CN112972657A

CN112972657A - Novel oral delivery composition containing liraglutide derivatives and application thereof

Info

Publication number: CN112972657A
Application number: CN202110172877.9A
Authority: CN
Inventors: 钱海; 石炜; 邱倩倩; 蔡衍
Original assignee: Nanjing Novin Haitai Biomedical Co Ltd
Current assignee: Nanjing Novin Haitai Biomedical Co Ltd
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2021-06-18

Abstract

The present invention relates to a class of oral delivery compositions comprising a liraglutide-derived polypeptide and N- (8- (2-hydroxybenzoyl) amino) caprylate; the mass ratio of the liraglutide derivative polypeptide to the N- (8- (2-hydroxybenzoyl) amino) caprylate is 1: 10-120; this patent is through changing the former fatty chain length of liraglutide and creative introducing the carboxyl at the fatty chain end, compares with the fatty chain that does not have the carboxyl end, and the fatty acid that has the carboxyl end has good oral bioavailability efficiency, can reduce the misery that patient's subcutaneous administration was many times, has the practicality, brings new breakthrough for diabetes treatment field.

Description

Novel oral delivery composition containing liraglutide derivatives and application thereof

Technical Field

The invention relates to a novel oral delivery composition containing liraglutide derivatives and application thereof, in particular to a composition containing liraglutide derivatives and N- (8- (2-hydroxybenzoyl) amino) caprylate and application thereof, including application thereof in the field of diabetes treatment, and belongs to the field of oral delivery compositions.

Background

Diabetes mellitus is the third most serious chronic non-infectious disease threatening human health after tumor and cardiovascular disease. Currently, about 3 million diabetics worldwide are predicted to increase to 5 million by 2025. Clinically, intensive insulin therapy is used to delay the progression of diabetes, but insulin injections risk hypoglycemia. The treatment effect is influenced by factors such as dosage, injection position, injection route and the like, the individual difference is large, and serious hypoglycemia side effect can occur due to the fact that insulin is used carelessly.

Glucagon-like peptide-1 (GLP-1) receptor agonists are a new treatment for type 2 diabetes and are gaining increasing attention because of their unique mechanism of glucose-dependent local stimulation of insulin secretion, along with the advantages of weight loss and cardiovascular benefits.

However, the clinical applicability of native GLP-1 as an anti-diabetic and anti-obesity therapy is limited by its short half-life (1-2 min). Since the Food and Drug Administration (FDA) approved Exenatide (Exenatide) to be marketed in 2005, various GLP-1 receptor agonists are on the market in succession, and the products mainly aim to overcome the defect that endogenous GLP-1 is rapidly cleared and prolong the half-life by adopting different strategies. Among them, liraglutide (liraglutide) developed by Novo Nordisk corporation was approved by the european union in 2009 and approved by the FDA in the united states for marketing in 2010. The liraglutide is based on endogenous GLP-1, Lys at position 34 is replaced by Arg, and a 16-carbon fatty acid side chain is conjugated to Lys at position 26. The fatty acid side chain can undergo non-covalent bond interaction with serum albumin, so that only 1-2% of the compound exists in the form of free polypeptide after the liraglutide is injected into circulation by subcutaneous injection. Therefore, the liraglutide has short blood sugar reduction duration, and can only continuously activate GLP-1 receptors within one day, so that the frequency of administration and the inconvenience of a subcutaneous administration mode are caused, and the compliance of patients is poor.

Somaloude company mixes Somaloutide with a compound named SNAC (sodium N- (8- [ 2-hydroxybenzoyl)]amino) caprylate) small molecule absorption enhancerMixing to form an oral somaglutide (semaglutide,

) It has been approved by the FDA for marketing in 2019, month 9.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a novel oral delivery composition containing liraglutide derivatives and application thereof, so as to solve the problems in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the present invention relates to a novel class of compositions comprising a liraglutide-derived polypeptide and N- (8- (2-hydroxybenzoyl) amino) caprylate in a mass ratio of liraglutide-derived polypeptide to N- (8- (2-hydroxybenzoyl) amino) caprylate of 1: 10-120.

Wherein, the N- (8- (2-hydroxybenzoyl) amino) caprylate is a compound shown as a general formula (1):

the liraglutide derivative polypeptide is a compound shown in a general formula (2) or a general formula (3):

n₁is an integer of 6 to 22;

or

n₂Is an integer of 1 to 20.

Further, as a preferable embodiment of the present invention,

the N- (8- (2-hydroxybenzoyl) amino) caprylate is a compound shown as a general formula (1):

n₁is an integer of 12 to 20;

or

n₂Is an integer of 3 to 10.

Further, as a preferred embodiment of the present invention, the liraglutide derivative polypeptide is any one of the following compounds:

further, as a preferred embodiment of the present invention, the liraglutide derivative polypeptide can also be any one of the following compounds:

in a second aspect, the present invention provides a pharmaceutical composition, wherein said composition is in the form of a solid, liquid or semisolid, and comprises one or more pharmaceutically acceptable excipients.

In a third aspect, the invention also provides a preparation method of the compound, wherein the liraglutide derivative polypeptide is efficiently and rapidly synthesized by adopting solid-phase synthesis and liquid-phase synthesis strategies to obtain the target compound.

In a fourth aspect, the invention also provides the use of the above composition in the preparation of a medicament for the treatment or prevention of diabetes.

The novel oral delivery composition prepared by the invention can be used for increasing the oral bioavailability of liraglutide.

On the basis of liraglutide molecules, the fat link length in 26 th Lys and 34 th Arg side chains is changed, and meanwhile, carboxyl is introduced into the tail end of the fat link, so that a liraglutide derivative is designed and synthesized; the combination of the N- (8- (2-hydroxybenzoyl) amino) caprylate and the liraglutide derivative can ensure that the liraglutide is partially absorbed in the stomach, can buffer the acidic environment in the stomach to resist the degradation of gastric peptidase, and can be easily broken when the non-covalent bond between the N- (8- (2-hydroxybenzoyl) amino) caprylate and the liraglutide derivative is exposed to blood, so that the liraglutide derivative is released into circulation to achieve the effect of improving the oral bioavailability.

The invention changes the length of the original fatty chain of liraglutide and creatively introduces carboxyl at the tail end of the fatty chain, and the invention has the advantages that the fatty acid with the tail end of the carboxyl has good oral bioavailability, can reduce the pain of multiple subcutaneous administrations of patients, has practicability and is a medicament with development prospect in the field of II type diabetes treatment.

