CN112316109A

CN112316109A - Application of polypeptide AT03 in medicine for treating diabetic nephropathy

Info

Publication number: CN112316109A
Application number: CN202011398008.XA
Authority: CN
Inventors: 张斌智; 刘琦
Original assignee: Shenzhen Turier Biotech Co ltd
Current assignee: Shenzhen Turier Biotech Co ltd
Priority date: 2020-12-02
Filing date: 2020-12-02
Publication date: 2021-02-05

Abstract

The invention discloses an application of polypeptide AT03 in a medicine for treating diabetic nephropathy, and belongs to the technical field of biological medicines. The amino acid sequence of the polypeptide AT03 is as follows: Val-His-Val-Val-R₁(ii) a Wherein R is₁＝‑NH₂. Research shows that the invention can obviously reduce blood sugar and improve kidney damage, and has application prospect in developing drugs for treating diabetic nephropathy.

Description

Application of polypeptide AT03 in medicine for treating diabetic nephropathy

Technical Field

The invention relates to application of a polypeptide, in particular to application of a polypeptide AT03 in a medicine for treating diabetic nephropathy, and belongs to the technical field of biochemistry.

Background

Diabetes is a chronic, systemic metabolic disease, has become one of chronic diseases of global concern, and is one of the leading causes of death worldwide. Diabetic nephropathy is one of the most common and serious microvascular complications of diabetes, and is a major risk factor for patients to progress to end-stage renal failure (zheng wen, panfeokang, liu dong wei, liu zhu. The main manifestations of diabetic nephropathy are the onset of early glomerular hyperfiltration, followed by the progression from microalbuminuria to macroalbuminuria, followed by a decrease in glomerular filtration rate. Diabetic nephropathy accounts for about 40% of diabetics, is the leading cause of chronic kidney disease worldwide, and is also the leading cause of end-stage renal disease (Liuyigai, Luoshuoming, Deng Min, Zhou Zhiguang. progress in diagnostics and treatment of diabetes neuropathology. Chin J Arterioscler, 2020, 1007 and 3949). The pathogenesis of the disease is mainly glomerular capillary vessel lesion, mainly the dilatation of the small artery entering the glomerulus, the increase of the pressure in the glomerulus and the increase of filtration rate caused by the glomerular capillary vessel lesion, and the clinical characteristics are that a large amount of continuous proteinuria is used. By the end stage, the nephron has a large number of necrotic locks, the glomerular filtration rate is reduced, the urine volume is reduced, proteinuria disappears, and uremia appears, and at the moment, the patient can only rely on kidney transplantation and hemodialysis to maintain life. A huge medical burden is generated to the patients and family members, and the quality of life of the patients is also seriously reduced. Research also shows that the expression of inflammatory factors such as NF-B, TNF-alpha, IL-6 and the like is higher in the diabetic nephropathy population than in the normal population. It is considered that it is possible that inflammatory factors form immune complexes to deposit on vascular endothelium, and intraglomerular vessels are stimulated by inflammatory factors for a long time, and repeated injury repair leads to irreversible injury of glomeruli (zhou wuhao, tian qiao, sun jun, yi, wang yan, wei yi. At present, the treatment of the diabetic nephropathy mainly comprises the treatment of reducing blood sugar and blood pressure, and no special medicine for treating the diabetic nephropathy exists.

An antidiabetic polypeptide is a natural polypeptide that is encrypted and inactivated in the parent protein and must function by enzymatic hydrolysis during digestion in the gastrointestinal tract in vivo or during food processing. The antidiabetic activity can be achieved by inhibiting glucagon secretion, promoting insulin secretion, enhancing beta cell function, and thus effectively inhibiting the hyperglycemic effect in mice (Shibu, Marthandam, Asokan, et al. short Tetra-peptide from source-protein carbohydrates in H9c2 cells and ICR semiconductor. journal of Food Biochemistry, 2018.). The polypeptide AT03 is a soybean protein hydrolysate antidiabetic polypeptide, and has high antidiabetic activity.

