ-glutamic acid.

[0025] The present invention discovers through research that the use of GLP-1 analog dimer can effectively treat diabetic complications.

[0026] In the present invention, the diabetic complications are diabetic retinopathy and / or diabetic nephropathy. The diabetic retinopathy includes retinal oxidative damage. The diabetic complications are diabetic retinopathy and / or diabetic nephropathy.

[0027] In the present invention, the dosage form of the medicament is preferably an ointment, an emulsion or a solution, and more preferably a solution. There is no special limitation on the preparation method of the GLP-1 analog dimer of the present invention, and a method well-known in the art can be adopted.

[0028] The present invention also provides a medicament for treating diabetic complications, which comprises the above-mentioned GLP-1 analog dimer and a pharmaceutically acceptable excipient.

[0029] The present invention provides an eye drops for treating diabetic retinopathy, which comprises the above-mentioned GLP-1 analog dimer and a pharmaceutically acceptable excipient.

[0030] In the present invention, the final concentration of the GLP-1 analog dimer is preferably 0.1 - 0.6 nM, and pH = 8.0. More preferably, it is 0.125 - 0.5 nM. The excipients preferably include an osmotic pressure regulator, a pH regulator, an antibacterial agent, a thickening agent, or a solubilizer. The osmotic pressure regulator includes one or more of sodium chloride, glucose, and mannitol; the pH regulator includes one or more of borax, boric acid, and phosphate buffer; the thickening agent includes one or more of hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and polyvinyl alcohol; the antibacterial agent includes one or more of chlorobutanol, methyl p-hydroxybenzoate, and ethyl p-hydroxybenzoate; the solubilizer includes one or more of water and polysorbate 80. The mass percentage of the GLP-1 analog dimer in the eye care solution is preferably 20% - 80%.

[0031] In the present invention, the GLP-1 analog dimer can be used as the sole active ingredient for treating diabetic complications.

[0032] In the present invention, unless otherwise specified, all raw material components are commercially available products or known products well-known to those skilled in the art.

[0033] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1 Preparation of the monomeric peptide and dimer of the GLP-1 analog

[0035] I. Solid-phase synthesis process of the monomeric peptide of the GLP-1 analog: Manual solid-phase polypeptide synthesis operation steps.

[0036] 1. Resin swelling: Put the amino resin (amide-terminated C-terminal sequence using amino resin) (purchased from Tianjin Nankai Synthetic Technology Co., Ltd.) into a reaction pot, add 15 mL / g of resin of dichloromethane (DCM, Dikma Technologies Inc.), and shake for 30 min. SYMPHONY type 12-channel polypeptide synthesizer (SYMPHONY model, software Version.201, Protein Technologies Inc.).

[0037] 2. Attachment of the first amino acid: Remove the solvent by suction filtration through a sintered glass funnel, add 3 molar equivalents of the first C-terminal Fmoc-AA amino acid (all Fmoc-amino acids are provided by Suzhou Tianma Pharmaceutical Group Fine Chemical Co., Ltd.), then add 10 molar equivalents of 4-dimethylaminopyridine (DMAP) and N,N'-dicyclohexylcarbodiimide (DCC), and finally add dimethylformamide (DMF) (purchased from Dikma Technologies Inc.) to dissolve, and shake for 30 min. Block with acetic anhydride.

[0038] 3. Deprotection: Remove the DMF, add 20% piperidine-DMF solution (15 mL / g), for 5 min, filter to remove the solvent, then add 20% piperidine-DMF solution (15 mL / g) again, for 15 min. Piperidine is provided by Shanghai Chemical Reagent Co., Ltd., Sinopharm Group.

[0039] 4. Detection: Remove the solvent by suction. Take a dozen resin beads, wash them three times with dichloromethane, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105 - 110 °C for 5 min, and a dark blue color change indicates a positive reaction.

[0040] 5. Washing the resin: Wash twice with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g) in sequence.

[0041] 6. Condensation: According to the specific synthesis conditions, the following methods can be used alone or in combination in polypeptide synthesis:

[0042] Method a: Three molar equivalents of the protected amino acid and three molar equivalents of 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, Suzhou Tianma Pharmaceutical Group Fine Chemical Co., Ltd.), both dissolved in the minimum amount of DMF, are added to the reaction flask. Immediately add ten molar equivalents of N-methylmorpholine (NMM, Suzhou Tianma Pharmaceutical Group Fine Chemical Co., Ltd.). React for 30 min and the detection shows negative.