Compared with the prior art, the invention has the following beneficial effects because the technology is adopted:

this patent is through changing the former fatty chain length of liraglutide and creative introducing the carboxyl at the fatty chain end, compares with the fatty chain that does not have the carboxyl end, and the fatty acid that has the carboxyl end has good oral bioavailability efficiency, can reduce the misery that patient's subcutaneous administration was many times, has the practicality, brings new breakthrough for diabetes treatment field.

Drawings

FIG. 1 is a graph of the effect of the composition on oral glucose tolerance in normal ICR mice; (a) represents the change in plasma glucose levels over time; (b) AUC showing blood glucose level_0-2h，

P is 0.01and p is 0.001.

FIG. 2 is the effect of the composition on Caco-2 cells in a monolayer transport assay, p.ltoreq.0.001.

Detailed Description

The present invention is illustrated by the following examples, which are not to be construed as limiting the invention in any way.

Example 1:

1. synthesis of chemically modified lysine

1.1, weighing 20g of Wang resin with the substitution degree of 1.0mmol/g, adding the Wang resin into a solid phase reaction column, washing the Wang resin with DMF for 2 times, swelling the resin with DMF for 30 minutes, dissolving 9.05g of Fmoc-Lys (alloc) -OH in DMF, adding 6.65mL of DIEA under ice water bath for activation, adding the mixture into the reaction column filled with the resin, reacting for 2 hours, and adding 10mL of anhydrous methanol for blocking for 1 hour. Washing with DMF 3 times, washing with DCM 3 times, blocking with anhydrous methanol for 30min, shrinking methanol and draining to obtain Fmoc-Lys (alloc) -Wang resin with a detection substitution of 0.605 mmol/g.

1.2, weighing 10.0g (6.0mmol) of Fmoc-Lys (alloc) -Wang resin with substitution degree of 0.605mmol/g, adding into the solid phase reaction column, washing with DMF for 2 times, swelling Fmoc-Lys (alloc) -Wang resin with DMF for 30 minutes, adding 30mL of dichloromethane, adding 8.8mL of phenylsilane, reacting for 3 minutes, adding 1.86g of Pd (PPh)₃)₄Reacting for 45 minutes at room temperature, pumping out reaction liquid, detecting the color of the resin by an indanthrone method, wherein the resin is colored, and indicating that Alloc is removed.

1.3, weighing 12.72g (30mmol) of Fmoc-Glu-OtBu, 13.05g (30mmol) of PyBOP and 4.88g (36mmol) of HOBt, dissolving the mixture with 30mL of DMF, adding 10.80mL of DIEA (60mmol) in an ice water bath for activation for 3 minutes, adding the mixture into a reaction column for reaction for 2 hours, detecting by an indantrione method to judge the reaction end point, removing Fmoc after the reaction is finished, and washing the mixture with DMF for 6 times.

1.4, 9.44g (30mmol) of monomethyl hexadecanedioate is weighed, dissolved in 30mL DMF, activated for 3 minutes by adding 4.75mL DIC (30mmol) in ice water bath, added to a reaction column for reaction for 2 hours, shrunk by methanol after the reaction is finished, dried overnight in vacuum, and 9.40g of side chain modified Lys intermediate resin is weighed.

1.5, 9.40g of the obtained side chain-modified Lys intermediate resin was transferred to a round-bottom flask, 145mL of a previously prepared lysate (TFE: DCM: 20:80 by volume) was added thereto, and the mixture was stirred at room temperature for 2 hours and then filtered, and the filtrate was collected; the resin was washed 2 more times with 10mL of DCM, the filtrates were combined, the filtrate was evaporated to dryness under reduced pressure and dried in vacuo to give 6.10g of the compound as a white solid.

1.6, purifying the obtained white solid compound by HPLC, wherein the purification conditions are as follows: octadecyl bonded silica gel is adopted as a stationary phase, and a mobile phase is as follows: phase A: 1% per mill TFA water; phase B: chromatographically pure acetonitrile, gradient: b%: 66-86% for 50-70 min. The white solid compound obtained by mass spectrometry detection, Ms, M/z 849.8(M + H) +, is a compound with the structure shown in formula I-1, the purity of the compound is 98.5%, and the mass of the compound is 2.56 g. The subsequent peptide chain synthesis is carried out by taking the formula I-1 as the 26 th amino acid.

2. Synthesis and purification of polypeptide peptide chain

2.1 swelling of the resin

Weighing 50mg of Fmoc-Rink amide-MBHA Resin (the substitution degree is 0.4mmol/g), swelling with 7mL of DCM for 30min, filtering off DCM by suction, swelling with 10mL of NMP for 30min, and finally washing with 7mL of NMP, DCM and NMP respectively.

2.2 removal of Fmoc protecting group

Putting the swelled resin into a reactor, adding 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt, reacting for 1min, and filtering the solution after the reaction is finished; then 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt was added, the reaction was carried out for 4min, and after completion, the solution was filtered off and washed with NMP. The resin was obtained with the Fmoc protecting group initially attached removed.

2.3 Synthesis of Fmoc-Gly-Rink amide-MBHA Resin

Fmoc-Gly-OH (8.0mg,0.04mmol), HBTU (15.1mg,0.04mmol), HOBt (5.4mg,0.04mmol) and DIPEA (13.9. mu.L, 0.08mmol) were dissolved in NMP 10mL, and this solution was added to the resin obtained in step 2.1, reacted for 7min, after which the reaction solution was filtered off and the resin was washed 3 times with 7mL each of DCM and NMP.

2.4 detection of coupling efficiency

Washing a small amount of resin particles with DMF, adding 3 drops of 1% bromophenol blue solution into a transparent vial, shaking at normal temperature for 3 minutes to obtain a positive resin with blue color, a negative resin with transparent color, and entering the next coupling cycle if the negative resin with blue color is obtained.

2.5 elongation of peptide chain

And (3) repeating the steps of deprotection and coupling (2.1-2.3) according to the sequence of the peptide chain, sequentially connecting corresponding amino acids (the 26 th position is I-1), and sequentially connecting the corresponding amino acids until the synthesis of the peptide chain is finished to obtain the resin connected with the polypeptide chain.

