An oral hypoglycemic composition and its application

Abstract

The present invention relates to the field of polypeptide technology, and discloses an oral hypoglycemic composition and its application. The composition of the present invention comprises a GLP-1 analog, sodium N-(8-(2-hydroxybenzoyl)amino)octanoate and fish oil. The composition has a high-efficiency and lasting hypoglycemic effect, can maintain a good hypoglycemic effect when taken orally, and can be used for the development of oral hypoglycemic drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to an oral hypoglycemic composition and application thereof. Background Art

[0002] Type 2 diabetes is a chronic metabolic disease caused by insufficient insulin secretion or insulin resistance in the body. Currently, it is estimated that there are 400 million diabetes patients worldwide. In recent years, GLP-1 receptor agonists are increasingly used to lower blood sugar. They enter the blood and bind to GLP-1 receptors, promote insulin synthesis and secretion in the pancreas, inhibit glucagon secretion, enhance the utilization of glucose by peripheral tissues, and reduce glycogen output, thereby lowering blood sugar.

[0003] Human glucagon-like peptide 1 (GLP-1) is an incretin produced by L cells in the ileum and colon. The activity of GLP-1 (1-37) is extremely low. After removing the N-terminal hexa-peptide, the biologically active GLP-1 (7-37) is formed. The sequence of human GLP-1 (7-37) is:

[0004]

[0005] However, human GLP-1(7-37) is easily degraded by DDP-IV and cleared by glomerular filtration after being secreted into the blood, with a half-life of only 3 to 5 minutes.

[0006] Currently available GLP-1 analogs are generally injected to work. However, the disadvantages of injections are quite obvious: first, they are inconvenient to use; second, injections usually cause obvious pain. Therefore, the development of an oral drug with efficient and long-lasting hypoglycemic effect will be of great application significance for the treatment of diabetes. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide an oral hypoglycemic composition and its application. Experiments show that the hypoglycemic composition still has an obvious hypoglycemic effect when taken orally.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] An oral hypoglycemic composition comprising a GLP-1 analog, sodium N-(8-(2-hydroxybenzoyl)amino)octanoate and fish oil;

[0010] The GLP-1 analogue is at least one of I) to III):

[0011] 1) a polypeptide having an amino acid sequence as shown in SEQ ID NO.1, wherein the cysteine ​​at position 2 and the cysteine ​​at position 13 of the sequence as shown in SEQ ID NO.1 form an intramolecular disulfide bond; or

[0012] II), a polypeptide having the same or similar function as I) obtained by replacing, deleting or adding one or more amino acids in the polypeptide described in I); or

[0013] III), a polypeptide having at least 90% homology with the amino acid sequence shown in I), and having the same or similar function as I).

[0014] Preferably, the mass ratio of the GLP-1 analog, sodium N-(8-(2-hydroxybenzoyl)amino)octanoate and fish oil is (7-15):(30-300):(20-84). In some specific embodiments, the mass ratio of the GLP-1 analog, sodium N-(8-(2-hydroxybenzoyl)amino)octanoate and fish oil is 10:30:20, 12:120:84, 7:35:21 or 15:300:30.

[0015] Preferably, the GLP-1 analog is: 6-7 additional 20 common amino acids are added to the C-terminus of the amino acid sequence shown in SEQ ID NO.1, and the cysteine ​​at the 2nd position and the cysteine ​​at the 13th position form an intramolecular disulfide bond.

[0016] In some specific embodiments of the present invention, the amino acid sequence of the GLP-1 analog is the amino acid sequence shown in any one of SEQ ID NO.2 or SEQ ID NO.3, and the cysteine ​​at position 2 and the cysteine ​​at position 13 of the sequence shown in any one of SEQ ID NO.2 or SEQ ID NO.3 form an intramolecular disulfide bond.

[0017] Experiments show that the GLP-1 analog provided by the present invention has been optimized and has a better ability to control blood sugar and a more lasting effect than existing products.

[0018] In the present invention, the GLP-1 analog is synthesized into a linear polypeptide by liquid phase synthesis, solid phase synthesis or solid-liquid combined synthesis process according to the amino acid sequence of the GLP-1 analog, and then oxidized to form a disulfide bond to obtain the GLP-1 analog.

