CN114685642B - Pharmaceutically acceptable salt of incretin analogue, and preparation method and application thereof - Google Patents

Abstract

The invention provides pharmaceutically acceptable salt of an incretin analogue, which has better stability and patentability, and is suitable for industrial production.

Description

Pharmaceutically acceptable salt of incretin analogue, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to pharmaceutically acceptable salts of an incretin analogue, a preparation method and application thereof.

Background

Hyperglycemia is caused by defective insulin secretion or impaired biological action, or both. Clinically, diabetes is generally classified into type one diabetes, type two diabetes, and the like. The diabetes mellitus has hyperglycemia symptoms for a long time, and various tissues, especially eyes, kidneys, hearts, blood vessels and nerves, are chronically damaged and dysfunctions caused by severe hyperglycemia, so that the health of human beings is threatened. Diabetes and its complications are becoming an important threat to human health, especially as lifestyle changes and aging populations become more and more advanced.

The main categories of clinical therapeutic drugs for type two diabetes in the prior art include: biguanide type diabetes drugs (metformin, or phenformin), sulfonylurea type diabetes drugs (glibenclamide, glipizide, glimepiride, or gliquidone), glucosidase inhibitor type drugs (acarbose, fu Geli-wave sugar, or miglitol), insulin sensitization type drugs (cyclic glitazone, troglitazone, rosiglitazone, or pioglitazone), aldose reductase inhibitor type drugs (aspartame, epanostat, or tolslata), insulinotropic type drugs (repaglinide), or nateglinide). Although various types of diabetes therapeutic drugs have been developed, there are many negative factors that limit the use of some drugs.

Among the currently marketed protein drugs, mainly human glucagon-like peptide-1 receptor (GLP-1R) agonists, such as Liraglutide (trade name SAXENDA) and Victoza) and semaglutin (trade name Ozempic), are used for the treatment of type II diabetes. Liraglutide is a chemically modified GLP-1 analogue, fatty acid (hexadecanoic acid) is connected to lysine at position 26 of GLP-1 protein skeleton through gamma-Glu, and the fatty acid can be combined with serum albumin, and is clinically administered once a day for two indications of reducing blood sugar and weight respectively. Structurally, the somalupeptide is composed of 8 Aib substituted Ala and 34 Arg substituted Lys on GLP-1 (7-37) chain, and 26 Lys connected with octadecane fatty acid chain. Compared with liraglutide, the fatty acid chain of the somalunin is longer, the affinity with serum albumin is higher, and the subcutaneous injection is clinically carried out 1 time per week.

The diabetics are generally obese, and the weight loss is obviously improved for diabetes. Therefore, weight loss is an important indicator for GLP-1 analogs. Liraglutide, while being approved for the treatment of obesity, actually loses approximately only 5.6 kg of weight. Whereas the average weight loss of the clinically treated groups of somalundin (0.5 mg), somalundin (1.0 mg) was 4.2kg and 5.5kg. The current drugs for obesity generally have a weight loss of about 5-10% (compared to placebo), i.e. the average weight loss ratio as a whole is not more than 10% of the patient's body weight (Rudolph L. Leibel et al, diabetes,64 (7): 2299-2309, 2015).

At present, although several pharmaceutical companies and scientific research institutions are developing multispecific drugs based on natural incretin sequences, there is still a lack of drugs that are clinically excellent, and further research and development is required in this field.

Because the polypeptide peptide sequence contains amino and carboxyl, the product can exist in the form of internal salt and/or combined salt root after the refining process. According to different peptide sequences, the solubility of the polypeptide can be increased after salt formation by combining with salt root, and the stability of the polypeptide and the preparation thereof is enhanced, so that the research on the salt formation of polypeptide medicines is necessary.

Disclosure of Invention

The invention aims to provide pharmaceutically acceptable salts of an incretin analogue, a preparation method and application thereof.

In a first aspect of the invention there is provided an incretin analogue comprising a glucagon-like polypeptide and a long chain fatty acid linked thereto; wherein the amino acid sequence of the glucagon-like polypeptide is shown as the formula (I):

YSEGTFTSDX ₁₀ SKYLDSQAAQDFVQWLLAGGPSSGAPPPSX ₄₀ (I)；

in the formula (I), X ₁₀ Is amino acid K, X ₄₀ Selected from the group OH or NH ₂ 。

In another aspect of the present invention, there is provided a pharmaceutically acceptable salt of an incretin analog, characterized in that the incretin analog has the amino acid sequence of formula (I):

YSEGTFTSDX ₁₀ SKYLDSQAAQDFVQWLLAGGPSSGAPPPSX ₄₀ (I)；

In the formula (I), X ₁₀ Is amino acid K, X ₄₀ Selected from the group OH or NH ₂ ；

Wherein "pharmaceutically acceptable salt" may be composed of a pharmaceutically acceptable non-toxic acid or base.

Further, the pharmaceutically acceptable salt of the incretin analogue has the amino acid sequence as follows:

or (b)

；

Wherein "pharmaceutically acceptable salt" may be composed of a pharmaceutically acceptable non-toxic acid or base.

Further, the acid includes inorganic acids and organic acids.

Further, the organic acid is one or more of acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, dichloroacetic acid, formic acid, fumaric acid, gluconic acid, glutamic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, trifluoroacetic acid, oxalic acid and p-toluenesulfonic acid.

Further, the alkali is one or more of alkali metal salt, alkaline earth metal salt, aluminum salt, fatty amine salt, aromatic amine salt and ammonium salt.

Fatty amines refer to alkyl primary, secondary and tertiary amines; aromatic amine refers to an amine having one aromatic substituent.

Further, the alkali is preferably one or more of sodium salt, potassium salt, calcium salt, magnesium salt, aluminum salt, triethylamine salt, dimethylamine salt, diethylamine salt, DIEA salt, tromethamine salt and ammonium salt.

Further, the base is more preferably a sodium salt or a potassium salt.

In another aspect of the invention, there is provided a process for the preparation of a pharmaceutically acceptable salt of an incretin analog, characterized in that it is chemically converted to the root salt.

In another aspect of the invention there is provided the use of a pharmaceutically acceptable salt of an incretin analogue in the preparation of a composition for: activating the human glucagon-like peptide-1 receptor, the glucose-dependent insulinotropic polypeptide receptor and/or the glucagon receptor; preventing, alleviating or treating metabolic disorders; or reducing food intake, reducing fat, reducing body weight, or reducing blood glucose.