[0043] Method b: Three molar equivalents of the protected amino acid FMOC-AA and three molar equivalents of 1-hydroxybenzotriazole (HOBt, Suzhou Tianma Pharmaceutical Group Fine Chemical Co., Ltd.), both dissolved in the minimum amount of DMF, are added to the reaction tube. Immediately add three molar equivalents of N,N'-diisopropylcarbodiimide (DIC). React for 30 min, and the detection shows negative.

[0044] 7. Washing the resin: Wash once with DMF (10 mL / g), twice with methanol (10 mL / g), and twice with DMF (10 mL / g) in sequence.

[0045] 8. Repeat steps 2 to 6, as shown in the GLP-1 peptide with side chain modification, and connect the corresponding amino acids from right to left in sequence.

[0046] 9. Synthesis of K 20

N-ε-(N-α-alkanoyl-L-γ-glutamyl)

N-ε-(N-α-palmitoyl)

N-ε-(palmitoyl)

[0047] 10. Subject the completed condensed polypeptide to DMF (10 mL / g) twice, DCM (10 mL / g) twice, DMF (10 mL / g) twice, and drain for 10 min. The ninhydrin test is negative.

[0048] 11. Remove the FMOC protecting group of the last N-terminal amino acid of the peptide chain. The test is positive, and the solution is drained for standby.

[0049] 12. Wash the resin according to the following method: DMF (10 mL / g) twice, methanol (10 mL / g) twice, DMF (10 mL / g) twice, DCM (10 mL / g) twice, and drain for 10 min.

[0050] 13. Cleaving the polypeptide from the resin: Prepare the cleavage solution (10 mL / g): 94.5% TFA (J.T. Baker Chemical Company); 2.5% water, 2.5% ethanedithiol (EDT, Sigma - Aldrich Chemistry), and 1% triisopropylsilane (TIS, Sigma - Aldrich Chemistry). Cleavage time: 120 min.

[0051] 14. Blowing dry and washing: Blow dry the lysate as much as possible with nitrogen, wash it six times with ether, and then volatilize it at room temperature.

[0052] 15. Purify the polypeptide by HPLC, identify it, and store it at -20°C in the dark using the following method.

[0053] The test method is as follows:

[0054] 1. Purifying the polypeptide by HPLC: Dissolve the crude peptide in pure water or with a small amount of acetonitrile added and purify it according to the following conditions: High - performance liquid chromatograph (analytical type; software Class - VP.Sevial System; manufacturer SHIMADZU, Japan) and Venusi MRC - ODS C18 chromatographic column (30×250 mm, Tianjin Bonna - Agela Technologies). Mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: 0.1% trifluoroacetic acid - 99.9% acetonitrile solution (acetonitrile purchased from Fisher Scientific Company). Flow rate: 1.0 mL / min, sample injection volume 30 μL, detection wavelength 220 nm. Elution program: 0 - 5 min: 90% A + 10% B; 5 - 30 min: 90% A / 10% B → 20% A / 80% B.

[0055] 2. Finally, lyophilize the purified effective solution using a lyophilizer (Freezone Plus 6 model, manufacturer LABCONCO) to obtain the finished product.

[0056] 3. Identification: Take a small amount of the finished polypeptide separately and perform HPLC analysis on its purity: Chromatographic column (4.6×150 mm). Mobile phase A: 0.1% trifluoroacetic acid aqueous solution, mobile phase B: 99.9% acetonitrile - 0.1% trifluoroacetic acid solution, flow rate: 1.0 mL / min, sample injection volume 10 μL, detection wavelength 220 nm. Elution program: 0 - 5 min: 100% A; 5 - 30 min: 100% A → 20% A / 80% B. The measured purity is required to be greater than 95%.

[0057] Identification of the molecular weight of the polypeptide by MS method: Take the polypeptide with qualified purity, dissolve it in water, add 5% acetic acid + 8% acetonitrile + 87% water to dissolve it, and measure the molecular weight by electrospray ionization mass spectrometry.

[0058] 4. Seal and package the powdery polypeptide and store it at -20°C in the dark.

[0059] Formation of the dimer: Dissolve the monomeric peptide with a unique cysteine at the C-terminus or within the peptide chain, which was finally prepared above at a concentration of 1 mg / mL, in an aqueous solution with pH = 9.5, and incubate at 37°C for 4 hours to form a 100% homologous dimeric peptide. The dimeric peptide was separated and identified by Sephadex G-25 chromatography (in a 2 × 60 cm G-25 chromatography column and at a natural flow rate, the dimeric component was the first peak and the residual impurity component was the second peak). The dimeric peptide can be identified by peptide PAGE electrophoresis or mass spectrometry without a sulfhydryl reducing agent - mercaptoethanol.

