CN113527505A

CN113527505A - A kind of polypeptide and pharmaceutical composition comprising the polypeptide and their application

Info

Publication number: CN113527505A
Application number: CN202010293860.4A
Authority: CN
Inventors: 龚珉
Original assignee: Borui Biotechnology Co ltd
Current assignee: Borui Biotechnology Co ltd
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2021-10-22

Abstract

The present application provides a polypeptide comprising S ¹ and S ² , wherein S ¹ and S ² are connected in series with peptide bonds along the N-terminal to C-terminal direction; the S ¹ is as shown in SEQ ID No: 1 Amino acid sequence or a functional variant thereof; the S ² is the amino acid sequence shown in SEQ ID No: 7 or a functional variant thereof.

Description

Polypeptide, pharmaceutical composition containing polypeptide and application of polypeptide and pharmaceutical composition

Technical Field

The application relates to the technical field of biological medicines, in particular to a polypeptide, a pharmaceutical composition containing the polypeptide and a biological medicine, and a preparation method and application of the polypeptide and the pharmaceutical composition.

Background

Biological drugs (especially nucleic acid or protein drugs) are widely used for treating various diseases, such as cancer, autoimmune diseases, AIDS and the like. However, biological drugs also have some outstanding problems, such as imperfect drug delivery systems, poor drug stability, short in vivo half-life and low bioavailability. Those skilled in the art will sometimes modify the pharmacokinetic properties of biological drugs to achieve half-life extension, but not all drugs may be modified and some may lose some or all activity during the modification. Therefore, there is an urgent need in the art to find a simple and stable pharmaceutical carrier.

Summary of The Invention

In a first aspect, the present application provides a polypeptide comprising S¹And S²Wherein

S along the direction from N terminal to C terminal¹And S²Are connected in series by peptide bonds;

said S¹Is an amino acid sequence shown as SEQ ID No. 1 or a functional variant thereof;

said S²Is the amino acid sequence shown as SEQ ID No. 7 or functional variant thereof.

In some embodiments, the polypeptide is the amino acid sequence shown in SEQ ID No. 2.

In some embodiments, the polypeptide comprises S¹、S²And S³Wherein

S along the direction from N terminal to C terminal¹、S²And S³Are connected in series by peptide bonds;

said S¹Is an amino acid sequence shown as SEQ ID No. 1 or the functionality thereofA variant;

said S²Is an amino acid sequence as shown in SEQ ID No. 7 or a functional variant thereof;

said S³Is the amino acid sequence shown as SEQ ID No. 8 or functional variant thereof.

In some embodiments, the polypeptide is the amino acid sequence shown in SEQ ID No. 3 or SEQ ID No. 4.

In some embodiments, the polypeptide comprises S¹、X¹And S²Wherein

S along the direction from N terminal to C terminal¹、S²、X¹Are connected in series by peptide bonds;

said X¹Is any amino acid.

In some embodiments, said X is¹Is lysine or arginine.

In some embodiments, said X is¹Is arginine.

In some embodiments, the polypeptide is other than X¹And S²Comprising in addition two S¹。

In some embodiments, the polypeptide is the amino acid sequence shown in SEQ ID No. 5 or SEQ ID No. 6.

In some embodiments, the polypeptide has increased solubility relative to the amino acid sequence set forth in SEQ ID No. 1.

In some embodiments, the polypeptide has a solubility that is about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, or more the solubility of the amino acid sequence set forth in SEQ ID No. 1.

In a second aspect, the present application provides a pharmaceutical composition comprising (1) a polypeptide according to the first aspect, and (2) an active agent.

In some embodiments, the polypeptide further comprises a pharmaceutically acceptable salt, ester, ether, amide, or mixture thereof.

In some embodiments, the active agent is a small molecule drug, a nucleic acid drug, or a proteinaceous drug.

In some embodiments, the active agent is selected from one or more of the following: penicillins, cephalosporins, GLP-1, EPO, Erylysin A, Exendin-4, PKA competitive polypeptide inhibitors, siRNA of EGFR, insulin, monoclonal antibody drugs or fragments thereof.