In another aspect of the invention, there is provided a composition comprising said one incretin analog pharmaceutically acceptable salt, and a carrier; the carrier is a pharmaceutically, dietetic or health-care acceptable carrier.

In another aspect of the invention, there is provided a method of non-therapeutically reducing food intake, reducing fat, reducing weight, or reducing blood glucose comprising administering to a subject in need of reduced food intake, reducing fat, reducing weight, or reducing blood glucose a pharmaceutically acceptable salt of an incretin analog, or a composition as defined herein.

In a preferred embodiment, the long chain fatty acid is a fatty acid having 14 to 20 carbons; preferably fatty acids containing 16 to 18 carbons; more preferably the long chain fatty acid is a straight chain saturated monocarboxylic acid; more preferably, the long chain fatty acid is palmitic acid.

In another preferred embodiment, the long chain fatty acid is linked to amino acid K of the peptide chain of the glucagon-like polypeptide; preferably, is connected to X ₁₀ Amino acids of (a); preferably, the linkage is cross-linking.

In a preferred embodiment, the glucagon-like polypeptide is linked to the long chain fatty acid via a linker; preferably, the linker is one that is reactive with amino acid K and with the reactive group of the long chain fatty acid.

In a preferred embodiment, the linker is a linker comprising at least 1 (e.g., 1-6) units of-gammaglutyl- (-gammaglut-).

In a preferred embodiment, the incretin analog has the structure of formula (II):

(II)。

in another aspect of the invention there is provided the use of said incretin analogue in the preparation of a composition for:

activating human glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor (GIPR) and/or glucagon receptor (GCGR);

Preventing, alleviating or treating metabolic disorders; or (b)

Reduced food intake, reduced fat, reduced weight, or reduced blood glucose.

In a preferred embodiment, the reduction of food intake, fat, weight or blood glucose may be a non-therapeutic situation.

In a preferred embodiment, the metabolic disease comprises: hyperglycemia-related metabolic diseases or hyperlipidemia-related metabolic diseases.

In another preferred embodiment, the hyperglycemia-related metabolic disease comprises: diabetes or metabolic syndrome associated with diabetes; preferably, the metabolic syndrome associated with diabetes includes insulin resistance, glucose intolerance.

In another preferred example, the hyperlipidemia-associated metabolic disease comprises: obesity, hyperlipidemia, fatty liver, hypertriglyceridemia, hypercholesterolemia, low HDL cholesterol, high LDL cholesterol; preferably, the fatty liver comprises non-alcoholic fatty liver disease (NAFLD), more preferably non-alcoholic steatohepatitis (NASH).

In another aspect of the invention, there is provided a composition comprising an incretin analog as described above, and a carrier; the carrier is a pharmaceutically, dietetic or health-care acceptable carrier.

In a preferred embodiment, the incretin analog is in an effective amount.

In another preferred embodiment, the composition includes, but is not limited to: pharmaceutical compositions, food compositions or nutraceutical compositions, etc.

In another aspect of the invention there is provided a pharmaceutically acceptable salt of a glucagon-like polypeptide for the preparation of an incretin analog as defined in claim 1, having the amino acid sequence of formula (I):

YSEGTFTSDX ₁₀ SKYLDSQAAQDFVQWLLAGGPSSGAPPPSX ₄₀ (I)；

in the formula (I), X ₁₀ Is amino acid K, X ₄₀ Selected from the group OH or NH ₂ ；

Wherein "pharmaceutically acceptable salt" may be composed of a pharmaceutically acceptable non-toxic acid or base.

In another aspect of the invention, polynucleotides encoding the glucagon-like polypeptides, expression vectors containing the polynucleotides and/or recombinant cells containing the polynucleotides are provided.

In another aspect of the present invention, there is provided a method for preparing the incretin analog, comprising: the glucagon-like polypeptide is linked to a long chain fatty acid.

In another aspect of the invention, there is provided a method of non-therapeutically reducing food intake, reducing fat, reducing body weight, or reducing blood glucose comprising administering to a subject in need of reduced food intake, reducing fat, reducing body weight, or reducing blood glucose the incretin analog, or the composition.

In another aspect of the present invention, there is provided a kit comprising: said incretin analog; or comprises said composition.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 is a graph showing the results of mass spectrometry analysis of the incretin analog P3 YELAN.

FIG. 2 is a graph showing the measurement of GLP-1R agonistic activity of the incretin analog P3YELAN in vitro; duraglutide was used as a control.

FIG. 3 is a graph showing the results of measurement of the in vitro GIPR agonistic activity of the incretin analog P3 YELAN.

FIG. 4 shows the results of an in vitro GCGR agonistic activity of the incretin analog P3 YELAN.

FIG. 5 is a graph showing the results of the in vitro serum stability of the incretin analog P3 YELAN; YELAN and Duraglutide were used as controls.

FIG. 6 shows the results of the in vitro serum stability of each of the salt roots of the incretin analog P3 YELAN.

FIG. 7 shows the results of random blood glucose changes in db/db mice with the incretin analog P3 YELAN; dolapride is used as a positive control; normal control (Normal control) is a Normal, non-diseased animal; the Negative control (Negative control) was a solvent control (no P3YELAN or positive control drug was given).

FIG. 8 shows the results of weight change of the incretin analog P3YELAN in DIO mice; liraglutide was used as a positive control; normal control (Normal control) is Normal uninduced obese animals; the Negative control (Negative control) was a solvent control (no P3YELAN or positive control drug was given).

FIG. 9 is the cumulative feeding change results of the incretin analog P3YELAN in DIO mice; liraglutide was used as a positive control; normal control (Normal control) is Normal uninduced obese animals; the Negative control (Negative control) was a solvent control (no P3YELAN or positive control drug was given).

FIG. 10 shows the results of fasting blood glucose detection of the incretin analog P3YELAN in DIO mice; liraglutide was used as a positive control; normal control (Normal control) is Normal uninduced obese animals; the Negative control (Negative control) was a solvent control (no P3YELAN or positive control drug was given).

Detailed Description

In view of the defects of the incretins drugs in the prior art, the inventor provides an incretins analogue which has GLP-1R/GIPR/GCGR agonistic activity and is a three-effect agonist, and the incretins analogue has remarkable effects on reducing blood sugar, fat and weight. The invention also provides a preparation method and application of the pharmaceutically acceptable salt.