Because the pathogenic mechanism and the treatment principle of diabetes and diabetic nephropathy are different, no medicine which can treat the two diseases simultaneously is found in the prior art, and a good treatment effect can be achieved. The inventor of the present invention is dedicated to research on the polypeptide AT03, develops a new application of the polypeptide AT03, provides an application of the polypeptide AT03 in a medicament for treating diabetic nephropathy, and provides a medicament for effectively treating diabetic nephropathy.

Disclosure of Invention

The invention aims to provide a new application of polypeptide AT03, provides an application of polypeptide AT03 in a medicine for treating diabetic nephropathy, and provides a medicine for effectively treating diabetic nephropathy.

The invention provides an application of a polypeptide AT03 in a medicine for treating diabetic nephropathy, wherein the amino acid sequence of the polypeptide AT03 is as follows: Val-His-Val-Val-R₁(ii) a Wherein R is₁＝-NH₂。

Val-His-Val-Val-R of the present invention₁Is a degradation product of soybean protein.

The invention also provides a pharmaceutical composition for treating diabetic nephropathy, which takes the polypeptide AT03 as an active ingredient.

The pharmaceutical composition of the invention also comprises pharmaceutically acceptable auxiliary materials.

The auxiliary materials of the invention are liquid auxiliary materials, solid auxiliary materials or semisolid auxiliary materials.

It will be appreciated by those skilled in the art that the pharmaceutical composition of the present invention comprises AT least one effective amount of the present polypeptide AT03 and AT least one pharmaceutically acceptable pharmaceutical carrier or adjuvant. The pharmaceutical composition of the present invention is suitable for various administration modes, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, and the like. The pharmaceutical composition of the present invention may be formulated into various suitable dosage forms depending on the administration mode employed.

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

The pharmaceutical compositions of the present invention may be formulated as solutions or lyophilized powders for parenteral administration, which powders may be reconstituted with a suitable solvent or other pharmaceutically acceptable carrier prior to use, typically in the form of buffers, isotonic and aqueous solutions.

The invention confirms the pharmacological activity of the polypeptide AT03 in the treatment of diabetic nephropathy through a series of animal experiments, confirms that the polypeptide AT03 can be applied to diabetes and diabetic nephropathy, provides a medicament for effectively treating diabetic nephropathy, and also provides an application of the polypeptide AT03 in the medicament for treating diabetic nephropathy. The polypeptide AT03 can obviously reduce blood sugar and improve kidney damage, and has an application prospect in developing drugs for treating diabetic nephropathy.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: is a comparison graph of the blood sugar reducing effect of 6 groups of mice.

FIG. 2: OGTT test control plots and OGTT-AUC histograms for 6 groups of mice (. about.. indicates within 99% confidence (p <0.0001) compared to control).

FIG. 3: the ITT test control plots and ITT-AUC histograms for 6 groups of mice (. x.: indicate within 99% confidence (p <0.0001) compared to control).

FIG. 4: the kidney weight comparison graph and the kidney weight/body weight comparison graph of 6 mice are obtained.

FIG. 5: the comparative figure for renal function evaluation of 6 groups of mice was obtained.

FIG. 6: h & E stained pathological section images of the kidney of 6 groups of mice.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. Unless otherwise indicated, reagents or equipment used are commercially available.

The results of the experiments of the present invention were analyzed using GraphPad Prism software, and the data are expressed as Mean ± standard error (Mean ± SEM) and evaluated by t-test. The model group was compared with the control group and denoted by P <0.05, # P <0.01, # P <0.001, and the administration group was compared with the model group and denoted by # P <0.05, # P <0.01, # P < 0.001. And analyzing the Western blot result by Tanon image analysis software.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The mice in the following examples were all healthy male C57BL/6J mice, age 6-8 weeks.

The compounds in the following examples were all formulated using sterile PBS as solvent.

Example 1 preparation of the polypeptide AT03

The amino acid sequence of the polypeptide AT03 is: Val-His-Val-Val-R₁(ii) a Wherein R is₁＝-NH₂. In this embodiment, the anti-diabetic polypeptide compound AT03 is synthesized from the carboxyl terminus to the amino terminus by Fmoc solid phase polypeptide synthesis, and the amino acids of the polypeptide AT03 are sequentially linked according to the aforementioned amino acid sequence.