In some embodiments, the molar ratio of the polypeptide to the active agent is from 10:1 to 1: 10.

In some embodiments, the pharmaceutical composition is in the form of a lyophilized powder or a solution injection.

In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutically acceptable excipient is one or more selected from the group consisting of: soluble filler, pH regulator, stabilizer, non-ionic surfactant and osmotic pressure regulator.

In a third aspect, the present application provides a method of preparing a pharmaceutical composition comprising the steps of:

the polypeptide is mixed with the active agent.

In some embodiments, the mixing is sonication for 1 to 15 minutes.

In some embodiments, the mixing is stirring for 60 to 180 minutes.

In some embodiments, the mixing is at room temperature for about 6 to 12 hours.

In some embodiments, the polypeptide is mixed with the active agent at a ratio of 10:1 to 1:10 on a molar basis.

In some embodiments, the polypeptide is mixed with the active agent in saline, pure water, or PBS buffer.

In a fourth aspect, the present application provides the use of a polypeptide according to the first aspect in the preparation of a pharmaceutical composition for the treatment of a disease.

In some embodiments, the disease is a bacterial infection, diabetes, obesity, anemia, or non-alcoholic fatty liver disease.

In a fifth aspect, the present application provides a nucleic acid encoding a polypeptide according to the first aspect.

Drawings

Embodiments of the present application will now be described in detail with reference to the accompanying drawings.

FIG. 1 shows a long-acting hypoglycemic test of a pharmaceutical composition comprising GLP-1 and SEQ ID No: 3.

FIG. 2 shows that a pharmaceutical composition comprising GLP-1 and SEQ ID No:5 improves the in vivo stability of GLP-1.

FIG. 3 shows that the pharmaceutical composition comprising GLP-1 and SEQ ID No:6 has a long-acting hypoglycemic effect.

FIG. 4 shows that a pharmaceutical composition comprising insulin and SEQ ID No:4 improves the serum stability of insulin.

FIG. 5 shows that the pharmaceutical composition comprising insulin and SEQ ID No:4 has an oral hypoglycemic effect.

FIG. 6 shows an oral hypoglycemic experiment comprising insulin and the pharmaceutical composition of SEQ ID No: 3.

FIG. 7 shows a solubility comparison of the polypeptides shown in SEQ ID Nos. 1-4.

DESCRIPTION OF THE SEQUENCES

SEQ ID No. 1 is S of the present application¹An example of an amino acid sequence is the sequence:

QQCTTGQLQCCESTSTANDPATSXLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV

wherein X is Glu (E) or Lys (K).

SEQ ID No. 2 is a peptide containing S¹And S²An example of the polypeptide of (1), which consists of SEQ ID No. 1 and SEQ ID No. 7 at the N-terminus of SEQ ID No. 1. Specifically, the sequence of SEQ ID No. 2 is:

GLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSXLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV

wherein X is Glu (E) or Lys (K).

SEQ ID No. 3 is a peptide containing S¹、S²And S³An example of the polypeptide of (1), which consists of SEQ ID No. 2 and SEQ ID No. 8 at the N-terminus of SEQ ID No. 2 and X in SEQ ID No. 2 is E. Specifically, the sequence of SEQ ID No. 3 is:

SSMATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV

SEQ ID No. 4 is a nucleotide sequence comprising S¹、S²And S³An example of the polypeptide of (1), which consists of SEQ ID No. 2 and SEQ ID No. 8 at the N-terminus of SEQ ID No. 2 and X in SEQ ID No. 2 is K. Specifically, the sequence of SEQ ID No. 4 is:

SSMATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSKLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV

SEQ ID No. 5 is a peptide containing 2S¹、X¹And S²An example of the polypeptide of (1), which consists of SEQ ID No:2 and the amino acids R (Arg, arginine) at the C-terminus of SEQ ID No:2 and SEQ ID No:1 and X in SEQ ID No:1 is E and X in SEQ ID No:2 is E. Specifically, the sequence of SEQ ID No. 5 is:

GLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNVRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV

SEQ ID No. 6 is a sequence containing 2S¹、X¹And S²An example of the polypeptide of (1), which consists of SEQ ID No:2 and the amino acids R (Arg, arginine) at the C-terminus of SEQ ID No:2 and SEQ ID No:1 and X in SEQ ID No:1 is K and X in SEQ ID No:2 is E. Specifically, the sequence of SEQ ID No. 6 is:

GLTEGLHGFHVHEFGDNTAGSTSAGPRQQCTTGQLQCCESTSTANDPATSELLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNVRQQCTTGQLQCCESTSTANDPATSKLLGLIGVVISDVDALVGLTCSPISVIGVGSGSACTANPVCCDSSPIGGLVSIGCVPVNV

the SEQ ID No. 7 is a polypeptide with the sequence as shown in the specification:

GLTEGLHGFHVHEFGDNTAGSTSAGPR

the SEQ ID No. 8 is a polypeptide with the sequence as shown in the specification:

SSMATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIK

Detailed Description

While this application contains many specifics, these should not be construed as limitations on the scope of the invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in the context of separate embodiments in this application can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.

Unless otherwise indicated, the terms herein have the same meaning as commonly understood by one of ordinary skill in the art, e.g., in reference to the starting materials and products, operating steps, process parameters, equipment and tools used, and units of values.

Definition of

As used herein, the terms "comprises" and "comprising" mean either open or closed. For example, the term "comprises" or "comprising" may mean that other elements or steps or other elements not listed may also be included or included, or that only the listed elements or steps or other elements may be included or included.

Herein, the term "about" (e.g., in component amounts and reaction parameters) is to be interpreted in a sense that is generally understood by those skilled in the art. In general, the term "about" may be understood as any value within plus or minus 5% of a given value, for example, about X may represent any value in the range of 95% X to 105% X.

In this context, two or more elements are in some way "substantially" identical to one another in the technical requirements and experience of a person skilled in the art in a particular practice. In general, the term "substantially" may be understood as meaning that two or more elements differ by no more than 5% in some way.

It should also be understood that specific values (e.g., in proportions, temperatures, and durations) given herein are not only to be understood as individual values, but are also to be construed as providing endpoints of a range and can be combined with other ranges. For example, where it is disclosed that the reaction (e.g., mixing) can be carried out for 60 minutes or 180 minutes, it is also correspondingly disclosed that the reaction can be carried out for 60 to 180 minutes. Further, particular numerical values given herein are also to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical values set forth herein are approximations that may vary depending upon the requirements. For example, a duration of 60 minutes may be understood as a duration of about 60 minutes, and a duration of 60-180 minutes may be understood as a duration of about 60 minutes to about 180 minutes or about 60-180 minutes.

Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

In the present application, amino acids may be represented by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission (Biochemical Nomenclature Commission).

In this application, S¹、S²And S³Represents a peptide fragment sequence in which amino acid residues are represented using one-letter abbreviations. GLTEGLHGFHVHEFGDNTAGSTSAGPR (SEQ ID No:7) for example is to be understood as representing a peptide stretch consisting of the amino acid residues in the single letter abbreviations.

In the present application, the term "tandem linkage" refers to the linkage of the C-terminus of one peptide chain to the N-terminus of another peptide chain. Optionally, the order of the interconnected peptide chains may be interchanged. For example, the C-terminus of one peptide chain may be linked to the N-terminus of another peptide chain, or the N-terminus of one peptide chain may be linked to the C-terminus of another peptide chain.

In the present application, X¹Represents a single arbitrary amino acid residue.

The abbreviation "GLP-1" refers to glucagon-like peptide-1.

The abbreviation "EPO" refers to erythropoietin.

The abbreviation "PKA" refers to protein kinase a.

The abbreviation "EGFR" refers to epidermal growth factor receptor.

With regard to amino acid residues present in the peptide sequences of the present application, L-type residues, i.e. the naturally occurring form, are to be understood, unless otherwise indicated.