Incretin analogues and preparation thereof

The invention provides a pharmaceutically acceptable salt of an incretin analog, which comprises glucagon-like polypeptide and long chain fatty acid connected with the glucagon-like polypeptide.

The sequence of the glucagon-like polypeptide used to create the incretin analog is (SEQ ID NO: 2):

YSEGT FTSDX ₁₀ SKYLD SQAAQ DFVQW LLAGG PSSGA PPPSX ₄₀ (I)

wherein X is ₁₀ = K，X ₄₀ Is OH or NH ₂ 。

Glucagon-like polypeptides of the invention may be recombinant polypeptides, synthetic polypeptides. It may be a product of chemical synthesis or produced from a prokaryotic or eukaryotic host (e.g., bacterial, yeast, higher plant, insect, and mammalian cells) using recombinant techniques.

The invention also includes fragments, derivatives and analogs of the glucagon-like polypeptide shown in SEQ ID NO. 2. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity of such glucagon polypeptides. The polypeptide fragment, derivative or analogue may be (i) a polypeptide having one or more (e.g. 1-5, 1-3 or 1-2) conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more (e.g. 1-5, 1-3 or 1-2) amino acid residues, or (iii) a polypeptide (e.g. a leader or secretory sequence or a pro-protein sequence for purification of the polypeptide, or a fusion protein) formed by fusion of an additional amino acid sequence to the polypeptide sequence. Such fragments, derivatives and analogs are within the purview of one skilled in the art in view of the definitions herein.

Also included in the present invention are polypeptides having at least 75% (preferably at least 80%,85%,90%, 95%) sequence identity (sequence identity) to the amino acid sequence of the glucagon-like polypeptide shown in SEQ ID NO. 2 and having the function of the glucagon-like polypeptide shown.

In the present invention, a polypeptide comprising one or more amino acids modified (usually without changing the primary structure) to increase the stability, half-life and promotion of the polypeptide is also included, which comprises: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Polypeptides modified to improve hydrolytic resistance or to optimize solubility are also included.

The invention also provides polynucleotide sequences encoding the glucagon-like polypeptides of the invention or conservative variations thereof. The polynucleotides of the invention may be in the form of DNA or RNA. The DNA may be a coding strand or a non-coding strand. That is, a "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and/or non-coding sequences.

The invention also relates to expression vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered with the expression vectors or the coding sequences for glucagon-like polypeptides, and methods for producing the polypeptides by recombinant techniques.

The invention also provides application of the glucagon-like polypeptide, which is used for preparing the incretin analogue.

The long chain fatty acid used to create the incretin analog is a fatty acid containing 14 to 20 carbons, preferably it is a fatty acid containing 16 to 18 carbons. In the present invention, esters, ethers or derivatives of the long chain fatty acids are also included, as well as salts (e.g., sodium salts) of the long chain fatty acids. In a preferred mode of the invention, the long chain fatty acid is a straight chain saturated monocarboxylic acid.

As a preferred mode of the invention, the linkage is chemical crosslinking. To achieve the purpose of chemical crosslinking, the present inventors introduced amino acids K or C based on the original sequence of the polypeptide, and crosslinked fatty acids to K or C.

After the glucagon-like polypeptide is crosslinked with long-chain fatty acid, the activity of the glucagon-like polypeptide is obviously improved, and the half life of the medicine in vivo is obviously prolonged. Preferably, Y at position 10 is mutated to K (in formula I: X ₁₀ =k), whereby fatty acid chains can be cross-linked to the incretin analog polypeptide fragment by K.

In a preferred embodiment of the present invention, the incretin analog provided by the present invention, wherein the fatty acid chain is palmitic acid (C16), is a linear monocarboxylic acid, and has the chemical structural formula shown as follows:

in the incretin analog provided by the invention, a joint can be arranged between the glucagon-like polypeptide fragment and the long-acting carrier. The linker may typically be reacted with lysine residue K and/or cysteine residue C on the glucagon-like polypeptide fragment, respectively, and an active group on the long-acting carrier (e.g., an active group such as carboxyl, maleimide, etc. may be included in the linker) such that both ends of the linker are attached to the long-acting carrier and the incretin analog polypeptide fragment, respectively, to effect cross-linking of the long-acting carrier and the incretin analog polypeptide fragment, e.g., various types of condensation reactions may be employed.

The linker may be any linker suitable in the art for linking the incretin analog polypeptide fragment and the long acting carrier, and in some embodiments of the invention, the linker may be-gammaglu- (-gammaglutamoyl-). A group of the formula:

As mentioned above, the C-terminal amino acid of the incretin analogs provided herein may be modified, such as amidated. The amidation generally refers to the conversion of the C-terminal-COOH group to-CONH ₂ Groups such as those of formula (I): x is X ₄₀ Is NH ₂ 。

The salt root provided by the invention comprises acid and alkali. Wherein the acid is acetic acid or trifluoroacetic acid. Acceptable base salts include sodium, potassium, ammonium salts.

In some embodiments of the invention, the specific sequence of the incretin analog is shown in Table 1 as SEQ ID NO. 6. In Table 1, the sequence alignment of the polypeptide fragment with Glucago (SEQ ID NO:1, abbreviated as GCG), GLP-1 (SEQ ID NO: 3) and GIP (SEQ ID NO: 4) is also presented.

TABLE 1

In Table 1, - γE-i.e. - γGlu (- γ -glutamyl-), "γE-C16" means that palmitoyl is conjugated to the epsilon nitrogen of lysine via a- γ -glutamyl-linker. "2xOEG" means 2-OEG- (-2- (2- (2-aminoethoxy) ethoxy) acetyl-) linkages.

The invention also provides a preparation method of the incretin analog, which comprises the steps of connecting the glucagon-like polypeptide with long-chain fatty acid and converting salt roots.

The preparation method can comprise the following steps: preparing the incretin analog by a chemical synthesis method; the preparation method can also comprise the following steps: culturing a proper host cell under proper conditions to express the incretin analog polypeptide fragment, separating and purifying to obtain the incretin analog polypeptide fragment, and chemically crosslinking the long-acting carrier to the incretin analog polypeptide fragment and converting the long-acting carrier into different salt roots in a chemical mode. The incretin analogs of the invention can be prepared by standard peptide synthesis methods, e.g., by standard solid or liquid phase methods, stepwise or by fragment assembly, and isolation and purification of the final incretin analog polypeptide fragments, incretin analog products, or by any combination of recombinant and synthetic methods, and conversion to different salt roots by chemical means.