[0060] The GLP-1 analog monomer and dimer were synthesized in this laboratory. The inventors of the GLP-1 analog dimer confirmed its structure by HPLC purity, ESI or laser time-of-flight mass spectrometry and cysteine oxidation. The amino acid sequence of the GLP-1 analog dimer synthesized in the present invention is as follows:

[0061]

[0062] Among them, the three-letter amino acids in the amino acid sequence are L-α-amino acids, "|" indicates that Cys-Cys is connected by a disulfide bond, β-Aib is β-aminoisobutyric acid, and Lys[ε-N(γE-palmitic acid)] is lysine modified with γ-glutamic acid-palmitic acid on the ε-amino group of the lysine side chain, and γE represents γ-glutamic acid.

[0063] The structure of Lys[ε-N(γE-palmitic acid)] is shown as follows:

[0064]

[0065] Example 2

[0066] Protective and reparative effects of GLP-1 analog dimer on oxidative damaged retinal cell model

[0067] Oxidative damaged retinal cell model:

[0068] Treat 661W or ARPE-19 cells with 0.05% H2O2 (v / v) for 12 h respectively to establish an oxidative damaged retinal cell model.

[0069] Experimental method for the repair of the oxidative damaged retinal cell model by GLP-1 analog dimer:

[0070] The 661W cells were treated with 0.05% H2O2 (volume percentage) for 12 h respectively to establish an oxidative damage retinal cell model. Then, the GLP-1 analog dimer prepared in Example 1 with final concentrations of 0.125, 0.25 or 0.5 nM was added to the 661W cells after treatment with 0.05% H2O2 (volume percentage), and the cells were cultured for 12 h. At the same time, the 661W cells treated with 0.05% H2O2 (volume percentage) for 12 h were used as the model group, and the 661W cells treated with normal saline for 12 h were used as the normal saline group, and the ROS fluorescence values in each cell were detected.

[0071] Figure 1 The results showed that compared with the normal saline group, the ROS fluorescence value of the model group was significantly increased, indicating that the retinal oxidative damage model was successfully constructed; compared with the oxidative damage retinal cell model, the GLP-1 analog dimer prepared in Example 1 with concentrations of 0.125, 0.25 or 0.5 nM had the effect of preventing retinal cells from generating oxidative stress, and the GLP-1 analog dimer could also significantly reduce the level of reactive oxygen species after cell oxidative damage, so it had an obvious repair effect on human retinal cells.

[0072] Example 3

[0073] Study on the Therapeutic Effect of GLP-1 Analog Dimer on Diabetic Kidney Disease in Mice

[0074] (1) Establishment of a diabetic nephropathy mouse model

[0075] C57B16 / J mice were placed in a SPF-level environment with a standard diet and allowed to drink water freely. All experimental operations were carried out in accordance with the guiding principles of the experimental animal ethics and use system. Five-week-old male C57B16 / J mice were fed a standard diet for 7 days.

[0076] Method for establishing a diabetic mouse model: After feeding the mice with a high-fat diet of 60 kcal% (D12492, Changzhou Mouse One Mouse Two Biotechnology Co., Ltd.) for 4 weeks, streptozotocin (STZ, Sigma Chemical Company, USA) was intraperitoneally injected once at a dose of 75 mg / kg. Three days later, STZ was intraperitoneally injected again at a dose of 50 mg / kg. Three weeks later, diabetic mice were obtained.

[0077] After successful model establishment, the diabetic nephropathy mice were randomly divided into 2 groups, with 10 mice in each group, namely the diabetic nephropathy control group (Group B) and the GLP-1 mimetic dimer group (Group C, abbreviated as the GLP-1 intervention group). At the same time, 10 healthy C57B16 / J male mice were used as the normal control group (Group A). In Group A, NaCl-PB solution (a solution adjusted to pH = 8.0 with disodium hydrogen phosphate) was subcutaneously injected, and the standard diet was maintained; in Groups B and C, the diabetic nephropathy mice were fed a 60 kcal% high-fat diet for 35 days; in Group C, 1.126 nmol of GLP-1 mimetic dimer was subcutaneously injected once every seven days for 35 days, and they were fed a 60 kcal% high-fat diet for 35 days.