(1) Polypeptide synthesis

The preparation condition of the invention adopts MBHAR (Amide-MBHA-Resin protected by Amide) or Wang Resin, and the Fmoc protective group is removed for synthesis by a method of activating lipid.

The indene detection reagent comprises the following components:

reagent 1: 20g phenol/5 ml ethanol;

reagent 2: 0.050.001M KCN (water)/2.5 ml pyridine;

reagent 3: 0.5g ninhydrin in 10ml ethanol;

the indene detection reagent is used in a ratio of reagent 1: reagent 2: reagent 3 ═ l:2: l (dropwise), and heated in boiling water for 3-10 min.

The preparation method of the polypeptide AT03 specifically comprises the following steps:

A. treating resin: weighing a certain amount of MBHA resin (1% crosslinking degree, 200-400 meshes, substitution value 0.5mmol/g) in a synthesizer, adding DCM, stirring for 30min, draining, washing with DMF for 4 times (2 min each time), picking a small amount of resin for indene detection, and if the color of the resin is not changed, indicating normal, using the resin.

B. Removing Fmoc protection: 20% piperidine (dissolved in DMF) was washed 4 times for 2min each, then 4 times for 2min each with DMF, after the 3 rd wash an indene test showing bluish purple color indicating that Fmoc had been removed and the next amino acid was accessible.

C. Condensation reaction: amino acid, HOBT and HBTU are sequentially added into a beaker, DMF is added to dissolve the amino acid, HOBT and HBTU, the DMF is added into the beaker as small as possible, DIEA is added to start reaction, and then the mixture is immediately added into a synthesizer, and then a small amount of DMF is used for washing the beaker and the wall of the synthesizer (the feeding amount is the resin substitution value (mmol/g). times.m (resin mass g). times.M (molecular weight of peptide g/mol). times.more than a few times), 3 times more than the amino acid, HBTU and HOBT are added, DIEA is liquid alkali to perform an activated ester reaction, and 6 times more than the amino acid, HBTU and HOBT are added, so that each step. The whole reaction system is protected by argon. The reaction was stirred for 1h, drained and washed 3 times with DMF for 2min each time with no change in indene detection, indicating that the amino acid had been attached to the resin.

D. Step B, C is repeated for each amino acid, until the peptide is completed.

E. Polypeptide cleavage:

a. after the amino acids of the polypeptide AT03 are completely connected, the F-moc is removed by 20% piperidine, washing is carried out for 4 times, each time is 2min, then washing is carried out for 4 times, each time is 2min, after indene detection and color development, washing is carried out for 2 times, each time is 3min, washing is carried out for 1 time by methanol, each time is 3min, washing is carried out for 1 time by DCM, each time is 3min, and washing is carried out for 2 times by methanol, each time is 3 min. After the treatment, the stirring rod is removed, the synthesizer is sealed by a rubber plug, and the resin is thoroughly pumped by a vacuum pump for at least more than 2 hours until the resin is completely dried and is powdery.

b. The cleavage agent (TFA: Tis: water: 95:2.5:2.5(V/V/V)) was added to the suction-dried synthesizer and reacted for 3h, stirring for 1min every 20min, collecting the cleavage agent with a round-bottom flask and washing 2 times with TFA, 5min each (5 ml/time), and the collected TFA was combined with the cleavage agent collected previously.

c. The resulting cleavage agent was then dried under reduced pressure on a rotary evaporator, and the pre-cooled ether (50-80ml) was added to a round-bottom flask, precipitated by vigorous shaking, and allowed to stand. And (4) after the white solid is precipitated to the bottom, sucking the supernatant by using a dropper, adding the supernatant into a separating funnel, adding water with the same volume, shaking (deflating the middle for several times), and standing. At this time, the liquid in the separatory funnel was separated into layers, and the lower aqueous phase was poured into a round-bottomed flask to dissolve the precipitate (if not dissolved, acetic acid was added to adjust the solubility).

d. The round bottom flask was gently shaken (to prevent emulsification) to allow complete dissolution, then poured into a separatory funnel and the aqueous phase was collected by flowing into a beaker and washing multiple times with each wash followed by collection of the aqueous phase.

e. Sealing the beaker with a preservative film, inserting the beaker into a hole, marking the beaker, storing the beaker overnight at-80 ℃, and then performing vacuum freeze drying treatment.