Use of incretin analogues

The invention also provides application of the incretin analog in preparing medicines for treating metabolic diseases and GLP-1R/GIPR/GCGR pleiotropic agonists. The metabolic disease may be specifically selected from diabetes, obesity, blood lipid disorders, non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH), other metabolic syndromes associated with diabetes, including hypertriglyceridemia, low HDL cholesterol and high LDL cholesterol, insulin resistance, obesity or glucose intolerance, etc.

The invention also provides a method for treating diseases, which comprises the following steps: administering to a subject an incretin analog provided in accordance with the first aspect of the invention. In the random blood sugar detection test, the blood sugar reducing effect and the weight reducing effect of the diabetes model mice to which the incretin analog of the invention is applied are obviously better than those of the control group samples.

In addition to being useful in the prevention, alleviation or treatment of disease, the incretin analogs of the present invention may also be useful in some non-therapeutic applications. Partial results according to embodiments of the present invention show that the incretin analogues are capable of very significantly reducing food intake, reducing fat, reducing body weight or reducing blood glucose. Thus, the incretin analogs of the present invention may also be applied to subjects who do not have disease characteristics in their bodies, but who have a need to control food intake, reduce fat, and reduce weight.

Although in the prior art, research and development of molecules with multiple agonistic activity have been carried out in the art, it is actually very difficult to obtain an ideal drug of this type. First is a safety issue, particularly an immunogenicity issue. The hypoglycemic weight-losing medicine needs to be used for a long time and has extremely high safety requirement. In order to design and obtain a polypeptide with high multiple agonistic activity and stable in vivo, the prior technical schemes often introduce more mutation sites and often introduce unnatural amino acids and other modifications. The introduction of these mutations and unnatural amino acids both increase the risk of potential immunogenicity. Safety is extremely important for drugs for treating diabetes, obesity, and the like. In addition, for small peptides of about 30 amino acids in length, such as GLP-1, glucoagon, changes in sequence are extremely sensitive to changes in activity; in the case of multiple active polypeptides, however, the change is more complex due to the fact that it involves agonism at a plurality of different receptors, and it is not predicted at all what the effect on receptor agonism will be upon any one amino acid change. For example, joseph Chabenne et al report (Joseph Chabenne et al, molecular Metabolism, 3:293-300, 2014) that GCGR agonistic activity was greatly reduced by mutations at positions 1, 2, 3, 4, 6-12, 14, 15, 22, 23, 25-27, 29 of GCG after alanine scanning (Ala scan) of GCG was performed, each site of GCG was independently replaced with alanine, and the relative residual activity remained spanning from 0.2% to 100% (Table 4 in the article). However, it can be seen from other reports that the mutation at one or several of these sites is performed simultaneously, and that the change in activity is not always consistent with the result of alanine scanning when substituted with other amino acids. As reported by Jonathan W Day et al (Jonathan W Day et al, nature Chemical Biology,5:749-757, 2009), the GCGR agonistic activity was instead improved by performing 16 S.fwdarw.G, 16 S.fwdarw.T, 16 S.fwdarw.H, 16 S.fwdarw.E on position 16 of GCG, which is completely contradictory to the alanine scanning results of Joseph channel. Second, the Joseph channel study suggests that substitution with alanine at position 23 will result in almost complete loss of GCGR agonist activity (retention of only 1.1%); however, jonathan W Day et al mutated position 23 to Ile and did not decrease GCG activity. For example, the alanine scanning result shows that the second position S is very important for retaining GCG activity (activity is only 1/3 of that of mutation to Ala), but Brian Finan et al report (Finan B et al, nat Med 2015; 21:27-36.) that the relative agonistic activity of GCGR is instead improved to 200% -640% after substitution mutation of 2S- & gtAib, 2S- & gtdSer, 2S- & gtG, 2S- & gtdAba on the second amino acid of GCG is carried out respectively and mutation of other sites is combined. It has also been found in the studies of the present inventors that when some combinations of mutations are introduced which are advantageous for increasing GLP-1, GCG or GIP activity, the effect is quite often not consistent with single site mutations. Furthermore, increasing or decreasing amino acids at both the N-and C-termini of polypeptides such as GLP-1, exendin-4, GCG or GIP affects the biological activity. If one or two amino acids are removed from the N-terminus, the agonistic activity of GLP-1, GCG, etc. is completely lost. For example, oxyntomodulin has only 8 more KRRNNIA than Glucoago C-terminal, and its GCGR agonistic activity is lost by about 90% (Alessandro Pocai et al, diabetes; 58 (10): 2258-2266, 2009;Henderson SJ et al, diabetes Obes Metab, 2016).

The incretin analog provided by the invention has extremely high GLP-1R and GIPR agonistic activities and slightly weak GCGR agonistic activities, and surprisingly, the in vitro activity of the incretin analog polypeptide is remarkably changed before and after fatty acid crosslinking.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Composition and method for producing the same

The present invention also provides a composition comprising an effective amount of an incretin analog of the present invention and a carrier; the carrier is a pharmaceutically, dietetic or health-care acceptable carrier. Such compositions include, but are not limited to: pharmaceutical compositions, food compositions or nutraceutical compositions, etc.

In the present invention, the pharmaceutical composition may contain the incretin analog in a weight ratio of 0.01-95% (e.g., 0.1%,1%,5%,10%,20%,30%,50%,80%, etc.).

As used herein, a "pharmaceutically, dietetic or nutraceutically acceptable" ingredient is one that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic response), commensurate with a reasonable benefit/risk ratio; such as pharmaceutical carriers or excipients commonly used in the art.

As used herein, "effective amount" or "effective dose" refers to that amount which is functional or active in and acceptable to humans and/or animals as used herein.

The dosage form of the pharmaceutical composition of the present invention may be various, as long as it is a dosage form capable of allowing the active ingredient to reach the mammalian body effectively. For example, it may be selected from: a gel, aerosol, tablet, capsule, powder, granule, syrup, solution, or suspension. The type of disease to be treated with the compounds of the invention can be selected by those skilled in the art as a convenient dosage form for use.

Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. A sufficient description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). The pharmaceutically acceptable carrier in the composition may contain a liquid such as water, phosphate buffer, ringer's solution, physiological saline, balanced salt solution, glycerin or sorbitol, etc. In addition, auxiliary substances such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers, such as albumin, may also be present in these carriers.