2.6 cleavage of the Polypeptides on the resin

Putting the obtained resin connected with polypeptide chain into a reaction bottle, adding 10mL of a cracking agent Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5, V/V), shaking at 0 ℃ for 30min, and reacting at normal temperature for 3 h; after the reaction is finished, carrying out suction filtration, adding a small amount of TFA and DCM for washing three times, and combining filtrates; adding the filtrate into a large amount of glacial ethyl ether to separate out white flocculent precipitate, freezing and centrifuging to obtain crude product of the target polypeptide, and finally obtaining crude product of the compound of 62.8mg with the yield of 95.2%.

2.7 purification of the polypeptide

Purification was performed using preparative liquid chromatography under the following chromatographic conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid/water (V/V), mobile phase B: 0.1% trifluoroacetic acid/acetonitrile (V/V); gradient of mobile phase: mobile phase B40% -80%, 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate is 5mL/min, and the detection wavelength is 214 nm; collecting the solution, distilling under reduced pressure to remove acetonitrile, and lyophilizing to obtain pure product. The theoretical relative molecular mass is 3795.2. ESI-MS M/z Calcd [ M +3H ]]³⁺1266.1，[M+4H]⁴⁺949.8；Found[M+3H]³⁺1266.8，[M+4H]⁴⁺950.6. The compound obtained was a compound of formula (4) with a purity of 97.3% and a mass of 25.6 mg.

3. Preparation of liraglutide derivative and N- (8- (2-hydroxybenzoyl) amino) caprylate composition

The chemically modified liraglutide derivative (compound of formula 4) was mixed with sodium N- [8- (2-hydroxybenzoyl) -amino ] caprylate (SNAC) in a mass ratio of 1:10, and a complex was prepared with the help of a weak non-covalent drug between the two.

Example 2:

1. synthesis of chemically modified lysine

1.1, weighing 20g of Wang resin with the substitution degree of 1.0mmol/g, adding the Wang resin into a solid phase reaction column, washing the Wang resin with DMF for 2 times, swelling the resin with DMF for 30 minutes, dissolving 9.05g of Fmoc-Lys (alloc) -OH in DMF, adding 6.65mL of DIEA under ice-water bath for activation, adding the mixture into the reaction column filled with the resin, reacting for 2 hours, and adding 10mL of anhydrous methanol for sealing for 1 hour; washing with DMF 3 times, washing with DCM 3 times, blocking with anhydrous methanol for 30min, shrinking methanol and draining to obtain Fmoc-Lys (alloc) -Wang resin with a detection substitution of 0.605 mmol/g.

1.3, weighing 12.72g (30mmol) of Fmoc-Glu-OtBu, 13.05g (30mmol) of PyBOP and 4.88g (36mmol) of HOBt, dissolving with 30mL of DMF, adding 10.80mL of DIEA (60mmol) in an ice water bath for activation for 3 minutes, adding into a reaction column for reaction for 2 hours, and detecting by an indetrione method to judge the reaction end point; after the reaction is finished, Fmoc is removed, and DMF is washed for 6 times.

1.4, weighing 10.26g (30mmol) of monomethyl octadecanedioate, dissolving with 30mL of DMF, adding 4.75mL of DIC (30mmol) under ice-water bath for activation for 3 minutes, adding into a reaction column for reaction for 2 hours, shrinking with methanol after the reaction is finished, drying overnight under vacuum, and weighing 8.11g of side chain modified Lys intermediate resin.

1.5, the obtained 8.11g side chain modified Lys intermediate resin was transferred to a round-bottom flask, 145mL of a previously prepared lysate (TFE: DCM ═ 20:80 by volume) was added, and the mixture was stirred at room temperature for 2 hours and then filtered, and the filtrate was collected; the resin was washed 2 more times with 10mL of DCM, and the filtrates were combined; the filtrate was evaporated to dryness under reduced pressure and dried in vacuo to give 5.30g of a white solid compound.

1.6, purifying the obtained white solid compound by HPLC, wherein the purification conditions are as follows: octadecyl bonded silica gel is adopted as a stationary phase, and a mobile phase is as follows: phase A: 1% per mill TFA water; phase B: chromatographically pure acetonitrile, gradient: b%: 66-86% for 50-70 min. The white solid compound obtained by mass spectrometry detection, Ms, M/z 877.5(M + H) +, is a compound with the structure shown in formula I-2, the purity of the compound is 96.5%, and the mass is 2.43 g. The subsequent peptide chain synthesis is carried out by taking the formula I-2 as the 26 th amino acid.

2. Synthesis and purification of polypeptide peptide chain

2.1 swelling of the resin

2.2 removal of Fmoc protecting group

Putting the swelled resin into a reactor, adding 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt, reacting for 1min, and filtering the solution after the reaction is finished; then adding 7mL of 25% piperidine/NMP (V/V) solution containing 0.1M HOBt, reacting for 4min, filtering the solution after the reaction is finished, and washing the solution by using NMP; the resin was obtained with the Fmoc protecting group initially attached removed.

2.3 Synthesis of Fmoc-Gly-Rink amide-MBHA Resin

2.4 detection of coupling efficiency

Washing a small amount of resin particles with DMF, putting into a transparent vial, adding 3 drops of 1% bromophenol blue solution, shaking at normal temperature for 3 minutes, and determining that the resin is positive when the resin is blue and transparent is negative; if negative, the next coupling cycle can be entered.

2.5 elongation of peptide chain

And (3) repeating the steps of deprotection and coupling (2.1-2.3) according to the sequence of the peptide chain, sequentially connecting corresponding amino acids (the 26 th position is I-2), and sequentially connecting the corresponding amino acids until the synthesis of the peptide chain is finished to obtain the resin connected with the polypeptide chain.

2.6 cleavage of the Polypeptides on the resin

Putting the obtained resin connected with polypeptide chain into a reaction bottle, adding 10mL of a cracking agent Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5, V/V), shaking at 0 ℃ for 30min, and reacting at normal temperature for 3 h; after the reaction is finished, carrying out suction filtration, adding a small amount of TFA and DCM for washing three times, and combining filtrates; adding the filtrate into a large amount of glacial ethyl ether to separate out white flocculent precipitate, and freezing and centrifuging to obtain a crude product of the target polypeptide; the final crude compound was obtained in 62.8mg with a yield of 95.2%.

2.7 purification of the polypeptide

Purification was performed using preparative liquid chromatography under the following chromatographic conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid/water (V/V), mobile phase B: 0.1% trifluoroacetic acid/acetonitrile (V/V); gradient of mobile phase: 40-80% of mobile phase B for 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate is 5ml/min, and the detection wavelength is 214 nm; collecting the solution, distilling under reduced pressure to remove acetonitrile, and lyophilizing to obtain pure product. Relative theoryThe molecular mass was 3823.2. ESI-MS M/z Calcd [ M +3H ]]³⁺1275.4，[M+4H]⁴⁺956.8；Found[M+3H]³⁺1275.9，[M+4H]⁴⁺957.6. The compound obtained was a compound of formula (5) with a purity of 98.4% and a mass of 21.6 mg.

