WO2011147253A1

WO2011147253A1 - Polypeptides used for preventing and treating diabetes and lipid metabolic disorder

Info

Publication number: WO2011147253A1
Application number: PCT/CN2011/073798
Authority: WO
Inventors: 陈正望; 杜仲夏
Original assignee: 华中科技大学; 江苏威洋正泰生物科技有限公司
Priority date: 2010-05-26
Filing date: 2011-05-09
Publication date: 2011-12-01
Also published as: CN102030820A

Abstract

Provided is a class of polypeptides which is used for preventing and treating diabetes, especially for type II diabetes, and lipid metabolic disorder. The polypeptides are originated from pea. The polypeptides can significantly reduce the level of triglyceride in blood and protect pancreas beta cells. The use of the polypeptides in treating diabetes will not result in hypoglycemia. The polypeptides are enzymolysis-resistant and suitable for the preparation of a medicament for oral administration.

Description

Prevention and treatment of diabetes and peptides with lipid metabolism disorders

[Technical Field]

The present invention belongs to the field of biomedicine and relates to a medicament for preventing and treating diseases of diabetes and lipid metabolism disorders.

【technical background】

Diabetes is caused by genetic factors, immune dysfunction, microbial infections and their toxins, free radical toxins, mental factors and other pathogenic factors that cause islet dysfunction, insulin resistance (IR), etc. A series of metabolic disorders such as sugar, protein, fat, water and electrolytes. Diabetes has become the third largest "health killer" in humans following cancer, cardiovascular and cerebrovascular diseases. As the economy develops, obesity increases, and the human structure ages, the incidence of diabetes continues to grow.

In 1980, the World Health Organization (WH〇) divided diabetes into: insulin-dependent diabetes mellitus (also known as type I), noninsulin-dependent diabetes mellitus (NIDDM, Also known as

Type II) and other types of diabetes. Type I patients often develop from childhood, also known as juvenile onset, which is caused by insufficient secretion of endogenous insulin and requires insulin therapy. Type II patients have more onset after the age of 40, sometimes called the onset of the disease, the patient's hyperglycemia is mainly due to insulin signaling pathway barrier, insulin stimulates the ability of cells to take up glucose. Due to the complex etiology of diabetes, the pathogenesis is not completely clear, which brings great difficulties to treatment.

At present, the treatment of diabetes is mainly administered by injection of insulin, and since insulin is a water-soluble polypeptide, it is easily degraded by enzymes in the gastrointestinal tract, and thus is not suitable for oral administration; Infusion therapy requires daily subcutaneous injection of insulin. Long-term frequent injection not only causes pain to the patient, but also may cause various adverse reactions such as subcutaneous fat contracture. In addition, long-term administration of insulin has the risk of causing hypoglycemia.

[Summary of the Invention]

SUMMARY OF THE INVENTION The object of the present invention is to provide a polypeptide for preventing and treating diabetes and a disorder of lipid metabolism and a preparation method thereof.

The technical solution for realizing the present invention is:

The polypeptide for preventing and treating diabetes and lipid metabolism disorder provided by the present invention has the following sequence

The amino acid sequence shown in any one of 1 to 4:

Sequence 1: ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG Sequence 2: ISCNGVCSPFDIPPCGTPLCRCIPAGLFVGKCRHPYG

Sequence 3: VSCNGVCSPFDIPPCGTPLCRCIPYGLFVGNCRHPYG

Sequence 4: VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRHPSG The polypeptides provided by the present invention are useful for the prevention and treatment of diabetes and disorders of lipid metabolism disorders. A medicament for preventing and treating diabetes and a disorder of a lipid metabolism can be prepared by using the polypeptide of the present invention as an active ingredient and a pharmaceutically acceptable carrier.

The polypeptide extraction process of the invention:

The peas (soybean) or the pressed soybean meal were soaked in 0.5 M acetic acid overnight, beaten, and the slurry was frozen and centrifuged. The supernatant was centrifuged with alginic acid, then eluted with 0.2 M hydrochloric acid, and the adsorption and elution operations were repeated 3 times. Desiccation is carried out for salting out, and the product after salting out is desalted by G-25 column. The desalted liquid is filtered through G-50 column, the filtrate is freeze-dried and concentrated, separated by HPLC, and different separation peak components are taken for pharmacodynamics. In the experiment, the peak component with hypoglycemic effect was finally selected for protein sequencing. The extraction process flow chart is shown in Figure 1. Yield: 11^ Soybean → Beating → Centrifugation → Alginic acid adsorption → Salting out (109, 1 %) → Desalting (6g, 0.6%) → G-50 column filtration (3g, 0.3%) → HPLC (500mg, 0.05% )

Identification of peptides:

1. Collect the separation liquids of each separation peak in HPLC (see Figure 2) and perform pharmacodynamic tests. The results show that the substances in peak 3, peak 4, peak 5, and peak 7 have hypoglycemic effects, and then respectively The substance was subjected to protein sequencing, and the sequencing result was:

Peptide 1 (peak 3) ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG ( 37 ) Peptide 2 (peak 4) ISCNGVCSPFDIPPCGTPLCRCIPAGLFVGKCRHPYG ( 37) Peptide 3 (peak 5) VSCNGVCSPFDIPPCGTPLCRCIPYGLFVGNCRHPYG (37) Peptide 4 (peak 7) VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRHPSG (37) where A-alanine, C- Cysteine, D-aspartic acid, E-glutamic acid, F-phenylalanine, G-glycine, H-histidine, I-isoleucine, K-lysine, L- Leucine, M-methionine, N-asparagine, P-valine, R-arginine, S-serine, T-threonine, V-valine, Y-tyrosine

2. Physical properties

The four compounds are all white in appearance and are easily soluble in polar solvents such as water. They are stable to heat and resistant to protease hydrolysis.

3. Naming

The patent application of the present invention names the four polypeptides together as legaglycins, Chinese name: amylin. Pharmacodynamics experimental data

1. Experimental drugs and experimental animals

Experimental use: The four polypeptides provided by the present invention were prepared by using physiological saline to prepare an oral liquid having a concentration of 2 mg/ml, which was used for experimental intragastric administration. Experimental animals: The animals in this experiment were all BABC mice, 10 in each group, randomized.

2. Experimental methods and results

1) Therapeutic effect of type II diabetes was established to establish an animal model of type 2 diabetes: 100 mg/kg - sub-dose injection of streptozotocin I (STZ), and fed with high-sugar and high-fat words to establish type II diabetes Mouse animal model. Diabetic mice were randomly divided into a peptide gavage group, a metformin (positive control) group, a model control group, and a blank control group, with 10 rats in each group. The drug was administered intragastrically every other day for 4 weeks, and the dose was 20 ug/g, and the blood glucose of the mice was determined weekly. The results are shown in Table 1. Table 1: Changes in blood glucose in mice

Blood sugar level (mmol. L- ¹ )

1 week 2 weeks 3 weeks 4 weeks Peptide 1 7.1 ± 0.6 7.3 ± 0 · 5 7.0 ± 0.4 6 · 9 ± 0 · 3

Peptide 2 7·2±0· 2 7·4±0· 6 7·2±0· 4 7.0±0.2

Peptide 3 7·1±0· 5 7.3±0· 3 7.0±0.2 6·9±0· 2

Peptide 4 7·2±0· 3 7·4±0· 5 7.2±0· 4 7.0±0· 3

Metformin 7·2±0· 3 7·4±0· 4 7·3±0· 5 7·2±0· 4

Blank control group 5.6±0.4 5.9±0· 2 5.7±0· 3 5.4±0· 4

Model control group 8.6±0.9 10.3±0·3 11.1±0·8 11.1±0· 5

Conclusion: The four peptides have obvious therapeutic effects on π-type diabetes, which is equivalent to or slightly better than dimethyl hydrazine.

2) Anti-sugar test after oral administration (OGTT) results In the hypoglycemic test, after 1 hour of the last administration, 2.5 g/kg glucose was administered to each mouse by intragastric administration, and the blood glucose level of the mice was measured at 0 h, 0.5 h, and 2 h. The results are shown in Table 2. Table 2 Sugar tolerance after oral administration of peptide (0GTT) Experimental results

Blood sugar level (mmol. L ^_1 )

Oh 0.5h 2h

Peptide 1 6·3±0· 3 14.1±0· 2 6.5±0· 4

Peptide 2 6·4±0· 2 14.6±0· 3 6.7±0· 3

Peptide 3 6·3±0· 4 14.0±0· 4 6.4±0· 4 Peptide 4 6.3 ± 0.2 14.1 ± 0.2 6.5 ± 0.2

Metformin 6.5±0.4 14.9±0.2 7.4±0.1

Model control group 9.9 ±0.6 22.1±1· 5 11.3±0· 7

Conclusion: The hypoglycemic effects of the four peptides are hyperglycemic and therefore safer.

3) Pepsin and trypsin digestion test

Four polypeptide samples were hydrolyzed by gastrointestinal enzymes for 12 h, and their degradation was examined by HPLC. The results showed that these were resistant to gastrointestinal enzymatic hydrolysis, and the results are shown in Fig. 3. No small fragments after hydrolysis were observed in the HPLC chart, indicating that the polypeptide was not hydrolyzed by pepsin. Conclusion: The four peptides can be resistant to gastric protein hydrolysis and can be developed into oral preparations.

4) Lipid lowering effect

The mice after the above glucose tolerance test were taken from the eyeballs, and then sacrificed, and the serum was subjected to four tests of blood lipids. The results showed that after administration, the total cholesterol in the peptide group and the metformin group was smaller than that in the model group, but there was no significant difference (P>0.05). The triglyceride level in the peptide group and the metformin group was significantly lower than that in the model group (P>0.05). <0.05) The results are shown in Table 3. Table 3 Changes in total cholesterol and triglycerides in BABC mice after administration

Total cholesterol (mg/dl) triglyceride (mg/dl)

Peptide 1 98±0.2 45±0.3

Peptide 2 99±0.3 45±0.2

Peptide 3 98±0.1 44±0.2

Peptide 4 97±0.3 44±0.3

Metformin 100±0.2 50±0.4

Model control group 118±0.4 1223±0.2

Conclusion: Four peptides have the ability to significantly reduce blood triglyceride levels and can be developed as a lipid-lowering drug.