The term "functional variant" refers to a variant having the same or similar biological function and properties as the parent. By way of non-limiting example, a "functional variant" may be obtained by substitution, deletion, addition (e.g., conservative substitution or substitution of a D-type amino acid residue) of one or more amino acid residues in the parent.

One skilled in the art will recognize that for an alteration, addition or deletion of a single or several amino acid residues in a sequence, a functional variant is considered if the alteration does not affect its original function. For example, an amino acid residue may be substituted with other amino acid residues that are chemically similar, such substitutions being well known in the art. For example, amino acids within the following groups may be substituted for each other, typically as conservative substitutions:

1) alanine (a), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T); and

8) cysteine (C), methionine (M).

In some embodiments, a functional variant of a sequence is a variant in which 1 amino acid in the sequence is conservatively substituted with another amino acid.

In some embodiments, a functional variant of a sequence is a variant in which 2 amino acids in the sequence are conservatively substituted with other amino acids.

In some embodiments, a functional variant of a sequence is a variant in which 3 amino acids in the sequence are conservatively substituted with other amino acids.

In some embodiments, a functional variant of a sequence is a variant in which a plurality of amino acids in the sequence are conservatively substituted with other amino acids.

In some embodiments, a functional variant of a sequence is a variant in which 1 amino acid in the sequence is substituted with another amino acid.

In some embodiments, a functional variant of a sequence is a variant in which 2 amino acids in the sequence are substituted with other amino acids.

In some embodiments, a functional variant of a sequence is a variant in which 3 amino acids in the sequence are replaced with other amino acids.

In some embodiments, a functional variant of a sequence is a variant in which a plurality of amino acids in the sequence are substituted with other amino acids.

In some embodiments, a functional variant of a sequence is a variant in which 1 amino acid in the sequence is substituted with the corresponding D-form amino acid.

In some embodiments, a functional variant of a sequence is a variant in which 2 amino acids in the sequence are substituted with the corresponding D-form amino acid.

In some embodiments, a functional variant of a sequence is a variant in which 3 amino acids in the sequence are substituted with the corresponding D-form amino acid.

In some embodiments, a functional variant of a sequence is a variant in which a plurality of amino acids in the sequence are substituted with the corresponding D-form amino acid.

First of allIn one aspect, the present application provides a polypeptide comprising S¹And S²Wherein

In some embodiments, the polypeptide comprises the structure:

S²-S¹or S¹-S²

In some embodiments, the structures are shown in order from N-terminus to C-terminus.

In some embodiments, the polypeptide is prepared by artificial fermentation, and can be obtained by fermentation of Escherichia coli or Pichia yeast, and the fermentation method is known in the art. The polypeptide may be purified using HPLC.

In some embodiments, having S²-S¹The polypeptides of the structure have a higher self-assembly capacity than those having S¹-S²A polypeptide of structure (la).

In some embodiments, having S²-S¹The self-assembly ability of a polypeptide of structure is S¹-S²About 2-fold greater than the self-assembly capability of the polypeptide of structure.

In some embodiments, the polypeptide comprises S¹、S²And S³Wherein

In some embodiments, the polypeptide comprises the structure:

S³-S²-S¹or S³-S¹-S²Or S¹-S²-S³Or S¹-S³-S²Or S²-S¹-S³Or

S²-S³-S¹

In some embodiments, the polypeptide comprises S¹、X¹And S²Wherein

said X¹Is any amino acid.

In some embodiments, said X is¹Is lysine or arginine.

In some embodiments, said X is¹Is arginine.

In some embodiments, the polypeptide comprises the structure:

S²-X¹-S¹or S¹-X¹-S²

In some embodiments, the polypeptide comprises 2S¹、X¹And S²。

In some embodiments, the polypeptide comprises the structure:

S²-S¹-X¹-S¹or S¹-X¹-S¹-S²Or S¹-S²-X¹-S¹Or S¹-X¹-S²-S¹Or X¹-S¹-S²-S¹Or S¹-S²-S¹-X¹

In some embodiments, the active agent is a polypeptide fragment of GLP-1 or a portion thereof.