3. Preparation of chemically modified liraglutide derivatives and N- (8- (2-hydroxybenzoyl) amino) caprylate compositions

The chemically modified liraglutide derivative (compound of formula (5)) was mixed with sodium N- [8- (2-hydroxybenzoyl) -amino ] caprylate (SNAC) in a mass ratio of 1:30 to prepare a complex with the help of a weak non-covalent drug therebetween.

Example 3:

1. synthesis of chemically modified lysine

1.4, 9.44g (30mmol) of the monomethyl eicosanedioate is weighed, dissolved in 30mL of DMF, activated for 3 minutes by adding 4.75mL of DIC (30mmol) in an ice-water bath, added to a reaction column for reaction for 2 hours, shrunk by using methanol after the reaction is finished, dried overnight in vacuum, and weighed to obtain 8.71g of side chain modified Lys intermediate resin.

1.5, the obtained 8.71g side chain modified Lys intermediate resin was transferred to a round-bottom flask, 145mL of a previously prepared lysate (TFE: DCM ═ 20:80 by volume) was added thereto, and the mixture was stirred at room temperature for 2 hours and then filtered, and the filtrate was collected; the resin was washed 2 more times with 10mL of DCM, and the filtrates were combined; the filtrate was evaporated to dryness under reduced pressure and dried in vacuo to give 6.10g of a white solid compound.

1.6, purifying the obtained white solid compound by HPLC, wherein the purification conditions are as follows: octadecyl bonded silica gel is adopted as a stationary phase, and a mobile phase is as follows: phase A: 1% per mill TFA water; phase B: chromatographically pure acetonitrile, gradient: b%: 66-86% for 50-70 min. Detecting the obtained white solid compound by mass spectrum, wherein Ms, M/z 891.5(M + H) +, and the obtained compound is a compound with a structure shown in a formula I-3, the purity of the compound is 97.5%, and the mass of the compound is 2.06 g. The subsequent peptide chain synthesis is carried out by taking the formula I-3 as the 26 th amino acid.

2. Synthesis and purification of polypeptide peptide chain

2.1 swelling of the resin

2.2 removal of Fmoc protecting group

Putting the swelled resin into a reactor, adding 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt, reacting for 1min, and filtering the solution after the reaction is finished; then 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt is added for reaction for 4min, and after the reaction is finished, the solution is filtered and washed clean by NMP to obtain the resin with the Fmoc protecting group removed from the initial connection.

2.3 Synthesis of Fmoc-Gly-Rink amide-MBHA Resin

2.4 detection of coupling efficiency

2.5 elongation of peptide chain

And (3) repeating the steps of deprotection and coupling (2.1-2.3) according to the sequence of the peptide chain, sequentially connecting corresponding amino acids (the 26 th position is I-3), and sequentially connecting the corresponding amino acids until the synthesis of the peptide chain is finished to obtain the resin connected with the polypeptide chain.

2.6 cleavage of the Polypeptides on the resin

The resin with polypeptide chain obtained above was placed in a reaction flask, 10mL of cleavage agent Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5, V/V) was added, shaken at 0 ℃ for 30min, and reacted at room temperature for 3 h. After the reaction is finished, carrying out suction filtration, adding a small amount of TFA and DCM for washing three times, and combining filtrates; adding the filtrate into a large amount of glacial ethyl ether to separate out white flocculent precipitate, and freezing and centrifuging to obtain a crude product of the target polypeptide; the final crude compound was obtained in 62.8mg with a yield of 95.2%.

2.7 purification of the polypeptide

Purification was performed using preparative liquid chromatography under the following chromatographic conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid/water (V/V), mobile phase B: 0.1% trifluoroacetic acid/acetonitrile (V/V); gradient of mobile phase: 40-80% of mobile phase B for 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate is 5ml/min, and the detection wavelength is 214 nm; collecting the solution, distilling under reduced pressure to remove acetonitrile, and lyophilizing to obtain pure product; the theoretical relative molecular mass is 3795.2. ESI-MS 3851.2M/z Calcd [ M +3H ]]³⁺1284.7，[M+4H]⁴⁺963.8；Found[M+3H]³⁺1285.8，[M+4H]⁴⁺964.6. The compound thus obtained was a compound of formula (6) having a purity of 98.3% and a mass of 29.6 mg.

Chemically modified liraglutide derivatives (compounds of formula (6)) and sodium N- [8- (2-hydroxybenzoyl) -amino ] caprylate (SNAC) in a mass ratio of 1: 50 and a complex is prepared with the help of a weak non-covalent drug between the two.

Example 4

1. Synthesis of chemically modified lysine

1.1.1, weighing 20g of Wang resin with the substitution degree of 1.0mmol/g, adding the Wang resin into a solid phase reaction column, washing the Wang resin for 2 times by using DMF, swelling the resin by using DMF for 30 minutes, dissolving 9.05g of Fmoc-Lys (alloc) -OH by using DMF, adding 6.65mL of DIEA under ice water bath for activation, adding the obtained product into the reaction column filled with the resin, reacting for 2 hours, and adding 10mL of anhydrous methanol for sealing for 1 hour; washing with DMF 3 times, washing with DCM 3 times, blocking with anhydrous methanol for 30min, shrinking methanol and draining to obtain Fmoc-Lys (alloc) -Wang resin with a detection substitution of 0.605 mmol/g.

1.1.2, weighing 10.0g (6.0mmol) of Fmoc-Lys (alloc) -Wang resin with substitution degree of 0.605mmol/g, adding into a solid phase reaction column, washing with DMF for 2 times, swelling the Fmoc-Lys (alloc) -Wang resin with DMF for 30 minutes, adding 30mL of dichloromethane, adding 8.8mL of phenylsilane, reacting for 3 minutes, adding 1.86g of Pd (PPh)₃)₄Reacting for 45 minutes at room temperature, pumping out reaction liquid, detecting the color of the resin by an indanthrone method, wherein the resin is colored, and indicating that Alloc is removed.