[0078] Collection of tissue specimens: 24-hour urine of mice was collected to detect the 24-hour urinary protein of mice in the three groups (Groups A - C). After blood collection from the tail vein of the mice, an incision was made in the middle of the abdomen to expose the kidneys, and bilateral kidneys were taken and stored in a -80°C refrigerator. Blood samples were used to detect glycated hemoglobin, urea nitrogen, and creatinine, and frozen sections of kidney tissues were stained with HE.

[0079] Table 1 Glycated hemoglobin and renal function of mice in different groups (mean ± standard deviation, n = 10)

[0080] Grouping A (Normal control group) B (Diabetic nephropathy control group) C (GLP-1 intervention group) Glycated hemoglobin (mmol / mL) 6.9±2.51 <![CDATA[23.1±5.0 a > <![CDATA[14.6±3.82 b > Blood urea nitrogen mmol / L 18.28±1.85 <![CDATA[37.52±2.75 a > <![CDATA[27.21±1.74 c > Creatinine mmol / L 19.61±2.72 <![CDATA[72.5±5.41 a > <![CDATA[49.2±3.12 b > 24-hour urinary protein (mg / 24h) 16.4±0.49 <![CDATA[96.2±4.66 a > <![CDATA[61.2±3.29 c >

[0081] a indicates P < 0.01 compared with the normal group; b indicates P < 0.01 compared with the nephropathy group; c indicates P < 0.05 compared with the nephropathy group.

[0082] The results in Table 1 showed that compared with the normal control group, the contents of glycated hemoglobin, urea nitrogen, creatinine, and 24-hour urinary protein in the diabetic nephropathy group were significantly increased, indicating that the diabetic nephropathy mice were successfully constructed. Compared with the diabetic nephropathy group, after treatment with GLP-1 mimetic dimer, the contents of glycated hemoglobin, urea nitrogen, creatinine, and 24-hour urinary protein in the mice were significantly decreased, indicating that GLP-1 mimetic dimer can treat diabetic nephropathy.

[0083] Figure 2 The results showed that the sizes and morphologies of glomeruli and renal tubular cells in the normal control group (Group A) were normal, and the basement membrane was intact, while the glomerular cells and renal tubular epithelial cells of the diabetic nephropathy mice were degenerated. The pathological condition of the kidney tissue treated with GLP-1 mimetic dimer was significantly improved compared with Group B. The glomeruli were enlarged, and the morphological structures of glomeruli and renal tubular cells were still clear. It indicates that GLP-1 mimetic dimer can treat diabetic nephropathy.

[0084] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a GLP-1 analog peptide dimer in the preparation of a medicament for preventing and / or treating diabetic complications, characterized in that, The amino acid sequence of the GLP-1 analog dimer is as follows: In the amino acid sequence, the three-letter amino acids are L-α-amino acids. "|" indicates that Cys-Cys is linked by a disulfide bond. β-Aib is β-aminoisobutyric acid, and Lys[ε-N(γE-palmitic acid)] is lysine modified with γ-glutamic acid-palmitic acid on the ε-amino group of the lysine side chain. γE represents γ-glutamic acid.

2. The application according to claim 1, characterized in that, The diabetic complication is diabetic retinopathy and / or diabetic nephropathy.

3. The application according to claim 1, wherein The lesions of the diabetic nephropathy include diabetes and kidney injury.

4. The application according to claim 1, wherein The diabetic retinopathy includes retinal oxidative damage.

5. The application according to any one of claims 1 to 4, characterized in that, The dosage form of the drug is an ointment, an emulsion or a solution.

6. A drug for treating diabetic complications, characterized in that, The drug includes the GLP-1 analog dimer described in claim 1 and a pharmaceutically acceptable excipient.

7. An eye care solution for treating diabetic retinopathy, characterized in that, The eye drops include the GLP-1 analog dimer described in claim 1 and a pharmaceutically acceptable excipient.

8. The eye care lotion according to claim 7, wherein The final concentration of the GLP-1 analog dimer is 0.1 to 0.6 nM, and the pH = 8.

0.

9. The eye care lotion according to claim 7, characterized in that, The excipient includes an osmotic pressure regulator, a pH regulator, an antibacterial agent, a thickening agent or a solubilizer.

10. The eye protection lotion according to claim 9, wherein The mass percentage of the GLP-1 analog dimer in the eye drops is 20% to 80%.

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