Preferred pharmaceutical compositions are solid compositions, especially tablets and solid filled or liquid filled capsules, from the standpoint of ease of preparation and administration. The compounds of the present invention or pharmaceutical compositions thereof may also be stored in a disinfecting device suitable for injection or instillation.

The effective dosage of the incretin analog of the present invention as an active ingredient may vary depending on the mode of administration and the severity of the condition to be treated, for example, at a dosage of about 0.00001-10mg/kg body weight per day. The administration time may also be adjusted, for example, in relation to administration in a slow release form, which may be administered once every day or several days apart. The dosage regimen can be adjusted to provide the optimal therapeutic response.

When the composition is applied to large animals and patients, the effective use dosage (including solid or solution dosage conversion) of the large animals or the human can be converted according to the use dosage of the small animals through corresponding professional conversion formulas. In particular embodiments of the invention, dosing regimens for animals such as mice are presented. The conversion from an animal, such as a mouse, to a dose suitable for human administration is readily made by a person skilled in the art, and can be calculated, for example, according to the Meeh-Rubner formula: meeh-Rubner formula: a=k' (W2/3)/10,000. Wherein A is the body surface area and calculated by m 2; w is body weight, calculated as g; k is a constant and varies with animal species, and generally, mouse and rat 9.1, guinea pig 9.8, rabbit 10.1, cat 9.9, dog 11.2, monkey 11.8, human 10.6. It will be appreciated that the conversion of the dosage administered may vary depending on the drug and clinical situation, as assessed by an experienced pharmacist.

The invention also provides a kit or kit comprising: said incretin analog; or a pharmaceutical composition as described. For ease of clinical application, other auxiliary accessories, such as syringes, etc., may also be included in the kits or kits of the present invention. Instructions for use may also be included in the kit or kit to facilitate proper use by those skilled in the art.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art literature and see, in particular, sambrook et al MOLECULAR CLONING: a LABORATORY MANUAL, second edition, cold Spring Harbor Laboratory Press,1989 and Third edition,2001; ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987 and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego, 1998; METHODS IN ENZYMOLOGY, vol.304, chromatin (p.m. wassman and a.p. wolffe, eds.), academic Press, san Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatin Protocols (p.b. becker, ed.) Humana Press, totowa,1999, etc.

Abbreviations referred to in the examples have the following meanings:

RT: room temperature;

DMF: n, N-dimethylformamide;

fmoc: 9H-fluoren-9-ylmethoxycarbonyl;

trt: a trityl group;

boc: t-butoxycarbonyl;

HOBt: 1-hydroxybenzotriazole;

tBu, t-butyl;

DCM: dichloromethane;

DBLK:20% N, N-dimethylformamide piperidine;

DIC: n, N' -diisopropylcarbodiimide;

MeOH: methanol;

TFA: trifluoroacetic acid;

Fmoc-Lys(Pal-Glu-OtBu)-OH：N ^α fmoc- (N) ^ε - (gamma-glutamyl (N) ^α -hexadecyl, α -tert-butyl)) lysine;

- γe-: - γ -glutamyl-;

-OEG-: 2- (2- (2-Aminoethoxy) ethoxy) acetyl-

DIEA: n, N-diisopropylethylamine;

MTBE: methyl tertiary butyl ether.

TFEA:2, 2-trifluoroethanol

Pd (PPh 3) 4: tetrakis (triphenylphosphine) palladium

Alloc: allyloxycarbonyl group

The commercial amino acids and amino acid fragments, and the commercial resins referred to in the examples, were manufactured and the commercial models were as follows:

fmoc protecting group amino acid raw material, 2-CTC resin and Rink amide MBHA resin are all conventional commercial reagents (protecting amino acid manufacturer: chengdu Zheng Yuan Biochemical technology Co., ltd., resin manufacturer: tianjin Nanka and Tech Co., ltd.);

the organic solvent and other raw material sources are all commercial products (manufacturer: national medicine group chemical reagent Co., ltd.; chemical purity).

In addition, the conditions of HPLC and mass spectrometry and the equipment model and manufacturer used are described as follows:

instrument: HPLC UltiMate3000; the detection conditions are shown in table 2 below.

TABLE 2

Preparing a liquid phase: beijing innovation is general, LC3000.

Mass spectrometry: the instrument model was 5800 MALDI-TOF-TOF (AB SCIEX), the analysis software was T0F/TOF Explorer, data Explorer, MS used Reflector Positive parameters: CID (OFF), mass range (700-6500 Da) Focus Mass (1200 Da) Fixed laser intensity (5600) digizer: bin Size (0.5 ns)

EXAMPLE 1 preparation of incretin analog

1. Preparation of the incretin analog P3YELAN

The structure of the incretin analog P3YELAN is as follows:

namely: YSEGT FTSDX ₁₀ SKYLD SQAAQ DFVQW LLAGG PSSGA PPPS-NH ₂

X ₁₀ =k (palmitoyl- γe). Synthesis of Fmoc-Ser (tBu) -Rink amide MBHA resin:

2.65g of Rink amide MBHA resin (Tianjin Nanking and Chemie Co., ltd.) having a substitution of 0.38mmol/g was weighed into a solid phase reaction column, 10mL of DCM was added to swell the resin for 30 minutes, and the resin was washed 3 times with 10mL of DMF. 15mL of DBLK solution was added to the reaction column, the reaction was performed for 5 minutes, suction filtration was performed, 20mL of DMF was used for washing once, 15mL of DBLK solution was added, and the reaction was performed for 10 minutes, and Kaiser detected positive. Suction filtration and washing with DMF 3 times, 20mL each.

1.91g of Fmoc-Ser (tBu) -OH and 0.81g of HOBt were dissolved in 10mL of LDMF, activated by adding 0.69g of DIC at 5-8deg.C for 5min, and then placed in the above resin-filled reaction column to react for 2 hours. After Kaiser detection was negative, it was used directly for the next peptide resin synthesis.