1.1.3, weighing 12.72g (30mmol) of Fmoc-Glu-OtBu, 13.05g (30mmol) of PyBOP and 4.88g (36mmol) of HOBt, dissolving with 30mL of DMF, adding 10.80mL of DIEA (60mmol) in an ice water bath for activation for 3 minutes, adding into a reaction column for reaction for 2 hours, and detecting by an indetrione method to judge the reaction end point; after the reaction is finished, Fmoc is removed, and DMF is washed for 6 times.

1.1.4, weighing 8.10g (30mmol) of monomethyl hexadecanoate, dissolving with 30mL of DMF, adding 4.75mL of DIC (30mmol) under ice-water bath for activation for 3 minutes, adding into a reaction column, and reacting for 2 hours; after the reaction is finished, shrinking the mixture by using methanol, and drying the mixture overnight in vacuum; the weight was taken to obtain 9.40g of side chain modified Lys intermediate resin.

1.1.5, 9.40g of the obtained side chain-modified Lys intermediate resin was transferred to a round-bottomed flask, 145mL of a previously prepared lysate (TFE: DCM ═ 20:80 by volume) was added thereto, and the mixture was stirred at room temperature for 2 hours and then filtered, and the filtrate was collected; the resin was washed 2 more times with 10mL of DCM, and the filtrates were combined; the filtrate was evaporated to dryness under reduced pressure and dried in vacuo to give 5.30g of a white solid compound.

1.1.6, purifying the obtained white solid compound by HPLC, wherein the purification conditions are as follows: octadecyl bonded silica gel is adopted as a stationary phase, and a mobile phase is as follows: phase A: 1% per mill TFA water; phase B: chromatographically pure acetonitrile, gradient: b%: 66-86% for 50-70 min; detecting the obtained white solid compound by mass spectrometry, wherein Ms, M/z 791.5(M + H) +, and the obtained compound is a compound with a structure shown in a formula I-4, the purity of the compound is 96.5%, and the mass of the compound is 1.83 g; the subsequent peptide chain synthesis is carried out by taking the formula I-4 as the 26 th amino acid; this was used for the synthesis of examples 5 and 6.

1.2.1, weighing 5g of Wang resin with the substitution degree of 1.0mmol/g, adding the Wang resin into a solid phase reaction column, washing the Wang resin for 2 times by using DMF, swelling the resin by using DMF for 30 minutes, dissolving 9.05g of Fmoc-Lys (alloc) -OH by using DMF, adding 6.65mL of DIEA under ice water bath for activation, adding the obtained product into the reaction column filled with the resin, reacting for 2 hours, and adding 10mL of anhydrous methanol for sealing for 1 hour; washing with DMF 3 times, washing with DCM 3 times, blocking with anhydrous methanol for 30min, shrinking methanol and draining to obtain Fmoc-Lys (alloc) -Wang resin with a detection substitution of 0.605 mmol/g.

1.2.2 weighing 5.0g (3.0mmol) of Fmoc-Lys- (alloc) -Wang resin with substitution degree of 0.605mmol/g, adding into solid phase reaction column, washing with DMF for 2 times, swelling Fmoc-Lys- (alloc) -2CTC resin with DMF for 30min, adding 30mL of dichloromethane, adding 8.8mL of phenylsilane, reacting for 3 min, adding 0.93g Pd (PPh)₃)₄Reacting for 45 minutes at room temperature, pumping out reaction liquid, detecting the color of the resin by an indanthrone method, wherein the resin is colored, and indicating that Alloc is removed.

1.2.3, weighing 1.98g (15mmol) of monomethyl succinate, 6.52g (15mmol) of PyBOP and 2.44g (18mmol) of HOBt, dissolving with 30mL of DMF, adding 5.40mL of DIEA (30mmol) in an ice water bath for activation for 3 minutes, adding into a reaction column for reaction for 2 hours, detecting by an indantrione method to judge the reaction end point, removing Fmoc after the reaction is finished, and washing with DMF for 6 times; the weight was taken to obtain 1.69g of side chain modified Lys intermediate resin.

1.3.1, 1.69g of the side chain-modified Lys intermediate resin obtained was transferred to a round-bottomed flask, 145mL of a previously prepared lysate (TFE: DCM ═ 0:80 by volume) was added thereto, and the mixture was stirred at room temperature for 2 hours and then filtered, and the filtrate was collected; the resin was washed 2 more times with 10mL of DCM, and the filtrates were combined; the filtrate was evaporated to dryness under reduced pressure and dried in vacuo to give 0.87g of a white solid compound.

1.3.2, purifying the obtained white solid compound by HPLC, wherein the purification conditions are as follows: octadecyl bonded silica gel is adopted as a stationary phase, and a mobile phase is as follows: phase A: 1% per mill TFA water; phase B: chromatographically pure acetonitrile, gradient: b%: 66-86% for 50-70 min. Detecting the obtained white solid compound by mass spectrometry, wherein Ms, M/z 468.2(M + H) +, and the obtained compound is a compound with a structure shown in a formula I-5, the purity of the compound is 98.5%, and the mass of the compound is 0.45 g; the subsequent peptide chain synthesis was performed as amino acid 34 of formula I-5.

2. Synthesis and purification of polypeptide peptide chain

2.1 swelling of the resin

2.2 removal of Fmoc protecting group

2.3 Synthesis of Fmoc-Gly-Rink amide-MBHA Resin

2.4 detection of coupling efficiency

2.5 elongation of peptide chain

And (3) according to the sequence of the peptide chain, repeating the steps (2.1-2.3) of deprotection and coupling, sequentially connecting corresponding amino acids (wherein the 26 th position is I-4, and the 34 th position is I-5), and sequentially connecting the corresponding amino acids until the peptide chain is synthesized to obtain the resin connected with the polypeptide chain.

2.6 cleavage of the Polypeptides on the resin

2.7 purification of the polypeptide

Purification was performed using preparative liquid chromatography under the following chromatographic conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid/water (V/V), mobile phase B: 0.1% trifluoroacetic acid/acetonitrile (V/V); gradient of mobile phase: mobile phase B40% -80%, 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate is 5ml/min, and the detection wavelength is 214 nm; collecting the solution, distilling under reduced pressure to remove acetonitrile, and lyophilizing to obtain pure product; the theoretical relative molecular mass is 3853.2. ESI-MS M/z Calcd [ M +3H ]]³⁺1285.4，[M+4H]⁴⁺964.3；Found[M+3H]³⁺1285.9，[M+4H]⁴⁺965.6. The compound obtained was a compound of formula (7) with a purity of 96.4% and a mass of 16.6 mg.

