WO2015180634A1 - Long-acting enterocrinin polypeptide analogue for treating type 2 diabetes and uses thereof - Google Patents

Abstract

Provided are a GLP-1 analogue and uses thereof. The GLP-1 analogue has the following general formula: HAEGTFTSDVSSYLEGQAAKEFIAWLX₂₇KX₂₉RX₃₁n₁YCZ, wherein X₂₇ is valine or cysteine, X₂₉ is glycine or cysteine, X₃₁ is glycine or cysteine, and at least one of X₂₇, X₂₉ and X₃₁ is cysteine; n₁Y represents n₁ Ys, n₁=1-10, and Y is glycine, alanine or valine; Z represents amino saturated fatty acid; and two cysteines contained in the general formula both form disulfide bonds. The GLP-1 analogue of the present invention can effectively increase the blood half-life period of GLP-1 and can be used for treating diabetes and adiposis.

Description

Long-acting enterosteroid polypeptide analogues for the treatment of type 2 diabetes and their applications Technical field

The present invention relates to the field of pharmaceutical technology, and in particular to a GLP-1 analogue and its use in the manufacture of a medicament for the treatment of diabetes, obesity and/or diabetes, obesity-related diseases and prevention of obesity.

Background technique

GLP-1 (glucagon-like peptide-1, hereinafter referred to as GLP-1) is a polypeptide consisting of 37 amino acids mainly secreted by small intestinal L cells, and its active form is GLP-1 (7-37). OH and GLP-1 (7-36) NH2 (Mojsov S, J Clin Invest. 1987 Feb; 79(2): 616-9). GLP-1 significantly reduces blood sugar after eating, stimulates insulin production, and also has a certain weight loss effect, and does not cause hypoglycemia (Drucker D J, Diabetes. 1998 Feb; 47 (2): 159-69 ). Recent studies have also shown that GLP-1 has pancreatic regeneration (Drucker D J, 2003 Dec; 144(12): 5145-8). In addition, since GLP-1 is a fully humanized polypeptide, it has a large safety advantage as a clinical drug. However, the serum half-life of GLP-1 (7-37) is only 3-5 minutes, and multiple injections per day are very inconvenient in clinical use.

At present, many studies have used GLP-1 analogue fusion protein technology to solve the retention time of GLP-1 analogues in vivo. However, the existing technology has a great distance from the ideal clinical target, and generally has not reached the clinical stage. Standard, Liraglutide produced by Novo Norisk recently is a GLP-1 analogue, which has been modified by palmitic acid for GLP-1 and marketed in the United States in 2009. However, liraglutide also has a short half-life problem, and its dosage form still needs to be injected daily.

Summary of the invention

The object of the present invention is to provide an analogue of GLP-1 having a longer half-life than the technical drawbacks existing in the prior art.

Another object of the invention is to provide an application of a GLP-1 analogue.

The technical solution adopted for achieving the object of the present invention is:

(1) GLP-1 analogues:

The GLP-1 analogue of the invention has the following general formula A:

HAEGTFTSDVSSYLEGQAAKEFIAWLX ₂₇ KX ₂₉ RX ₃₁ n ₁ YCZ,

In the above formula A, X ₂₇ is valine or cysteine, X ₂₉ is glycine or cysteine, X ₃₁ glycine or cysteine, and at least one of X ₂₇ , X ₂₉ and X ₃₁ Is cysteine; n ₁ X ₁ represents n ₁ Y, n ₁ = 1-10, and Y is glycine, alanine or valine; Z represents an amino-saturated fatty acid; Both cysteines form disulfide bonds.

In the general formula A, preferably n ₁ = 1-5, and Y is glycine, alanine or valine; Z is preferably aminobutyric acid, aminovaleric acid, aminocaproic acid, aminoheptanoic acid, aminooctanoic acid, Any of aminoguanidine and aminodecanoic acid.

The GLP-1 analog having the general formula A is as follows:

SEQ ID NO 1: HAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGGCZ;

SEQ ID NO 2: HAEGTFTSDVSSYLEGQAAK EFIAWLVKCRGAACZ;

SEQ ID NO 3: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRCVVVVVC Z.

(2) Application of GLP-1 analogues:

The above-described GLP-1 analogue is useful in the preparation of a medicament for the treatment of diabetes, obesity and/or diabetes, obesity-related diseases, and prevention of obesity.