In some embodiments, the active agent is a polypeptide fragment of EPO or a portion thereof.

In some embodiments, the active agent is a polypeptide fragment of eryysin a or a portion thereof.

In some embodiments, the active agent is insulin or a polypeptide fragment of a portion thereof.

In some embodiments, the active agent is a nucleic acid agent.

In some embodiments, the active agent is an siRNA.

In some embodiments, the active drug is a polar small molecule drug.

In some embodiments, the active agent is a penicillin drug.

In some embodiments, the active agent is amoxicillin.

In some embodiments, the molar ratio of the polypeptide to the active agent is from 3:1 to 1: 4.

In some embodiments, the molar ratio of the polypeptide to the active agent is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any range between the above values.

In some embodiments, the water soluble filler is one or more selected from the group consisting of: mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose and galactose.

In some embodiments, the pH adjusting agent is one or more selected from the group consisting of: non-volatile acids, physiologically acceptable organic acids, physiologically acceptable inorganic acids, physiologically acceptable bases and salts thereof.

In some embodiments, the pH adjusting agent is one or more selected from the group consisting of: citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, potassium hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, potassium bicarbonate, and ammonium bicarbonate.

In some embodiments, the stabilizing agent is one or more selected from the group consisting of: EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, polyethylene glycol 4000, sodium dodecyl sulfate and tris (hydroxymethyl) aminomethane.

In some embodiments, the stabilizing agent is one or more selected from the group consisting of: sodium metabisulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000 and tris (hydroxymethyl) aminomethane.

In some embodiments, the nonionic surfactant is a poloxamer.

In some embodiments, the osmolality adjusting agent is one or more selected from the group consisting of: sodium chloride and potassium chloride.

the polypeptide is mixed with the active agent.

In some embodiments, the mixing is sonication for 1 to 15 minutes.

In some embodiments, the mixing is sonication for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 minutes or any range between the above values.

In some embodiments, the mixing is stirring for 60 to 180 minutes.

In some embodiments, the mixing is stirring for 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 minutes or any range between the above values.

In some embodiments, the mixing is at room temperature for about 6 to 12 hours.

In some embodiments, the mixing is at room temperature for about 6, 7, 8, 9, 10, 11, 12 hours or any range therebetween.

In some embodiments, the polypeptide and the active agent are mixed at a molar ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any range therebetween.

In some embodiments, the polypeptide is formulated with other drugs in different molar ratios of the pharmaceutical compositions, the different molar ratios of the pharmaceutical compositions having different release profiles.

In some embodiments, an appropriate amount of the active agent is weighed and dissolved in an appropriate amount of physiological saline, pure water, PBS buffer, or the above solution containing a certain proportion (less than 20%) of an organic solvent, in terms of molar proportions. Weighing appropriate amount of polypeptide, dissolving in appropriate amount of phosphate buffer solution with different pH values, and mixing at 0-5 deg.C for 1-15 min by ultrasound or stirring for 1-3 hr or standing overnight. In addition, the pH value of the mixed solution can be adjusted, and the solution can be directly added with auxiliary materials to prepare the preparation, or the mixed solution can be frozen and dried to prepare the preparation together with other auxiliary materials.

In some embodiments, the method of preparing a pharmaceutical composition comprises the steps of:

taking a proper amount of active medicine solution and polypeptide solution, adding a water-soluble filler, a stabilizer, an osmotic pressure regulator and the like, adding a proper amount of water for injection, adjusting the pH value to 1-7, dissolving, adding water to dilute to a proper concentration, adding 0.1-0.5% of activated carbon, stirring at 0-10 ℃ for 10-20 minutes, removing the activated carbon, adjusting the pH value to 7, then filtering and sterilizing by adopting a microporous filter membrane, subpackaging the filtrate, preparing a white loose block by adopting a freeze-drying method, and sealing for later use.