Chemically modified liraglutide derivatives (compounds of formula (7)) and sodium N- [8- (2-hydroxybenzoyl) -amino ] caprylate (SNAC) in a mass ratio of 1: 80 are mixed together and a complex is prepared with the help of a weak non-covalent drug between the two.

Example 5

1. Synthesis of chemically modified lysine

1.1, weighing 5g of Wang resin with the substitution degree of 1.0mmol/g, adding the Wang resin into a solid phase reaction column, washing the Wang resin with DMF for 2 times, swelling the resin with DMF for 30 minutes, dissolving 9.05g of Fmoc-Lys (alloc) -OH in DMF, adding 6.65mL of DIEA under ice-water bath for activation, adding the mixture into the reaction column filled with the resin, reacting for 2 hours, and adding 10mL of anhydrous methanol for sealing for 1 hour; washing with DMF 3 times, washing with DCM 3 times, blocking with anhydrous methanol for 30min, shrinking methanol and draining to obtain Fmoc-Lys (alloc) -Wang resin with a detection substitution of 0.605 mmol/g.

1.2, 5.0g (3.0mmol) of Fmoc-Lys (alloc) -Wang resin with substitution degree of 0.605mmol/g was weighed, added to a solid phase reaction column, washed with DMF for 2 times, after swelling the Fmoc-Lys (alloc) -2CTC resin with DMF for 30 minutes, 30mL of dichloromethane was added, 8.8mL of phenylsilane was added, reaction was carried out for 3 minutes, and 0.93g of Pd (PPh) was added₃)₄Reacting for 45 minutes at room temperature, pumping out reaction liquid, detecting the color of the resin by an indanthrone method, wherein the resin is colored, and indicating that Alloc is removed.

1.3, weighing 1.20g (15mmol) of monomethyl adipate, 6.52g (15mmol) of PyBOP and 2.44g (18mmol) of HOBt, dissolving with 30mL of DMF, adding 5.40mL of DIEA (30mmol) in an ice water bath for activation for 3 minutes, adding into a reaction column for reaction for 2 hours, and detecting by an indetrione method to judge the reaction end point; after the reaction is finished, removing Fmoc, and washing with DMF for 6 times; the weight was taken to obtain 1.56g of side chain modified Lys intermediate resin.

1.4, 1.56g of the obtained side chain-modified Lys intermediate resin was transferred to a round-bottomed flask, 145mL of a previously prepared lysate (TFE: DCM: 20:80 by volume) was added thereto, and the mixture was stirred at room temperature for 2 hours and then filtered, and the filtrate was collected; the resin was washed 2 more times with 10mL of DCM and the filtrates combined. The filtrate was evaporated to dryness under reduced pressure and dried in vacuo to give 0.66g of a white solid compound.

1.5, purifying the obtained white solid compound by HPLC, wherein the purification conditions are as follows: octadecyl bonded silica gel is adopted as a stationary phase, and a mobile phase is as follows: phase A: 1% per mill TFA water; phase B: chromatographically pure acetonitrile, gradient: b%: 66-86% for 50-70 min. Detecting the obtained white solid compound by mass spectrometry, wherein Ms, M/z 496.2(M + H) +, and the obtained compound is a compound with a structure shown in a formula I-6, the purity of the compound is 97.5%, and the mass of the compound is 0.33 g; the subsequent peptide chain synthesis was performed as amino acid 34 of formula I-6.

2. Synthesis and purification of polypeptide peptide chain

2.1 swelling of the resin

2.2 removal of Fmoc protecting group

2.3 Synthesis of Fmoc-Gly-Rink amide-MBHA Resin

2.4 detection of coupling efficiency

2.5 elongation of peptide chain

And (3) according to the sequence of the peptide chain, repeating the steps (2.1-2.3) of deprotection and coupling, sequentially connecting corresponding amino acids (wherein the 26 th position is I-4, and the 34 th position is I-6), and sequentially connecting the corresponding amino acids until the peptide chain is synthesized to obtain the resin connected with the polypeptide chain.

2.6 cleavage of the Polypeptides on the resin

2.7 purification of the polypeptide

Purification was performed using preparative liquid chromatography under the following chromatographic conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid/water (V/V), mobile phase B: 0.1% trifluoroacetic acid/acetonitrile (V/V); gradient of mobile phase: mobile phase B40% -80%, 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate is 5ml/min, and the detection wavelength is 214 nm; collecting the solution, distilling under reduced pressure to remove acetonitrile, and lyophilizing to obtain pure product; the theoretical relative molecular mass is 3881.2. ESI-MS M/z Calcd [ M +3H ]]³⁺1294.7，[M+4H]⁴⁺971.3；Found[M+3H]³⁺1295.2，[M+4H]⁴⁺972.6. The compound obtained was a compound of formula (8) with a purity of 98.4% and a mass of 24.6 mg.

Chemically modified liraglutide derivatives (compounds of formula (8)) and sodium N- [8- (2-hydroxybenzoyl) -amino ] caprylate (SNAC) in a mass ratio of 1: 100 and a complex is prepared with the help of a weak non-covalent drug between the two.

Example 6

1. Synthesis of chemically modified lysine

1.3, weighing 2.32g (15mmol) of monomethyl suberate, 6.52g (15mmol) of PyBOP and 2.44g (18mmol) of HOBt, dissolving with 30mL of DMF, adding 5.40mL of DIEA (30mmol) in an ice water bath for activation for 3 minutes, adding a reaction column for reaction for 2 hours, and detecting by an indetrione method to judge the reaction end point; after the reaction is finished, removing Fmoc, and washing with DMF for 6 times; the weight was taken to obtain 1.31g of side chain modified Lys intermediate resin.

1.4, 1.31g of the side chain-modified Lys intermediate resin obtained was transferred to a round-bottom flask, 145mL of a previously prepared lysate (TFE: DCM ═ 20:80 by volume) was added thereto, and the mixture was stirred at room temperature for 2 hours and then filtered, and the filtrate was collected; the resin was washed 2 more times with 10mL of DCM and the filtrates combined. The filtrate was evaporated to dryness under reduced pressure and dried in vacuo to give 0.93g of a white solid compound.

1.5, purifying the obtained white solid compound by HPLC, wherein the purification conditions are as follows: octadecyl bonded silica gel is adopted as a stationary phase, and a mobile phase is as follows: phase A: 1% per mill TFA water; phase B: chromatographically pure acetonitrile, gradient: b%: 66-86% for 50-70 min; detecting the obtained white solid compound by mass spectrum, wherein Ms, M/z 424.2(M + H) +, and the obtained compound is a compound with a structure shown in a formula I-7, the purity of the compound is 98.5%, and the mass of the compound is 0.53 g. Subsequent peptide chain synthesis was performed as amino acid 34 of formula I-7.