Synthesis of peptide resin:

the resin Fmoc-Ser (tBu) -Rink amide MBHA resin (1.0 mmol) was weighed and added to the reaction column and swollen with 20mL DCM for 30 minutes before washing 3 times with 20mL each. After the washing was completed, 10mL of DBLK solution (20% piperidine/DMF (V/V)) was added to the reaction column, the reaction was performed for 5 minutes, suction filtration was performed, 20mL of DMF was used for washing once, 10mL of DBLK solution (20% piperidine/DMF (V/V)) was added, the reaction was performed for 10 minutes, and the Kaiser test was positive. Suction filtration and washing with DMF 3 times, 20mL each. Fmoc-Pro-OH (1.69 g,5.0 eq) and HOBt (0.81 g,6.0 eq) were dissolved in 10mL DMF and DIC (0.69 g,5.5 eq) was added at 5-8deg.CAfter 5min of incubation, the reaction was carried out for 1 hour, and the Kaiser was negative, and the reaction was complete, washed 3 times with 20mL portions of DMF. Repeating the deprotection and coupling operations to sequentially complete the coupling of other amino acids according to the sequence of the peptide, wherein X ₁₀ Coupling was performed using Fmoc-Lys (Pal-Glu-OtBu) -OH (Chengdu Zheng Yuan Biochemical technologies Co., ltd.). After the final amino acid coupling was completed, deprotection was performed as described above, and after the deprotection was completed, DMF was washed 2 times, meOH was washed 2 times, DCM was washed 2 times and MeOH was washed 2 times in sequence, with 20mL of solvent for each wash. And (5) receiving materials, and drying at normal temperature under reduced pressure to obtain the target peptide resin.

Cleavage of crude peptide:

5.02g of the peptide resin is weighed and slowly added into 60mL of lysate (trifluoroacetic acid: phenylthioether: anisole: ethanedithiol=90:5:3:2) at 20-30 ℃ to react for 2 hours after the addition. After the completion of the reaction, the resin was removed by filtration, and the filtrate was poured into methyl tertiary ether (600 mL) pre-cooled in advance with vigorous stirring, and the resulting mixed solution was allowed to stand in a refrigerator for sedimentation for 2 hours. The supernatant was removed and washed 5 times with 400mL of pre-chilled methyl tertiary ether. After completion, the crude peptide (2.23 g) was obtained by collecting the material and drying it under reduced pressure at room temperature.

Purification of crude peptide:

crude peptides were refined by multi-step purification using the preparation liquid phase (Beijing innovation, LC 3000): the first step: stationary phase: c18 (Daisogel: sp-120-40/60-C18-RPS), mobile phase 0.1% TFA, acetonitrile; and a second step of: stationary phase: c8 (Daisogel: sp-120-10-C8-P), mobile phase: 0.5% phosphoric acid, acetonitrile, third step: stationary phase: c8 (Daisogel: sp-120-10-C8-P), mobile phase: 50mM ammonium acetate, 0.3% acetic acid, acetonitrile, and finally freeze-dried (Freeze-dryer Beijing boqikang, FD-2A) to obtain refined peptide (98.0%). And finally, determining the precise molecular weight of the refined peptide by adopting MS: m/z 4455.69 (M+H) ⁺ . The MS is shown in fig. 1.

2. Preparation of glucagon derivative P5 YELAN:

The glucagon derivative P5YELAN has the structural formula:

namely: YSEGT FTSDX ₁₀ SKYLD SQAAQ DFVQW LLAGG PSSGA PPPS-NH2

X ₁₀ =k (((octadecanedioic acid monoacyl) - γe) -2 xOEG)

Synthesizing branched protected amino acid W1 by a solid phase method: alloc-Lys ((Octadecanedioic Acid mono-tert-butyl) -Glu-OtBu) -OEG-OEG) -OH, has the structure:

synthesis of W1:

20g of 2-CTC resin with a substitution degree of 1.0mmol/g was weighed, added to a solid phase reaction column, added to the solid phase reaction column, washed 1 time with DMF, swelled with DMF for 30 minutes, then 8.53g of Alloc-Lys (Fmoc) -OH (20 mmol) was dissolved in DMF, 7.5ml of DIEA (45 mmol) was added under ice water bath to activate, then added to the above resin-filled reaction column, reacted for 2 hours, then 30ml of anhydrous methanol was added to block for 1 hour, and washed 3 times with DMF. Fmoc protection was removed with a mixed solution of DMF: pyridine in a volume ratio of 4:1, and then washed 6 times with DMF, 15.42g of [2- [2- (Fmoc-amino) ethoxy ] was weighed out]Ethoxy group]Acetic acid, 5.41g HOBt and DMF were added for dissolution, 6.2ml DIC was added under ice water bath for activation, and then the mixture was put into the above resin-filled reaction column and reacted at room temperature for 2 hours. Repeating the steps of removing Fmoc protection and adding corresponding materials for coupling, and sequentially completing the [2- [2- (Fmoc-amino) ethoxy ] according to the sequence of branched chain fragments ]Ethoxy group]Acetic acid, fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester. After coupling, the resin was washed 3 times with DMF, 5 times with MeOH and dried. The resin was added to 400ml of TFEA/dcm=1:4 and reacted at room temperature for 4h. After filtration of the resin, the filtrate was taken up in DCM and precipitated in 500ml MTBE, which was dried by centrifugation to give the title compound 19.43g, M/Z1242.51 (M+H) ⁺ 。

Polypeptide synthesis with P3YELAN, X ₁₀ Coupling was performed using W1 and the Alloc groups were removed using Pd (PPh 3) 4. The crude peptide obtained was purified by RP-HPLC and finally lyophilized to give the refined peptide (96.5%). M is MS, accurate molecular weight determination is carried out on the spermatid: m/z 4804.13 (M+H) ⁺ 。

3. Preparation of glucagon derivative P9 YELAN:

the glucagon derivative P9YELAN has the structural formula:

namely: YSEGT FTSDX ₁₀ SKYLD SQAAQ DFVQW LLAGG PSSGA PPPS-NH ₂

X ₁₀ =k (((eicosadioic monoacyl) - γe) -2 xOEG)

Synthesis of branched protected amino acid and P5YELAN, solid phase process to synthesize branched protected amino acid W2: alloc-Lys ((Eicosanedioic Acidmono-tert-butyl) -Glu-OtBu) -OEG) -OH (wherein fatty acid coupling employs mono-tert-butyl eicosanoate) has the following structure:

then polypeptide synthesis is carried out, wherein the polypeptide synthesis is identical to P3YELAN, and X ₁₀ Coupling was performed using W2 and the Alloc groups were removed using Pd (PPh 3) 4. The crude peptide obtained was purified by RP-HPLC to give the refined peptide (97.1%). MS determination of precise molecular weight for spermatid: m/z 4832.50 (M+H) ⁺ 。

Example 2, salt-transfer example

In example 1, the P3YELAN essential peptide was acetate, the acetic acid content was measured to be 2.2%, and the P3YELAN essential peptide was converted into 4 other salt forms: sodium salt, potassium salt, ammonium salt, trifluoroacetate salt, specific examples are as follows:

1. preparation of sodium salt

1.70g of P3YELAN refined peptide is weighed, dissolved in 340ml of purified water, loaded into a reversed phase polymer column, subjected to ion exchange by using 50mM sodium bicarbonate solution 95% + acetonitrile 5%, eluted by purified water 40% + acetonitrile 60% to obtain P3YELAN sodium salt solution, and lyophilized to obtain 1.63g of finished product. The sodium content of the finished product was measured to be 1.7%.