The polypeptides of the present application have the ability to form physico-chemically stable complexes with more polar biopharmaceutical molecules (e.g., DNA, RNA, or protein-based drugs). The formation of the stable compound can provide effective protection for the active medicament in the compound, thereby reducing the degradation of the active medicament by physiological conditions such as protease, acid and alkali and the like, and finally increasing the stability and half-life of the active medicament.

The pharmaceutical composition formed by the polypeptide and other active drugs can effectively improve the half-life and stability of the active drugs in blood. Still further, certain active agents requiring frequent injections over an extended period of time may even be administered orally, under the protection of the polypeptides of the present application.

An object of the present application is to provide a polypeptide capable of forming a complex with a polar drug, aiming at the short in vivo retention time of the polar drug (especially, a biological drug), so as to increase the stability of the drug to degradation factors in organisms and prolong the half-life of the drug in vivo.

The treatment direction of the pharmaceutical composition is consistent with that of the active pharmaceutical ingredient, and the pharmaceutical composition can reduce the administration times or enable the active pharmaceutical ingredient to be orally administered, thereby improving the compliance of patients.

In particular, the pharmaceutical compositions of the present application may be administered in intravenous, intramuscular, subcutaneous injection or oral form. Although the dosage varies depending on the subject to be treated, the mode of administration, the symptoms and other factors, the pharmaceutical composition of the present application is effective over a relatively wide dosage range. The actual dosage should be determined by a physician in light of the relevant circumstances, including the physical condition of the subject, the route of administration, the age, body weight, individual response of the patient to the drug, the severity of the patient's symptoms, and the like, and therefore, the above dosage ranges are not intended to limit the scope of the application in any way.

The application has at least the following advantages:

1. the polypeptide can prolong the concentration duration of an active drug in vivo and provides a slow release function;

2. the polypeptide of the application can form a stable compound with an active drug, and the pore size of the compound is about 60-120nm and can be absorbed through intestinal tract;

3. pharmaceutical compositions formed using the polypeptides of the present application help to reduce drug uptake, thereby reducing medical costs.

Examples

The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. Experimental procedures without specific conditions noted in the examples below are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages are by mass and ratios between components are in molar ratios. Unless defined otherwise, all terms of art or science used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are exemplary only.

Experimental materials:

EGFR siRNA lyophilized powder was purchased from Santa Cruz Biotechnology;

microporous filtration membranes were purchased from Millipore, Inc;

SD rats were purchased from the shanghai SLAC animal center;

diabetic rats were purchased from the Shanghai SLAC animal center;

the GLP-1EIA kit was purchased from Phoenix Pharmaceuticals, INC;

EPO was purchased from shenyang sansheng pharmaceutical limited liability company;

kunming mice were purchased from Shanghai laboratory animals center of Chinese academy of sciences;

beagle dogs were purchased from Kunming animal research institute, Chinese academy of sciences.

Example 1: preparation of polypeptides

The polypeptides referred to in the present application (e.g., SEQ ID Nos: 1-6) can be prepared by fermentation using E.coli as a host cell. The cDNA sequence encoding the polypeptide was ligated into the pET15b plasmid by digestion and subsequently transfected into E.coli. The obtained positive clones were subjected to primary fermentation, and then the primary fermentation broth was transferred to a secondary fermentor (500L) and IPTG induction was performed while culturing to OD 2-5. After protein expression, mycelia were collected and cell disruption was performed. And collecting and re-suspending the mycelium crushed liquid, purifying by using a C8 liquid phase column, and freeze-drying for later use.

Example 2: preparation of a pharmaceutical composition comprising a Small molecule drug and SEQ ID No. 3

Amoxicillin is weighed, dissolved in physiological saline to form a 1mmol/L solution and mixed well, 0.2mmol of polypeptide shown in SEQ ID No. 3 is weighed, dissolved in phosphate buffer (pH 3.0) and mixed well, and amoxicillin and polypeptide shown in SEQ ID No. 3 are mixed according to the molar ratio of 5: 1. Sonication at 4 ℃ for 5 minutes followed by adjustment of the pH to 7.0-7.3 resulted in a pharmaceutical composition solution, which was lyophilized for use.