2. Synthesis and purification of polypeptide peptide chain

2.1 swelling of the resin

2.2 removal of Fmoc protecting group

2.3 Synthesis of Fmoc-Gly-Rink amide-MBHA Resin

2.4 detection of coupling efficiency

2.5 elongation of peptide chain

And (3) according to the sequence of the peptide chain, repeating the steps (2.1-2.3) of deprotection and coupling, sequentially connecting corresponding amino acids (wherein the 26 th position is I-4, and the 34 th position is I-7), and sequentially connecting the corresponding amino acids until the peptide chain is synthesized to obtain the resin connected with the polypeptide chain.

2.6 cleavage of the Polypeptides on the resin

2.7 purification of the polypeptide

Purification was performed using preparative liquid chromatography under the following chromatographic conditions: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid/water (V/V), mobile phase B: 0.1% trifluoroacetic acid/acetonitrile (V/V); gradient of mobile phase: mobile phase B40% -80%, 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate is 5ml/min, and the detection wavelength is 214 nm; collecting the solution, distilling under reduced pressure to remove acetonitrile, and lyophilizing to obtain pure product; the theoretical relative molecular mass is 3909.2. ESI-MS M/z Calcd [ M +3H ]]³⁺1304.1，[M+4H]⁴⁺978.3；Found[M+3H]³⁺1305.2，[M+4H]⁴⁺979.6. The compound obtained was a compound of formula (9) with a purity of 98.4% and a mass of 24.6 mg.

Chemically modified liraglutide derivatives (compounds of formula (9)) and sodium N- [8- (2-hydroxybenzoyl) -amino ] caprylate (SNAC) in a mass ratio of 1: 120, and a complex is prepared with the help of a weak non-covalent drug between the two.

Example 7: pharmacological experimental methods and results for the oral delivery compositions prepared by the present invention:

1. test for receptor agonistic Activity of GLP-1 analogs

HEK293 cells are co-transfected with cDNA encoding GLP-1R, the cell lines express and Western Blot is used for detecting the protein level of GLP-1R in the constructed HEK293 cells so as to investigate whether stable and high-expression GLP-R-HEK293 cell strains are established; in the receptor agonism experiment, firstly, cells are planted in a 96-well plate, after 2 hours, a compound is dissolved by DMSO, diluted to different times by using a culture medium containing 0.1% bovine serum albumin, and added into the co-transfected GLP-1R-HEK293 cells; incubationAfter 20min, the corresponding cAMP values were detected using an ELISA kit from Cisbo, and the EC of the compound was calculated after non-linear regression₅₀Numerical values.

TABLE 1 EC for oral delivery compositions₅₀

As shown in table 1, all compounds retained agonistic activity towards GLP-1R, with a significant improvement compared to liraglutide. The agonistic activity of the oral delivery composition prepared in example 1 on GLP-1R is similar to that of endogenous GLP-1, and is improved by about 10 times compared with liraglutide.

2. Normal hour oral glucose tolerance test

The Oral Glucose Tolerance Test (OGTT) is mainly used for measuring the load capacity of an organism to glucose so as to reflect the blood sugar regulation level and the pancreatic islet function of the organism; the experiment used normal ICR mice for OGTT to screen for preferred compounds with potential in vivo hypoglycemic activity.

The instrument comprises the following steps: blood glucose meter (sano biosensing ltd, changsha), blood glucose test paper (sano biosensing ltd, changsha), elbow scissors, mouse gavage needle, 1mL medical syringe, AUY120 type electronic precision analytical balance (shimadzu, japan).

Materials: 18-22g of clean male ICR mice (comparative medical center of Yangzhou university), basal diet (Qinglongshan animal breeding center, Nanjing), glucose (Shanghai Lingfeng Chemicals Co., Ltd.).

Animal breeding environment: randomly cage-dividing 18-22g of clean male ICR mice, feeding sufficient food and drinking water, and simultaneously feeding the mice under the condition of 12h day/night circulating illumination, keeping the indoor temperature of an animal room at 23-25 ℃, keeping the indoor relative humidity at 70 +/-10%, and maintaining good indoor ventilation conditions; except for special instructions, the gavage solvent adopts 0.5% carboxymethyl cellulose water solution, and the compound to be tested is prepared into solution with a certain concentration according to the administration dosage.

The experimental method comprises the following steps: after being adaptively fed for one week, 18-22g of clean-grade male ICR mice were randomly divided into a normal control group, a liraglutide intraperitoneal administration group, a liraglutide oral administration group, and a test compound administration group (oral delivery composition prepared in examples 1-6), each group consisting of 6 mice; before the experiment, the mice are weighed after fasting for 12 hours without water prohibition, tail tip blood sampling is carried out to determine the fasting blood glucose level, a normal control group is administered with a menstruum by gastric lavage, the abdominal cavity and oral administration groups are both administered with the drugs to be determined with corresponding concentrations according to the body weight, the blood glucose level of each group is determined after administration for 30min, 3g/kg glucose aqueous solution is immediately injected into the abdominal cavity respectively, and the

blood glucose levels

15, 30, 45, 60 and 120min after administration of glucose are determined; collecting experimental results, drawing a line graph by using GraphPad 7.0 and using time as an abscissa and blood glucose values as an ordinate, and calculating the area AUC under each group of blood glucose curves.

As shown in fig. 1, after glucose was administered by gavage for 15min, the blood glucose levels of the mice in each group were significantly increased and reached the peak value at 30min, and the blood glucose levels of the mice in the liraglutide intraperitoneal administration control group were significantly different from those of the blank group and the liraglutide oral administration group.

3. Transport experiments across Caco-2 cell monolayers

The purpose of this experiment was to determine the penetration enhancing effect of different compositions on the transepithelial absorption of GLP-1 peptides in Caco-2 monolayers.