[0019] In a specific embodiment of the present invention, the GLP-1 analog is synthesized by solid phase synthesis, the linear polypeptide is purified by HPLC, and the oxidation to form disulfide bonds is performed by ammonium bicarbonate or DMSO.

[0020] The composition of the present invention uses a specific GLP-1 analog as an active ingredient, and reasonably combines it with specific components N-(8-(2-hydroxybenzoyl)amino) sodium caprylate and fish oil in a specific ratio. The hypoglycemic experimental results show that the obtained composition has a long-lasting and effective hypoglycemic effect after oral administration; based on this excellent technical effect, the present invention proposes the use of the oral hypoglycemic composition in the preparation of a drug for treating or preventing diabetes.

[0021] A drug for preventing or treating diabetes, comprising the oral hypoglycemic composition of the present invention. As a further preferred embodiment, it may also include pharmaceutically acceptable adjuvants and / or drug ingredients for treating other diseases that do not affect the activity of the GLP-1 analog.

[0022] The drug of the present invention can be prepared into any clinical dosage form, mainly including oral dosage forms or injection dosage forms, wherein the injection dosage form includes a drug dosage form in the form of an infusion, and these dosage forms include but are not limited to tablets, granules, pills, oral liquids, lyophilized powders, injection solutions, capsules, and microcapsules.

[0023] The composition of the present invention comprises a GLP-1 analog, sodium N-(8-(2-hydroxybenzoyl)amino)octanoate and fish oil. The composition has a highly effective and lasting hypoglycemic effect, can maintain a good hypoglycemic effect when taken orally, and can be used for the development of oral hypoglycemic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The hypoglycemic effect of the GLP-1 analog in the hypoglycemic composition of the present invention;

[0025] Figure 2 It is a line graph showing the average blood sugar level of the composition of Example 4 of the present invention changing over time. DETAILED DESCRIPTION

[0026] The embodiments of the present invention disclose an oral hypoglycemic composition and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The oral hypoglycemic composition and its application described in the present invention have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the oral hypoglycemic composition and its application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0027] In the comparative experiments in the specific embodiments of the present invention, except for the differences in the settings of each group, other experimental conditions, raw materials, reagents, etc. not explicitly mentioned are kept consistent to ensure the comparability of the comparative experiments.

[0028] In the present invention, the amino acid sequence shown in SEQ ID NO.1 is:

[0029] HCEGTFTSDVSSCYLEKQAAKEFIAXLVK.

[0030] Among them, the 26th amino acid X=Trp or Tyr.

[0031] The GLP-1 analog provided by the present invention is further optimized based on the amino acid sequence shown in SEQ ID NO.1, and the obtained polypeptide exhibits a highly effective and lasting hypoglycemic effect and can be widely used in the development of diabetes drugs.

[0032] In some embodiments, the GLP-1 analog provided by the present invention has 6-7 additional 20 common amino acids added to the C-terminus of the amino acid sequence shown in SEQ ID NO.1, and the cysteine ​​at position 2 and the cysteine ​​at position 13 form an intramolecular disulfide bond.

[0033] In some specific embodiments, the GLP-1 analogue has 7 additional common amino acids added to the C-terminus of the amino acid sequence shown in SEQ ID NO.1, and the obtained polypeptide amino acid sequence is shown in SEQ ID NO.2, and the cysteine ​​at position 2 and the cysteine ​​at position 13 form an intramolecular disulfide bond.

[0034] The sequence of SEQ ID NO.2 is as follows:

[0035] HCEGTFTSDVSSCYLEKQAAKEFIAWLVKAECHYGR.

[0036] In some specific embodiments, the GLP-1 analogue has 6 additional common amino acids added to the C-terminus of the amino acid sequence shown in SEQ ID NO.1, and the obtained polypeptide amino acid sequence is shown in SEQ ID NO.3, and the cysteine ​​at position 2 and the cysteine ​​at position 13 form an intramolecular disulfide bond.

[0037] The sequence of SEQ ID NO.3 is as follows:

[0038] HCEGTFTSDVSSCYLEKQAAKEFIAYLVKFSQERG.

[0039] The following is a further description of a GLP-1 analog provided by the present invention and its preparation method and application.