Example 3: preparation of pharmaceutical composition comprising siRNA and SEQ ID No. 3

Weighing 3nmol of EGFR siRNA freeze-dried powder, dissolving the EGFR siRNA freeze-dried powder in 1mL of phosphate buffer (pH is 3.0), fully mixing the EGFR siRNA freeze-dried powder and the phosphate buffer, weighing 9 mu M of polypeptide freeze-dried powder shown in SEQ ID No. 3, dissolving the polypeptide freeze-dried powder in the phosphate buffer containing the EGFR siRNA freeze-dried powder, fully mixing the polypeptide freeze-dried powder and the phosphate buffer, stirring the mixture at 4 ℃ for 1 hour, and freeze-drying the mixture to obtain the solid powder of the pharmaceutical composition.

Example 4: preparation of a pharmaceutical composition comprising GLP-1 and SEQ ID No. 3

Weighing the polypeptide freeze-dried powder shown in SEQ ID No. 3, dissolving the polypeptide freeze-dried powder in a phosphate buffer solution (pH is 3.0), then weighing GLP-1 freeze-dried powder, adding the GLP-1 freeze-dried powder into the solution containing the polypeptide shown in SEQ ID No. 3 to ensure that the molar ratio of the polypeptide shown in SEQ ID No. 3 to GLP-1 in the solution is about 6:1, fully mixing, stirring for 3 hours at 4 ℃, and freeze-drying to obtain the solid powder of the pharmaceutical composition.

Example 5: preparation of a pharmaceutical composition comprising GLP-1 and SEQ ID No. 5

Weighing the polypeptide freeze-dried powder shown in SEQ ID No. 5, dissolving the polypeptide freeze-dried powder in a phosphate buffer (pH 7.4), then weighing GLP-1 freeze-dried powder, adding the GLP-1 freeze-dried powder into the solution to ensure that the molar ratio of the polypeptide shown in SEQ ID No. 5 to the GLP-1 is about 1:2.5, fully and uniformly mixing, stirring at 4 ℃ for 3 hours, and freeze-drying to obtain the solid powder of the pharmaceutical composition.

Example 6: preparation of a pharmaceutical composition comprising GLP-1 and SEQ ID No. 6

Weighing the polypeptide freeze-dried powder shown by SEQ ID No. 6, dissolving the polypeptide freeze-dried powder in a phosphate buffer (pH 7.4), then weighing GLP-1 freeze-dried powder, adding the GLP-1 freeze-dried powder into the solution to ensure that the molar ratio of the polypeptide shown by SEQ ID No. 6 to the GLP-1 is about 1:5, fully mixing the components uniformly, stirring the mixture at 4 ℃ for 3 hours, and freeze-drying the mixture to obtain the solid powder of the pharmaceutical composition.

Example 7: preparation of a pharmaceutical composition comprising insulin and SEQ ID No. 3

Weighing an insulin raw material, dissolving the insulin raw material in water (adjusting the pH value of the water to be 2.0 by using HCl), then adding a polypeptide freeze-dried powder shown by SEQ ID No. 3 to ensure that the molar ratio of the polypeptide shown by SEQ ID No. 3 to insulin in the solution is about 1:1, violently shaking for 1-3 minutes and using ultrasonic treatment for 10 minutes, adjusting the pH value of the solution to be 6.5-7.3, centrifuging to obtain a supernatant, namely the pharmaceutical composition of insulin and hydrophobin, and freeze-drying the supernatant for later use.

Example 8: preparation of a pharmaceutical composition comprising insulin and SEQ ID No. 4

Dissolving insulin lyophilized powder in phosphate buffer (pH 3.0), then weighing polypeptide lyophilized powder shown in SEQ ID No. 4, adding the polypeptide lyophilized powder into the solution to enable the molar ratio of the polypeptide shown in SEQ ID No. 4 to insulin in the solution to be about 1:2, shaking or ultrasonically mixing the polypeptide and insulin sufficiently, adjusting the pH to 6.5-7.3 at 4 ℃, then stirring for 3 hours, and freeze-drying the filtered filtrate to obtain the pharmaceutical composition solid powder.