5) Safety evaluation

After one year of observation, the test animals were observed, no adverse drug reactions were found, no side effects were observed, no toxicity, carcinogenicity, teratogenicity, allergic reaction, and the medicinal safety of the four polypeptides was demonstrated. 6) Protection of islet cells

The rat pancreatic β cells were isolated, primary cultured, and the polypeptide of the present invention was added to the culture medium. The results showed that the cells to which the polypeptide was added grew vigorously, and the survival time was four days longer than that of the control cells without the polypeptide, as shown in Fig. 4. It is indicated that the polypeptide of the present invention has a protective effect on pancreatic β cells, and is suitable for administration to diabetic patients.

7) Improve and promote the secretion of pancreatic β-cells Through patch-clamp experiments, we found that peptides can inhibit the _sputum channels of pancreatic β-cells and induce extracellular calcium influx like insulin, thereby improving and promoting the secretion of pancreatic β-cells. The result is shown in Figure 5. Patch-clamp experiments confirmed that the polypeptide of the present invention induces extracellular calcium influx with Insulin-like to promote insulin secretion by pancreatic β cells. This result indicates that these polypeptides can promote the growth and reproduction of pancreatic cells and the recovery of physiological functions in vivo, so that the pancreas that loses normal function can be repaired.

Based on the above pharmacodynamic results, it is indicated that the four polypeptides provided by the present invention are indeed a safe and effective hypoglycemic and lipid-lowering drug, and the four polypeptides are equivalent in efficacy. Due to its resistance to enzymatic hydrolysis, it is suitable for oral preparation and can be used for oral administration. The hypoglycemic effect is obvious. The hypoglycemic effect occurs on the basis of hyperglycemia. It depends on glucose to lower the concentration of high blood sugar and does not cause hypoglycemia. It has good safety and has a certain protective effect on pancreatic β cells. In addition, pharmacological experiments have also found that the series of peptides have a significant effect on lowering blood triglycerides and regulating blood lipids. Therefore, the polypeptide of the present invention can be used as a medicament for the treatment and prevention of diseases of diabetes and disorders of lipid metabolism.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an extraction process diagram of four polypeptides;

Figure 2 is a HPLC chromatogram in the process flow of the present invention;

Figure 3 HPLC chromatogram of polypeptide after hydrolysis by pepsin;

Figure 4 Isolation of pancreatic β-cells from rats, primary culture, addition of polypeptides to the medium, protection of pancreatic β-cells by the peptides (arrows represent viable beta cells), and the results showed that the cells added with the polypeptide of the present invention grew vigorously and survived. The time was up to four days longer than the control cells without the addition of the polypeptide.

Figure 5 Patch clamp experiment of the polypeptide of the invention. 【detailed description】

Example 1

Preparation of the polypeptide of the present invention

The peas were soaked in 0.5 M acetic acid overnight, beaten, and the slurry was frozen and centrifuged. The supernatant was centrifuged with alginic acid, then eluted with 0.2 M hydrochloric acid, and the adsorption and elution operations were repeated three times. The eluate was subjected to salting out and salting out. After the product is desalted by G-25 column, the desalted liquid is filtered through G-50 column, the filtrate is freeze-dried and concentrated, and separated by HPLC (see Figure 2), taking peak 3, peak 4, peak 5, peak 7 The substance, that is, the polypeptide of the present invention. The obtained polypeptide was subjected to protein sequencing, and the results were as follows:

Peptide 1 (peak 3) ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG ( 37 ) Peptide 2 (peak 4) ISCNGVCSPFDIPPCGTPLCRCIPAGLFVGKCRHPYG ( 37) Peptide 3 (peak 5) VSCNGVCSPFDIPPCGTPLCRCIPYGLFVGNCRHPYG (37) Peptide 4 (peak 7) VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRHPSG (37)

Claims

Rights request

A polypeptide for preventing and/or treating diabetes and/or a disorder of lipid metabolism, which comprises the amino acid sequence shown in any one of the following sequences 1 to 4:

Sequence 1 : ASCNGVCSPFEMPPCGSSACRCIPVGLVVGYCRHPSG

Sequence 2: ISCNGVCSPFDIPPCGTPLCRCIPAGLFVGKCRHPYG

Sequence 3: VSCNGVCSPFDIPPCGTPLCRCIPYGLFVGNCRHPYG

Sequence 4: VSCNGVCSPFEMPPCGSSACRCIPYGLVVGNCRHPSG

2. Use of the polypeptide of claim 1 for the manufacture of a medicament for the prevention and/or treatment of diabetes and/or lipid metabolism disorders.

An agent for preventing and/or treating diabetes and/or a disorder of lipid metabolism, which comprises an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable carrier.