(2) Crude peptide desalination

Since the crude peptide contains byproducts, salts and some other impurities generated during the reaction process, the crude peptide is desalted to primarily remove the impurities. Firstly, weighing a certain amount of sephadex G-25 (about 50-100 meshes), adding 5-10 times of deionized water, heating and swelling for 3h, and filling into a column. After natural sedimentation is completed, balancing the column with 10% acetic acid for about 2h, then loading the sample, eluting with 10% acetic acid, collecting the solution with an absorption peak at 220nm of the nucleic acid protein ultraviolet detector, sealing the beaker with a preservative film, inserting the beaker into a hole, marking the beaker, storing the beaker overnight at-80 ℃, and then carrying out vacuum freeze-drying treatment.

(3) Purification and purity analysis of desalted product

Purification by high performance liquid chromatography: the preparative column was a C18 reverse phase preparative column of specification xbridgetbeh 130 Prep C18(10 μm, 19 × 250mm), elution system 20% -80% acetonitrile/water/0.1% trifluoroacetic acid for 60min, flow rate: 8ml/min, collect the main peak. After vacuum freeze-drying, mass spectrometry (ESI-MS) was performed for verification.

Purity analysis of the purified product: the analytical column was a C18 reverse phase preparative column, specification xbridgetbeh 130 Prep C18(10 μm, 4.6 × 250mm), elution system 10% -90% acetonitrile/water/0.1% trifluoroacetic acid for 30min, flow rate: 1ml/min, and calculating the purity according to the integration of a 220nm chromatogram.

The synthesized polypeptides were purified by high performance liquid chromatography (purity > 95%) and characterized by high resolution mass spectrometry.

Based on the above synthesis steps, the polypeptide AT03 of the invention is synthesized: VHVV.

Example 2 study of the therapeutic Effect of the polypeptide AT03 on diabetic and diabetic nephropathy mice

2.1 diabetic polypeptide Compound AT03 and the diabetes-ameliorating action of the Positive drug on diabetic mice

Obtaining db/db mouse diabetes model (purchased from Nanjing university-Nanjing biomedical research institute, mice about 12 weeks, and measuring blood sugar and body weight to ensure smooth proceeding of subsequent experiments), randomly dividing model mice into 5 groups (normal saline group, AT03 low dose group, AT03 high dose group, MED group (MEDI0382, a GLP-1/GCG receptor dual agonist) and SAR group (SAR425899, a GLP-1/GCG receptor dual agonist)), each group having 6 mice each having no difference in basic body weight and blood sugar, each group of mice was subcutaneously injected with the compounds AT03 (60. mu.g/kg), AT03 (120. mu.g/kg), MED (120. mu.g/kg), SAR (120. mu.g/kg), and physiological saline, respectively, daily (DN group: littermate normal mice; DC group: db/db mouse control group). After the mice were dosed daily, they were fasted every other day for 6h to measure blood glucose and body weight, and water intake and food intake were measured once a week. OGTT is measured at week 4, insulin tolerance (ITT) is measured at week 5, mouse metabolism conditions are measured by using mouse metabolism cages at weeks 6-7, materials are taken at week 8, and various serological indexes and pathological indexes are measured.

2.1.1 hypoglycemic action of polypeptide AT03 on diabetic mouse

A model of type 2 diabetes characterized by: obesity, insulin resistance, hyperglycemia, dyslipidemia, liver steatorvacuole-like degeneration, and the like.

As can be seen from the comparison of the blood sugar-reducing effects of the 6 groups of mice in figure 1, the blood sugar-reducing effect is as follows: SAR (120ug/kg) > AT03(120ug/kg) > AT03(60ug/kg) > MED (120 ug/kg); SAR (120ug/kg) and MED (120ug/kg) dropped to essentially normal levels on day 4, AT03(60ug/kg) and AT03(120ug/kg) dropped to essentially normal levels on day 8.