The GLP-1 analog of the present invention can be used in the preparation of a therapeutic drug for diabetes. Specifically, the composition of the present invention can be administered in the form of intravenous, intramuscular or subcutaneous injection. While the dosage varies depending on the subject, mode of administration, symptoms and other factors, the GLP-1 analogs of the invention are effective over a relatively wide dosage range. In the treatment of adults, the dose ranges from 5 μg/person to -1 mg/person, once daily or once every few days. The actual dose should be determined by the doctor according to the relevant circumstances, including the physical condition of the subject, the route of administration, age, weight, individual response of the patient to the drug, the severity of the patient's symptoms, etc., so the above dose range is The scope of the invention is not limited in any way.

(3) Pharmaceutical composition containing GLP-1:

The pharmaceutical composition of the present invention containing the above GLP-1 analogue, including the above GLP-1 analogue And one or more pharmaceutically acceptable excipients. These excipients include: water-soluble fillers, pH adjusters, stabilizers, water for injection, osmotic pressure regulators, and the like.

The water-soluble filler adjuvant according to the present invention is one or more selected from the group consisting of mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose, galactose and the like.

The pH adjusting agent is one or more selected from the group consisting of non-volatile acids such as capric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, and potassium hydroxide, sodium hydroxide or potassium hydroxide or ammonium hydroxide, and sodium carbonate. Or physiologically acceptable organic or inorganic acids and bases and salts such as potassium carbonate or ammonium carbonate, sodium hydrogencarbonate or potassium hydrogencarbonate or ammonium hydrogencarbonate.

The stabilizer is one or more selected from the group consisting of EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium hydrogencarbonate, sodium carbonate, arginine, glutamic acid, polyethylene. Alcohol 6000, polyethylene glycol 4000, sodium lauryl sulfate or trishydroxymethylaminomethane. Preferred are sodium metabisulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000, and trishydroxymethylaminomethane.

The osmotic pressure adjusting agent is one or a combination of two of sodium chloride and potassium chloride.

The pharmaceutical composition of the present invention can be administered by intramuscular, intravenous, subcutaneous injection or the like, and the pharmaceutical composition of the present invention is preferably an injection such as a lyophilized powder or a solution for injection.

Preparation method of freeze-dried preparation of lyophilized powder injection: take appropriate amount of the above GLP-1 analog solution, add water-soluble filler, stabilizer, osmotic pressure regulator, etc., add appropriate amount of water for injection, adjust pH to 4-8 to make it Dissolve, dilute to the appropriate concentration with water, add 0.1-0.5% activated carbon, stir at 0-10 °C for 10-20 minutes, decarburize, filter and sterilize by microporous membrane, the filtrate is divided, and the method is freeze-drying. A white loose mass is obtained, which is sealed, and each specification contains a GLP-1 analogue at 5 μg to 1 mg.

Preparation method of solution injection: take the above GLP-1 analog solution or lyophilized powder, add water-soluble filler, stabilizer, osmotic pressure regulator, etc., add appropriate amount of water for injection, adjust pH to 4-8 to dissolve Dilute to the appropriate concentration with water, add 0.1-0.5% activated carbon, stir at 0-10 °C for 10-20 minutes, decarburize, filter and sterilize by microporous membrane filtration, the filtrate is divided and sealed, and each specification is obtained. Contains GLP-1 analog at 5 μg to 1 mg.

Compared with the prior art, the beneficial effects of the present invention are:

The GLP-1 analog of the present invention overcomes the problem of short half-life of GLP-1, and the GLP-1 class provided The half-life of the analogy in the body can reach 72-96 hours or more, which is significantly longer than the half-life (half-life of only 3-5 minutes) of GLP-1 administered alone, which greatly facilitates its clinical promotion and application.

DRAWINGS

Figure 1 is a schematic view showing the structure of the general formula A;

Figure 2 shows the hypoglycemic test of the GLP-1 analogue (SEQ ID NO 1-3) in Example 2;

Figure 3 is a graph showing the stability of the GLP-1 analogue in Example 3 in human serum.

detailed description

The invention will be further described in detail below with reference to the drawings and specific embodiments.

The GLP-1 analogue of the invention has the following general formula A:

HAEGTFTSDVSSYLEGQAAKEFIAWLX ₂₇ KX ₂₉ RX ₃₁ n ₁ YCZ,

In the above formula A, X ₂₇ proline or cysteine, X ₂₉ is glycine or cysteine, X ₃₁ glycine or cysteine, and at least one of X ₂₇ , X ₂₉ and X ₃₁ is Cysteine; n ₁ Y means n ₁ Y, n ₁ = 1-10, and Y is glycine, alanine or valine; Z represents an amino-saturated fatty acid; Each cysteine forms a disulfide bond.