2. Preparation of Potassium salt

1.70g of P3YELAN refined peptide is weighed, dissolved in 340ml of purified water, loaded into a reversed phase polymer column, subjected to ion exchange by using 50mM potassium bicarbonate solution 95% + acetonitrile 5%, eluted by purified water 40% + acetonitrile 60%, and the P3YELAN potassium salt solution is obtained, and the finished product of 1.61g is obtained after freeze-drying. The potassium content in the finished product was measured to be 2.5%.

3. Preparation of ammonium salts

1.70g of P3YELAN refined peptide is weighed, dissolved in 340ml of purified water, loaded into a reversed phase polymer column, subjected to ion exchange by using 0.1% ammonia water solution 95% + acetonitrile 5%, eluted by purified water 40% + acetonitrile 60%, and obtained into P3YELAN ammonium salt solution, and the product is 1.63g after freeze-drying. The ammonium content of the finished product was measured to be 1.6%.

4. Preparation of trifluoroacetate salt

1.70g of P3YELAN refined peptide is weighed and dissolved in 340ml of purified water, the solution is loaded into a reversed phase polymer column, 0.1% trifluoroacetic acid solution is used, the solution is subjected to ion exchange by 5% of acetonitrile, then the solution is eluted by 40% of 0.1% trifluoroacetic acid solution and 60% of acetonitrile to obtain P3YELAN acetate solution, and the product of 1.68g is obtained after freeze drying. The trifluoroacetic acid content in the finished product was measured to be 2.3%.

5. Different salt root stability

The above different salt roots were allowed to stand at different temperatures (2-8deg.C, 25deg.C, 40 ℃) for 6 months, and their stability was examined.

Wherein the basic salt root exhibits more stable properties than the acidic salt root. Wherein the stability of sodium salt and potassium salt is optimal.

Example 3 in vitro cytological Activity assay

Measurement of GLP-1R agonistic Activity

GLP-1R agonistic activity was detected using the luciferase reporter assay (Jonathan W Day et al: nat Chem biol. 2009 Oct;5 (10): 749-57). The human GLP-1R gene is cloned into a mammalian cell expression plasmid pCDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GLP-1R, and simultaneously, the full-length Luciferase (Luciferase) gene is cloned into a pCRE plasmid to obtain a pCRE-Luc recombinant plasmid. pcDNA3.1-GLP-1R and pCRE-Luc plasmids are transfected into CHO-K1 cells according to the molar ratio of 1:10, and stable transgenic expression strains are screened.

Cells were cultured in 9-cm cell culture dishes with DMEM/F12 medium containing 10% FBS and 300. Mu.g/ml G418, and when the confluence reached around 90%, the culture supernatant was discarded, and after digestion with 2ml of pancreatin for 3min, 2ml of DMEM/F12 medium containing 10% FBS and 300. Mu.g/ml G418 was added for neutralization, transferred to a 15ml centrifuge tube, centrifuged at 1000rpm for 5min, the supernatant was discarded, and 2ml of DMEM/F12 medium containing 10% FBS and 300. Mu.g/ml G418 was added for resuspension, and counted. The cells were diluted to 1X 10 with DMEM/F12 medium containing 10% FBS ⁵ Each well of a 96-well plate was plated with 100. Mu.l, i.e., 1X 10,/ml ⁴ After attachment, the wells were replaced with DMEM/F12 medium containing 0.2% FBS. After the supernatant was discarded from the cells plated in the 96-well plate, the purified glucagon derivative (positive control: dulaglutide (trade name Trulicity) was diluted to a series of prescribed concentrations with DMEM/F12 medium containing 1% BSA, added to the cell culture well, 100. Mu.l/well, and assayed after 6 hours of stimulation. The detection was carried out according to the instructions of Lucifersae reporter kit (Ray Biotech, cat: 68-LucIR-S200). The activity measurement for each sample was repeated 3 times.

(II) GIPR agonistic Activity detection method

GIPR agonistic activity assays also employ luciferase reporter assays. Cloning human GIPR gene into mammal cell expression plasmid pcDNA3.1 to constitute recombinant expression plasmid pcDNA3.1-GIPR, transfecting CHO-K1 and stably transferring cell strain. The assay procedure was the same as above (positive control: native human GIP peptide), and the assay was repeated 3 times for each sample.

(III) GCGR (GCGR) agonistic activity detection method

The GCGR agonistic activity assay was also performed using the luciferase reporter assay. Cloning the humanized GCGR gene into a mammalian cell expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GCGR, transfecting CHO-K1, and screening and constructing stable transgenic cell strains. The assay procedure was as above (positive control: natural human GCG peptide) and the assay was repeated 3 times for each sample.

(IV) measuring the results of the Loose

FIGS. 2, 3 and 4 are graphs showing the results of GLP-1R, GIPR and GCGR agonistic activity, respectively, and the specific EC50 is shown in Table 3.

TABLE 3 Table 3

The results show that the P3YELAN has very high GLP-1R and GIPR agonistic activity and also has remarkable GCGR agonistic activity. However, P5YELAN and P9YELAN having the same peptide sequence were significantly reduced in cell agonistic activity due to the difference in the linked fatty acid chains. GLP-1R, GIPR of P3YELAN with different salt forms and GCGR agonistic activity are basically consistent

Example 4 serum stability

In this example, the serum stability of the incretin analog P3YELAN was determined using the following procedure:

(1) The incretin analog P3YELAN and different saline samples, YELAN and control (Dulaglutide) are prepared into 1.0mg/ml solution by using 5mM Tris-HCl, pH8.5 and 0.02% TWEEN-80 solution, and the solution is sterilized, filtered (0.22 mu m, millipore SLGP033 RB), diluted 10 times by rat serum, mixed evenly and packaged into a sterile centrifuge tube;

(2) The samples are respectively frozen at-20 ℃ for 3 tubes as a control, and the rest are placed in a 37 ℃ incubator, and the samples are sampled and tested for activity in 0 hours, 12 hours, 24 hours and 72 hours;

(3) GLP-1R agonistic activity is detected. Relative activity: the activity value at 0 hours was taken as 100%, and the values measured at the subsequent time points were compared with the activity value at 0 hours.