[0040] Example 1: Preparation of GLP-1 analogs

[0041] The solid phase peptide synthesis method of the Fmoc strategy was used to synthesize using a peptide synthesizer according to the manufacturer's instructions. The amino acid sequence shown in SEQ ID NO.2 was synthesized in sequence, and the protecting groups and resin were removed to obtain a crude GLP-1 analog intermediate. The crude GLP-1 analog intermediate was dissolved in water, purified by preparative HPLC, C8 column, acetonitrile / water (V / V) = 90:10, concentrated, and freeze-dried to obtain a pure GLP-1 analog intermediate with free sulfhydryl groups. The pure GLP-1 analog intermediate was dissolved in water, oxidized with ammonium bicarbonate or DMSO to form a disulfide bond, and purified to obtain a GLP-1 analog. The sequence of the obtained GLP-1 analog is as follows:

[0042]

[0043] The dashed line indicates the intramolecular disulfide bond formed by two cysteines.

[0044] SEQ ID NO.2:

[0045] HCEGTFTSDVSSCYLEKQAAKEFIAWLVKAECHYGR

[0046] Example 2 Preparation of GLP-1 Analogs

[0047] The solid phase peptide synthesis method of the Fmoc strategy was used to synthesize using a peptide synthesizer according to the manufacturer's instructions. The amino acid sequence shown in SEQ ID NO.3 was synthesized in sequence, and the protecting groups and resin were removed to obtain a crude GLP-1 analog intermediate. The crude GLP-1 analog intermediate was dissolved in water, purified by preparative HPLC, C8 column, acetonitrile / water (V / V) = 90:10, concentrated, and freeze-dried to obtain a pure GLP-1 analog intermediate with free sulfhydryl groups. The pure GLP-1 analog intermediate was dissolved in water, oxidized with ammonium bicarbonate or DMSO to form a disulfide bond, and purified to obtain a GLP-1 analog. The obtained GLP-1 analog sequence is as follows:

[0048]

[0049] The dashed line indicates the intramolecular disulfide bond formed by two cysteines.

[0050] SEQ ID NO.3:

[0051] HCEGTFTSDVSSCYLEKQAAKEFIAYLVKFSQERG.

[0052] Example 3: Blood sugar lowering experiment

[0053] Grouping:

[0054] Twenty-four normal mice were divided into 4 groups: blank group, positive control group, drug group 1, and drug group 2, with 6 mice in each group.

[0055] Dosage regimen:

[0056] The blank group was injected with normal saline;

[0057] The positive control group received liraglutide as the injected drug;

[0058] The injection drug of drug group 1 is the GLP-1 analogue prepared in Example 1 of the present invention;

[0059] The injection drug of drug group 2 is the GLP-1 analogue prepared in Example 2 of the present invention.

[0060] Each group of mice was fasted for 12 hours before administration, and the fasting blood glucose of each group of mice was measured. Then, the blank group was subcutaneously injected with 200 μl of normal saline, the positive control group was injected with 200 μl of 0.2 mg / ml liraglutide, the drug group 1 was injected with 200 μl of 0.2 mg / ml of the GLP-1 analogue prepared in Example 1 of the present invention, and the drug group 2 was injected with 200 μl of 0.2 mg / ml of the GLP-1 analogue prepared in Example 2 of the present invention. Each group of mice was gavaged with 200 mg of glucose immediately after administration, and the gavage of 200 mg of glucose was repeated every 12 hours. The glucose tolerance was measured at 6h, 24h, 36h, and 48h after administration, the blood glucose levels at 15min, 30min, and 60min were measured, and the blood glucose value AUC (mg / dL.min) was calculated. The results are shown in Figure 1 .

[0061] Blood glucose measurement method: blood glucose meter.

[0062] Figure 1 illustrate:

[0063] After 4 h of administration, drug group 1 and drug group 2 had hypoglycemic effects;

[0064] After 24 h of administration, the positive control group had no hypoglycemic effect;

[0065] After 48 hours of administration, drug group 1 and drug group 2 still had significant hypoglycemic effects, especially drug group 2.