Figure 1 is a schematic view showing the structure of a GLP-1 analogue according to the present invention, wherein the chain structure is a GLP-1 molecule, the letter C is a cysteine abbreviation and forms a disulfide bond, and (CH ₂ ) _n2 COOH refers to An amino-saturated fatty acid having a different carbon chain length, the C-terminal is coupled with an amino-saturated fatty acid, n ₂ = 4-10.

Among them, HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG is a human GLP-1 sequence, and the GLP-1 analog in the above formula A is an artificial sequence, and is modified only on the 27th, 29th or 31th amino acid, respectively, and replaced by cysteine. Some amino acids. And in the above amino acid formula, Y may be Gly, Ala or Val, and n ₁ = 1-10.

Example 1: Solid phase synthesis of polypeptide

The synthesis of the polypeptide of the present invention was carried out using a solid phase polypeptide synthesis method of the Fmoc strategy using a CS 336X type instrument manufactured by CSBio. The method of synthesis is carried out according to the manufacturer's instructions. The "Fmoc strategy" as used herein refers to a method for synthesizing a polypeptide by sequentially condensing an amino terminal Fmoc-protected amino acid in the presence of a coupling reagent using a polymer resin as a solid phase reaction substrate. For the specific method, see Fmoc solid phase peptide synthesis: a practical approach, 2000, Oxford University Press.

The prepared polypeptide was purified using an HPLC C18 semi-preparative column, and the mobile phase was acetonitrile. The polypeptide lyophilized powder is obtained by desalting and freeze-drying. The polypeptides encompassed by the present invention all contain disulfide bonds, ammonium bicarbonate or other oxidizing agents used to form disulfide bonds in the polypeptide.

Example 2: Hypoglycemic function of GLP-1 analogues

In this embodiment, the polypeptides used are as follows:

SEQ ID NO 4: HAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGGC - aminocaproic acid;

SEQ ID NO 5: HAEGTFTSDVSSYLEGQAAK EFIAWLVKCRGAAC-aminooctanoic acid;

SEQ ID NO 6: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRCVVVVVC - amino citric acid

One mg of the above polypeptide was dissolved in 1 ml of physiological saline to prepare a polypeptide solution, and the polypeptide solution was subcutaneously injected into mice (200 μl/f, 6/group, purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences), and 400 minutes after administration for 30 minutes. Micrograms of glucose were injected into each mouse. The blood glucose of the mice was measured at 2 hours, 24 hours, 48 hours, 72 hours, and 96 hours after the glucose injection (Note: the same dose of glucose was again given two hours before each blood glucose measurement), and the results are shown in FIG. The control (blank) in the present example was that subcutaneous injection of physiological saline into mice was carried out in an amount of 1 ml, and the injection of glucose was the same as in the other test groups.

The results of the glucose tolerance test of the GLP-1 analog shown in Figure 2 show that SEQ ID No 4, SEQ ID No 5 and SEQ ID No 6 have good long-term glycemic regulation ability.

Example 3: Determination of stability of GLP-1 analogues in human serum

In this embodiment, the polypeptides used are as follows:

SEQ ID NO 4: HAEGTFTSDVSSYLEGQAAKEFIAWLCKGRGGC - aminocaproic acid;

SEQ ID NO 5: HAEGTFTSDVSSYLEGQAAK EFIAWLVKCRGAAC-aminooctanoic acid;

SEQ ID NO 6: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRCVVVVVC - amino citric acid

(1) Preparation of serum: Three blood samples of volunteers were taken with a vacuum lancet (BD Biosciences, Franklin Lakes, NJ), and immediately centrifuged at 13,000 rpm for 20 minutes in a centrifuge, and the supernatant was taken for use.

(2) Dissolve the above three peptides and GLP-1 standard (purchased from Shanghai Shenggong Co., Ltd., the sequence is: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG) in 0.1mg of physiological saline, fully dissolved After the solution, they were separately added to 1 ml of rat serum (purchased from Invitrogen), and the serum was labeled as a blank group, and the No 1 experimental group (0.1 mg of the peptide having the sequence of SEQ ID No 4 was dissolved in 0.5 ml of serum), No 2 experiment The group (0.1 mg of the peptide having the sequence of SEQ ID No 5 was dissolved in 0.5 ml of serum) and the No. 3 experimental group (0.1 mg of the peptide having the sequence of SEQ ID No 6 was dissolved in 0.5 ml of serum). After incubating at 37 ° C for 0, 0.1, 0.3, 2.5, 5, 10, 15 and 24 h, 50 μl of the serum mixture was taken, and the serum of each group was detected by GLP-1 enzyme-linked immunosorbent assay kit (purchased from ALPCO Immunoassays). Absorbance values were read on a SpectraMax M5 plate reader to calculate the concentration of GLP-1 analogues at different time points in different serum samples. The results are shown in FIG. The circle represents the blank group, the inverted triangle represents the No 1 experimental group, the positive triangle represents the No 2 experimental group, and the square represents the No3 experimental group. The results indicate that the half-lives of SEQ ID No 4, SEQ ID No 5 and SEQ ID No 6 are >24 h, and the results demonstrate that the long-acting GLP-1 analog greatly prolongs the stability of human serum.