2.1.2 insulin-ameliorating Effect of the polypeptide AT03 on diabetic mice

Oral Glucose Tolerance Test (OGTT) and insulin resistance test (ITT) were performed AT 4 and 5 weeks of polypeptide AT03 and positive drug treatment in diabetic mice, respectively. The test results are shown in fig. 2 and 3.

As can be seen from the results of the OGTT test of the 6 groups of mice in FIG. 2, the polypeptide AT03 exhibited certain sugar tolerance improving effects AT the use concentrations of 60ug/kg and 120ug/kg, and exhibited concentration dependence, wherein AT03(120ug/kg) exhibited the same effect as SAR (120 ug/kg).

As can be seen from the results of the ITT test of the 6 groups of mice in FIG. 3, the polypeptide AT03 shows certain insulin resistance improving effect AT the use concentration of 60ug/kg and 120ug/kg, and shows concentration dependence, wherein AT03(60ug/kg) shows the same effect as MED, and AT03(120ug/kg) and SAR (120ug/kg) have the same effect.

2.2 therapeutic Effect of polypeptide AT03 and Positive drugs on diabetic nephropathy in diabetic mice

2.2.1 amelioration of Kidney atrophy in db/db model mice by the polypeptide AT03

As can be seen from the kidney weight comparison of 6 groups of mice in FIG. 4, AT03, MED and SAR all have the effect of improving kidney hypertrophy, and AT03(120ug/kg) and MED have more remarkable effects. From the results of the kidney weight/body weight ratio of 6 mice, it was shown that the diabetes model had a certain relative atrophy of the kidney, while AT03 had the function of improving the relative atrophy of the kidney.

2.2.2 improvement of kidney function in db/db model mice by the polypeptide AT03

As can be seen from the comparative graph of the renal function evaluation of the 6 groups of mice in the figure 5, both AT03(120ug/kg) and SAR (120ug/kg) can reduce the Cre (serum creatinine) content in the serum, and the effect of SAR is more obvious. In addition, AT03(60ug/kg), AT03(120ug/kg), MED (120ug/kg) and SAR (120ug/kg) all can reduce the content of Bun (urea nitrogen) in serum, which indicates that these polypeptides all have the function of improving renal function.

2.2.3 improvement of kidney fibrotic lesions in db/db model mice by the polypeptide AT03

As can be seen from FIG. 6, compared to DN group, DC group mice showed glomerular and tubulointerstitial fibrosis, and glomerular adhesion, AT03(60ug/kg), AT03(120ug/kg), MED (120ug/kg), SAR (120ug/kg) all inhibited renal fibrosis in diabetic mice, and improved renal damage.

In conclusion, the polypeptide AT03 has the effects of improving renal atrophy, renal function and renal fibrosis, and the polypeptide AT03 has a good treatment effect in the medicine for treating diabetic nephropathy. The polypeptide AT03 can obviously reduce blood sugar and improve kidney damage, and has an application prospect in developing drugs for treating diabetic nephropathy.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore intended that all such changes and modifications as fall within the true spirit and scope of the invention as defined by the appended claims be interpreted in accordance with the breadth to which they are fairly, if not explicitly recited herein.

Claims

1. The application of the polypeptide AT03 in the medicine for treating diabetic nephropathy is characterized in that the amino acid sequence of the polypeptide AT03 is as follows: Val-His-Val-Val-R₁；

Wherein R is₁＝-NH₂。

2. The use according to claim 1, wherein said Val-His-Val-R₁Is a degradation product of soybean protein.

3. A pharmaceutical composition for treating diabetic nephropathy, which comprises the polypeptide AT03 of any one of claims 1 or 2 as an active ingredient.

4. The pharmaceutical composition of claim 3, further comprising a pharmaceutically acceptable excipient.

5. The pharmaceutical composition of claim 4, wherein the excipient is a liquid excipient, a solid excipient, or a semi-solid excipient.

6. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is in a pharmaceutically acceptable dosage form.

7. The pharmaceutical composition of claim 6, wherein the dosage form is at least one of a tablet, a capsule, a sugar-coated tablet, a granule, an oral solution, a syrup, an ointment and patch for skin surface, an aerosol, a nasal spray, and a sterile solution for injection.