[0066] Example 4 Preparation of a composition containing a GLP-1 analog

[0067] (1) Preparation of Composition A

[0068] Table 1 Composition of composition A

[0069] GLP-1 analogues prepared in Example 1 10mg Sodium N-(8-(2-hydroxybenzoyl)amino)caprylate 30mg fish oil 20mg

[0070] Preparation method:

[0071] 10 mg of the GLP-1 analogue prepared in Example 1, 30 mg of sodium N-(8-(2-hydroxybenzoyl)amino)octanoate, and 20 mg of fish oil were mixed, loaded into a capsule, and coated. Eudragit L30D, talcum powder, and polyethylene glycol were weighed, dissolved with dichloromethane and isopropanol to obtain a coating solution, and the coating solution was sprayed onto the surface of the capsule to obtain an enteric-coated capsule. The coating solution flow rate was 1.5 ml / min, the coating temperature was 35°C, and the coating weight gain was 10%. Among them, Eudragit L30D: talcum powder: polyethylene glycol = 9:1:1 (weight ratio), dichloromethane: isopropanol = 10:1 (volume ratio).

[0072] (2) Composition B

[0073] Table 2 Composition of composition B

[0074] GLP-1 analogues prepared in Example 1 12mg Sodium N-(8-(2-hydroxybenzoyl)amino)caprylate 120mg fish oil 84mg

[0075] Preparation method:

[0076] 12 mg of the GLP-1 analog prepared in Example 1, 120 mg of sodium N-(8-(2-hydroxybenzoyl)amino)octanoate, and 84 mg of fish oil were mixed, placed in a capsule, and coated. Eudragit L30D, talcum powder, and polyethylene glycol were weighed, dissolved with dichloromethane and isopropanol to obtain a coating solution, and the coating solution was sprayed onto the surface of the capsule to obtain an enteric-coated capsule. The coating solution flow rate was 1.2 ml / min, the coating temperature was 33° C., and the coating weight gain was 10%.

[0077] (3) Composition C

[0078] Table 3 Composition of composition C

[0079] GLP-1 analogues prepared in Example 2 7mg Sodium N-(8-(2-hydroxybenzoyl)amino)octanoate 35mg fish oil 21mg

[0080] Preparation method:

[0081] 7 mg of the GLP-1 analog prepared in Example 2, 35 mg of sodium N-(8-(2-hydroxybenzoyl)amino)octanoate, and 21 mg of fish oil were mixed, placed in a capsule, and coated. Eudragit L30D, talcum powder, and polyethylene glycol were weighed, dissolved with dichloromethane and isopropanol to obtain a coating solution, and the coating solution was sprayed onto the surface of the capsule to obtain an enteric-coated capsule. The coating solution flow rate was 0.5 ml / min, the coating temperature was 30° C., and the coating weight gain was 8%.

[0082] (4) Composition D

[0083] Table 4 Composition of composition D

[0084] GLP-1 analogues prepared in Example 2 15mg Sodium N-(8-(2-hydroxybenzoyl)amino)caprylate 300mg fish oil 30mg

[0085] Preparation method:

[0086] 15 mg of the GLP-1 analogue prepared in Example 2, 300 mg of sodium N-(8-(2-hydroxybenzoyl)amino)octanoate, and 30 mg of fish oil were mixed, loaded into a capsule, and coated. Eudragit L30D, talcum powder, and polyethylene glycol were weighed, dissolved with dichloromethane and isopropanol to obtain a coating solution, and the coating solution was sprayed onto the surface of the capsule to obtain an enteric-coated capsule. The coating solution flow rate was 0.8 ml / min, the coating temperature was 45° C., and the coating weight gain was 5%.

[0087] Comparative Example 1 Preparation of Composition E (Placebo)

[0088] Table 5 Composition of composition E

[0089] GLP-1 analogues prepared in Example 2 0mg Sodium N-(8-(2-hydroxybenzoyl)amino)caprylate 30mg fish oil 20mg

[0090] Preparation method:

[0091] 30 mg of sodium N-(8-(2-hydroxybenzoyl)amino)octanoate and 20 mg of fish oil were mixed and loaded into a capsule for coating. Eudragit L30D, talcum powder, and polyethylene glycol were weighed and dissolved with dichloromethane and isopropanol to obtain a coating solution, which was sprayed onto the surface of the capsule to obtain an enteric-coated capsule. The coating solution flow rate was 1.0 ml / min, the coating temperature was 40°C, and the coating weight gain was 7%.