Example 4: Preparation of lyophilized powder injection by freeze-drying:

Take GLP-1 analogue to make a proper amount of solution, add water-soluble filler, stabilizer, osmotic pressure regulator, etc., add appropriate amount of water for injection, adjust the pH to 4-8 to dissolve it, dilute with water to the appropriate concentration, add 0.1 -0.5% activated carbon, stirred at 0-10 ° C for 10-20 minutes, decarburization, filtration using a microporous membrane filter, the filtrate is divided, and the freeze-drying method is used to obtain a white loose mass, which is obtained by sealing. Each specification contains a GLP-1 analog at 5 μg to 1 mg.

Example 5 Preparation method of solution injection:

Take GLP-1 analogue solution or lyophilized powder, add water-soluble filler, stabilizer, osmotic pressure regulator, etc., add appropriate amount of water for injection, adjust the pH to 4-8 to dissolve, dilute with water to the appropriate concentration, Add 0.1-0.5% activated carbon, stir at 0-10 °C for 10-20 minutes, decarburize, filter and sterilize by using microporous membrane filter, filtrate is divided and sealed, and each sample contains GLP-1 analogue. 5 μg - 1 mg.

The GLP-1 analogue of the present invention can effectively increase the blood half-life of GLP-1 and overcome the current situation that it cannot be used clinically because of its short half-life, and has application prospects in the field of diabetes and obesity therapeutic drugs.

Claims (6)

1. A GLP-1 analogue characterized by having the following formula A:

   HAEGTFTSDVSSYLEGQAAKEFIAWLX ₂₇ KX ₂₉ RX ₃₁ n ₁ YCZ,

   In the above formula A, X ₂₇ is valine or cysteine, X ₂₉ is glycine or cysteine, X ₃₁ glycine or cysteine, and at least one of X ₃₃ , X ₃₅ and X ₃₇ Is cysteine; n ₁ X ₁ represents n ₁ Y, n ₁ = 1-10, and Y is glycine, alanine or valine; Z represents an amino-saturated fatty acid; Both cysteines form disulfide bonds.
2. The GLP-1 analogue according to claim 1, wherein, in the formula A, n ₁ = 1-5, and Y is glycine, alanine or valine. Z is any one of aminobutyric acid, aminovaleric acid, aminocaproic acid, aminoheptanoic acid, aminooctanoic acid, aminodecanoic acid, and aminodecanoic acid.
3. Use of the GLP-1 analogue of claim 1 or 2 for the manufacture of a medicament for the treatment of diabetes, obesity and/or diabetes, obesity-related diseases, and prevention of obesity.
4. A pharmaceutical composition comprising the GLP-1 analogue of claim 1 or 2, comprising the GLP-1 analogue of claim 1 or 2 and one or more pharmaceutically acceptable Accessories.
5. A method for producing a pharmaceutical composition comprising the GLP-1 analogue according to claim 1 or 2, characterized in that the GLP-1 analogue according to claim 1 or 2 is made into a solution, and a water-soluble filling is added. Agent, stabilizer, osmotic pressure regulator and water for injection, adjust the PH value to 4-8 to dissolve the GLP-1, dilute with water, add 0.1-0.5% activated carbon, and stir at 0-10 ° C for 10-20 minutes. Decarburization, filtration and sterilization by microporous membrane filtration, fractionation of the filtrate, and freeze-drying method to obtain a white loose mass, which is obtained by sealing, and each specification contains the GLP-1 analogue at 5 μg to 1 mg.
6. A method for preparing a pharmaceutical composition comprising the GLP-1 analogue according to claim 1 or 2, wherein the GLP-1 analogue is prepared as a solution, a water-soluble filler, a stabilizer, and an infiltration are added. Pressure regulator and water for injection, adjust the pH to 4-8 to dissolve the GLP-1, dilute with water, add 0.1-0.5% activated carbon, stir at 0-10 ° C for 10-20 minutes, decarburize, use micro The membrane was sterilized by filtration, and the filtrate was dispensed and sealed, and each of the specifications contained the GLP-1 analogue at 5 μg to 1 mg.

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