FIG. 5 shows the residual activity of the incretin analog P3YELAN over time. The results show that P3YELAN retains a higher GLP-1R agonistic activity over a longer period of time than YELAN and Dulaglutide.

FIG. 6 shows the residual activity of various salts of the incretin analog P3YELAN over time. The results showed that the stability of the different saline roots in serum was substantially consistent.

Example 5, random blood glucose detection after db/db mice were dosed

Leptin receptor deficient glucose lowering experiments in type two diabetes (db/db) mice. db/db mice were screened and balanced for three criteria, mainly body weight, non-fasting blood glucose, pre-drug OGTT response, 6 per group, excluding oversized or undersized individuals, with non-fasting blood glucose greater than 15mM. P3YELAN was dissolved in 50 mM phosphate buffer (pH 7.4), 5% sorbitol, 0.02% v/v Tween-80, subcutaneously injected with either Duraglutide or P3YELAN (multiple doses) at a dose of 10nmol/kg/4d, P3YELAN was low (1 nmol/kg/d), medium (3 nmol/kg/d), high (6 nmol/kg/d). day0-day4 all animals were monitored for random blood glucose, followed by a random blood glucose measurement every 4 days, with the measurement dates being arranged at day6, 10, 14, 18, 22, 26, 30, 34.

The trend of blood glucose change is shown in fig. 7. The results showed that the blood glucose level of the test animals given P3YELAN was much lower than that of dolapride (10 nmol/kg/4 d) at the doses of 3nmol/kg/d or 6 nmol/kg/d.

Example 6 weight loss experiments in Diet Induced Obese (DIO) mice

Preparation of DIO murine model: male C57BL/6J mice of about 7 weeks of age were fed high fat diet (60% kcal from fat) for about 16 weeks (total 23 weeks) and tested at a weight of about 45 g. DIO mice were randomly divided into groups of 6, with no difference in basal body weight and weighed daily. Subcutaneous injections of P3YELAN, liraglutide or PBS. The dose of Liraglutide (Liraglutide) is 40nmol/kg/d; glucagon derivatives were used at low (5 nmol/kg/d) and high doses (20 nmol/kg/d). The first dosing Day begins weighing body weight and continues until Day30 at the end of the experiment, recording food intake and weighing daily, keeping consistency. After the experiment is finished, blood fat and fasting blood glucose are detected. Day28 fasted in the evening and Day29 measured fasting blood glucose.

The results of the measurement of the body weight change of the test animals are shown in fig. 8, and the results show that the P3YELAN of the three dose groups can significantly reduce the body weight of the test animals, and the effect of reducing the body weight is better than that of the liraglutide.

The results of the ingestion assay for the test animals are shown in fig. 9, and the results show that P3YELAN of the three dose groups can significantly reduce the food intake of the test animals, and the effect of reducing the food intake is better than that of liraglutide.

Results of fasting blood glucose measurements in the test animals are shown in fig. 10, and the results show that P3YELAN in the three dose groups can significantly reduce fasting blood glucose in the test animals, and the effect of reducing fasting blood glucose is superior to that of liraglutide.

In conclusion, the P3YELAN peptide effectively overcomes some technical defects in the prior art, and has good industrial application value. And the alkaline salt root such as sodium salt and potassium salt has better stability and patentability than the acidic salt root, and is suitable for industrial production.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Sequence listing

<110> Zhejiang and Zea medical science and technology Co., ltd

Hangzhou hezekunyuan Pharmaceutical Co.,Ltd.

ZHEJIANG DOER BIOLOGICS Corp.

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Claims (8)

1. A pharmaceutically acceptable salt of an incretin analog, wherein the incretin analog consists of a glucagon-like polypeptide and a long chain fatty acid linked thereto; wherein the amino acid sequence of the glucagon-like polypeptide is shown as the formula (I):

YSEGTFTSDX ₁₀ SKYLDSQAAQDFVQWLLAGGPSSGAPPPSX ₄₀ (I)；

in the formula (I), X ₁₀ Is amino acid K, X ₄₀ Selected from the group OH or NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the The long chain fatty acid is linked to X ₁₀ ；

Wherein, the "pharmaceutically acceptable salt" is pharmaceutically acceptable non-toxic acetate or basic salt.

2. A pharmaceutically acceptable salt of an incretin analog according to claim 1, wherein the incretin analog has the amino acid sequence:

wherein, the "pharmaceutically acceptable salt" is pharmaceutically acceptable non-toxic acetate or basic salt.

3. The pharmaceutically acceptable salt of an incretin analog according to claim 1 or 2, wherein the basic salt is one or more of an alkali metal salt, an alkaline earth metal salt, an aluminum salt, an aliphatic amine salt, an aromatic amine salt and an ammonium salt.

4. A pharmaceutically acceptable salt of an incretin analog according to claim 3, wherein the basic salt is one or more of sodium, potassium, calcium, magnesium, aluminum, triethylamine, dimethylamine, diethylamine, DIEA, tromethamine, ammonium salts.

5. A pharmaceutically acceptable salt of an incretin analog according to claim 3, wherein the basic salt is a sodium or potassium salt.

6. A process for the preparation of a pharmaceutically acceptable salt of an incretin analog according to claim 1 or 2, which is converted chemically to the root salt.

7. Use of a pharmaceutically acceptable salt of an incretin analog according to any one of claims 1 to 5 in the preparation of a composition for: activating the human glucagon-like peptide-1 receptor, the glucose-dependent insulinotropic polypeptide receptor and/or the glucagon receptor; preventing, alleviating or treating metabolic disorders; or reducing food intake, reducing fat, reducing body weight, or reducing blood glucose.

8. A composition comprising a pharmaceutically acceptable salt of an incretin analog of any one of claims 1-5, and a carrier; the carrier is a pharmaceutically acceptable carrier.