[0092] Example 5 Oral Composition Hypoglycemic Experiment

[0093] Category:

[0094] Healthy beagles were randomly divided into 5 groups. A total of 15 beagles weighing 10-12 kg were used. Double cross-administration was used, with 6 dogs in each group. The wash-out period was 1-2 weeks. The groups were: (1) Group A; (2) Group B; (3) Group C; (4) Group D; (5) Group E.

[0095] Dosage:

[0096] Group A orally administered composition A prepared in Example 4;

[0097] Group B orally administered composition B prepared in Example 4;

[0098] Group C orally administered composition C prepared in Example 4;

[0099] Group D orally administered composition D prepared in Example 4;

[0100] Group E orally administered composition E prepared in Comparative Example 1.

[0101] Blood Glucose Monitoring:

[0102] Each group of animals fasted for 12 hours overnight, and their fasting blood sugar was measured. Then, each group took one capsule of the corresponding group orally. 30 minutes after administration, each group of animals was gavaged with 8g / kg of glucose. The blood sugar levels of each group of animals were measured at 0h, 0.5h, 1h, and 1.5h after administration of glucose. Blood sugar was measured using a blood glucose meter. Each blood sugar value on the coordinate axis is the average blood sugar value of 6 beagles in the corresponding group at the corresponding time. The experimental results are shown in the figure below. Figure 2 .

[0103] in conclusion:

[0104] Composition A, composition B, composition C, and composition D all showed hypoglycemic effects.

[0105] The above description is only used to understand the method and core idea of ​​the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made to the present invention without departing from the principles of the present invention. These improvements and modifications also fall within the scope of protection of the rights of the present invention. Sequence Listing <110> Shanghai Haishen Biotechnology Co., Ltd. Hefei Tianhui Anchao Incubation Technology Co., Ltd. <120> An oral hypoglycemic composition and its application <130> MP21025484 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 29 <212> PRT <213> Artificial Sequence <220> <221> UNSURE <222> (26)..(26) <223> Xaa = Trp or Tyr <220> <221> UNSURE <222> (26)..(26) <223> The 'Xaa' at location 26 stands for Gln, Arg, Pro, or Leu. <400> 1 His Cys Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Cys Tyr Leu Glu 1 5 10 15 Lys Gln Ala Ala Lys Glu Phe Ile Ala Xaa Leu Val Lys 20 25 <210> 2 <211> 36 <212> PRT <213> Artificial Sequence <400> 2 His Cys Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Cys Tyr Leu Glu 1 5 10 15 Lys Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Ala Glu Cys 20 25 30 His Tyr Gly Arg 35 <210> 3 <211> 35 <212> PRT <213> Artificial Sequence <400> 3 His Cys Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Cys Tyr Leu Glu 1 5 10 15 Lys Gln Ala Ala Lys Glu Phe Ile Ala Tyr Leu Val Lys Phe Ser Gln 20 25 30 Glu Arg Gly 35

Claims

1. An oral hypoglycemic composition, It is characterized in that These include GLP-1 analogs, sodium N-(8-(2-hydroxybenzoyl)amino)octanoate, and fish oil; The amino acid sequence of the GLP-1 analog is shown in SEQ ID NO. 2 or 3, and the cysteine ​​at position 2 and the cysteine ​​at position 13 of the amino acid sequence form an intramolecular disulfide bond.

2. The oral hypoglycemic composition according to claim 1, It is characterized in that The mass ratio of the GLP-1 analog, sodium N-(8-(2-hydroxybenzoyl)amino)octanoate and fish oil is (7-15): (30-300): (20-84).

3. Use of the oral hypoglycemic composition according to any one of claims 1 to 2 in the preparation of a drug for treating or preventing diabetes.

4. A drug for preventing or treating diabetes, It is characterized in that The invention comprises the oral hypoglycemic composition according to any one of claims 1 to 2.

5. The drug according to claim 4, It is characterized in that It also includes pharmaceutically acceptable excipients and / or pharmaceutical ingredients used to treat other diseases and that do not affect the activity of the GLP-1 analogs.

6. The drug according to claim 4 or 5, It is characterized in that The medicine is in oral dosage form or injection form.

7. The drug according to claim 6, It is characterized in that The oral dosage forms include tablets, granules, pills, oral liquids, lyophilized powders, capsules and microcapsules.

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