Cell culture: human colon adenocarcinoma Caco-2 cell line ATCC # HTB-37, purchased from ATCC (American Type Culture Collection, Rockville, Md., USA), is frozen and stored 29 th generation Caco-2 cells are taken out from liquid nitrogen, and cultured for 2 generations by DMEM-20 and 3 generations by DMEM-15, and then cultured by DMEM-10 after the growth rate of the cells is normal; culturing the recovered cells by DMEM-10, changing the liquid every other day after changing the liquid on the first day, and carrying out passage every 4-5 days (the cell confluence rate is about 80%), wherein the passage ratio is 1: 5.

establishment of Caco-2 cell monolayer: after 80% cell confluence, cells were digested from the flask wall with 0.25% trypsin-EDTA, digestion was stopped with DMEM-10 and the cell suspension was diluted to 2.5X 10⁵Cell mL^-1. DMEM-10 medium was added to the bottom of a 12-well Transwell (BL)1.5mL, 0.5mL of the well-mixed cell suspension was added to the apical side (AP), and the mixture was cultured in a 37 ℃ incubator; changing the culture solution at the top end and the bottom end on the 2 nd day; changing the liquid at the top and the bottom every other day from day 3 to day 7; changing the liquid from the top every day from 8 th to 17 th; changing the top and bottom liquid every day after 18 days; the top liquid change is 0.5mL, and the bottom liquid change is 1.5 mL.

Investigation of Caco-2 cell monolayer integrity: caco-2 cells were cultured in a Transwell chamber for 21 days and detected to form a monolayer model (TEER)>200Ω/cm²) Starting a transmembrane transport assay; the fluorescein is a Caco-2 cell side transport marker, can be used for detecting the integrity of a Caco-2 cell monolayer model, and the fluorescein Papp should be used when cells form tight junction<5.0*10^-7cm/s。

Fluorescent yellow penetration experiment: before the experiment, the basal side and the basal side are respectively cleaned for 2 times by using preheated HBSS, and the basal side is changed to 400ul to be added with a solution of 100ug/ml of fluorescent yellow; at 37 ℃ 5% CO₂Sampling 100ul of the culture medium from the base side after culturing for 1h in the incubator, and detecting the fluorescence intensity by using a fluorescence microplate reader under the conditions of 428nm of excitation wavelength and 519nm of emission wavelength; the apparent permeability coefficient (Papp) is calculated as follows: papp ═ (dQ/dt)/(a × C0), where dQ/dt is the rate of penetration of insulin per unit time and a is the area of the polycarbonate membrane (0.33 cm for a 24-well plate)²) And C0 is the initial concentration of drug.

Papp values of 2.6X 10 were determined during the experiment^-7cm/s, less than 5.0X 10^-7cm/s, indicating that a dense tight junction has been formed, and a transmembrane transport experiment can be performed, and the experimental result is shown in fig. 2.

It is shown that the oral delivery composition prepared in example 1and the oral delivery composition prepared in example 4 can significantly increase the trans-cellular transport and apparent permeability coefficient of the corresponding derived peptide without significant effect on the electrical resistivity, which indicates that the increase in trans-cellular transport is not achieved by a paracellular route, but by increasing the trans-cellular absorption of the target derived peptide.

4. Pharmacokinetic experiments in rats

SD rats are adaptively fed for 2d before experiment, fasted for 12h before administration without water supply, and fasted for 2h after administration without water supply. The target composition is perfused into stomach (ig) or injected into tail vein (iv) at a dose of 46 mg/kg, blood is taken from tail vein after administration for 0, 5, 10, 20, 30, 45, 60min and 1.5, 2, 3, 5, 8, 10, 12h (within 2h after administration, physiological saline with the same blood taking amount is supplemented by intraperitoneal injection every 0.5h, and free drinking water is allowed after 2 h), plasma is separated, sealed and stored in a refrigerator at-80 ℃ for later use.

Sample preparation: plasma samples or plasma diluted samples were taken at 100. mu.L, and 5. mu.L (1000 ng. mL) of internal standard solution was added^-1. ) Mixing, adding 0.3% phosphoric acid-methanol solution 400 μ L, vortex mixing for 4min to precipitate protein, 10000 r.min^-1Centrifuging for 10min, collecting 450 μ L supernatant, centrifuging at room temperature, concentrating to dry, dissolving with 20% methanol water 100 μ L, and performing 12000 r min^-1Centrifuging for 10min, and collecting supernatant for UPLC-MS-MS analysis.

Preparation of a standard solution: precisely weighing appropriate amount of danshensu reference substance, adding 1.5% phosphoric acid water solution to obtain 0.860 mg/mL solution^-1The stock solution of (4) is stored at 4 ℃; before use, appropriate amount of stock solution of reference substance is diluted to 0.200 mg/mL^-1And then diluted by fold with rat blank plasma to the desired concentration.

Internal standard solutions were then prepared and pharmacokinetic parameters for different groups at different routes of administration were calculated by UPLC-MS with the results shown in the following table:

table 2 pharmacokinetic parameters for oral delivery compositions prepared in example 1 under different modes of administration

Experiments show that the bioavailability of liraglutide derivative polypeptide can be remarkably improved by constructing an oral delivery composition, and theoretical basis is provided for further developing oral GLP-1 receptor agonist drugs.

The above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention should be defined by the claims, and equivalents including technical features of the claims, i.e., equivalent modifications within the scope of the present invention.

Claims

1. A class of oral delivery compositions characterized by: including liraglutide derivative polypeptides and N- (8- (2-hydroxybenzoyl) amino) caprylate; the mass ratio of the liraglutide derivative polypeptide to the N- (8- (2-hydroxybenzoyl) amino) caprylate is 1: 10-120;

n₁is an integer of 6 to 22; n is₂Is an integer of 1 to 20.

2. A novel class of oral delivery compositions according to claim 1, characterized by: in the compound represented by the general formula (2), n₁Is an integer of 12 to 20; in the compound represented by the general formula (3), n₂Is an integer of 3 to 10.

3. A novel class of oral delivery compositions according to claim 1, wherein the liraglutide derived polypeptide is any one of the following compounds:

4. a novel class of oral delivery compositions according to claim 1, wherein the liraglutide derived polypeptide is any one of the following compounds:

5. the novel oral delivery composition according to any one of claims 1 to 4, characterized in that: the composition comprises one or more pharmaceutically acceptable excipients.

6. The novel oral delivery composition according to any one of claims 1 to 4, characterized in that: the composition is in the form of a solid, liquid or semi-solid.

7. The novel oral delivery composition according to any one of claims 1 to 4, characterized in that: the application of the composition in preparing a medicament for treating or preventing diabetes.

8. A process for preparing the novel oral delivery composition of any one of claims 1 to 4, characterized in that: the liraglutide derivative polypeptide is prepared by liquid phase synthesis and solid phase synthesis.