Example 9: preparation of a pharmaceutical composition comprising insulin and SEQ ID No. 4

Dissolving the polypeptide freeze-dried powder shown by SEQ ID No. 4 in a phosphate buffer solution (pH 7.0), then adding the insulin freeze-dried powder into the solution to ensure that the molar ratio of the polypeptide shown by SEQ ID No. 4 to insulin in the solution is about 5:1, fully mixing and stirring for 3 hours, and freeze-drying to obtain the pharmaceutical composition solid powder.

Example 10: preparation of a pharmaceutical composition comprising EPO and SEQ ID No. 3

Weighing the polypeptide freeze-dried powder shown in SEQ ID No. 3, dissolving the polypeptide freeze-dried powder in a phosphate buffer solution (pH 7.0), then weighing EPO freeze-dried powder, adding the EPO freeze-dried powder into the solution containing the polypeptide shown in SEQ ID No. 3 to ensure that the molar ratio of the polypeptide shown in SEQ ID No. 3 to the EPO in the solution is about 1:1, fully mixing the mixture, stirring the mixture for 3 hours at 4 ℃, and freeze-drying the mixture to obtain the solid powder of the pharmaceutical composition.

Example 11: preparation of a pharmaceutical composition comprising EPO and SEQ ID No. 4

Weighing the polypeptide freeze-dried powder shown in SEQ ID No. 4, dissolving the polypeptide freeze-dried powder in a phosphate buffer solution (pH 7.0), then weighing EPO freeze-dried powder, adding the EPO freeze-dried powder into the solution containing the polypeptide shown in SEQ ID No. 4 to ensure that the molar ratio of the polypeptide shown in SEQ ID No. 4 to the EPO in the solution is about 10:1, fully mixing the mixture, stirring the mixture at 4 ℃ for 3 hours, and freeze-drying the mixture to obtain the solid powder of the pharmaceutical composition.

Example 12: preparation of a pharmaceutical composition comprising EPO and SEQ ID No. 4

Weighing the polypeptide freeze-dried powder shown in SEQ ID No. 4, dissolving the polypeptide freeze-dried powder in a phosphate buffer solution (pH 8.0), then weighing EPO freeze-dried powder, adding the EPO freeze-dried powder into the solution containing the polypeptide shown in SEQ ID No. 4 to ensure that the molar ratio of the polypeptide shown in SEQ ID No. 4 to the EPO in the solution is about 1:1, fully mixing the mixture, then stirring the mixture for 3 hours at 4 ℃, and freeze-drying the mixture to obtain the solid powder of the pharmaceutical composition.

Example 13: preparation of an injectable preparation (taking the pharmaceutical composition prepared in example 3 as an example)

0.05g of poloxamer, 0.2g of mannitol, 0.1g of lactose and 3mL of water for injection are placed in a 10mL container, stirred to dissolve, and then 1mol/L of citric acid or sodium hydroxide is added to adjust the pH of the solution to 3.0, and the solution is cooled to 5 ℃. 5mL of the pharmaceutical composition solution prepared in example 3 was added, the pH was adjusted to 3.0, and water was added to make up to 10 mL. Adding 10mg of activated carbon, stirring at 5 ℃ for 20 minutes, filtering to remove the activated carbon, filtering with a microporous filter membrane (Millipore, Inc) for sterilization, subpackaging the obtained filtrate by 0.2mL per filter, pre-freezing for 2 hours, freeze-drying under reduced pressure for 12 hours, drying for 2 hours after the temperature of a sample is reduced to 5 ℃ to obtain a white loose block, sealing to obtain a medicinal composition, placing the medicinal composition into a prefilled syringe with the specification of 100 ug/piece, storing at 4 ℃, dissolving with 200uL of water for injection before injection, and then administering.

Example 14: preparation of an injectable preparation (taking the pharmaceutical composition prepared in example 4 as an example)

20mg of NaCl, 10mg of citric acid and 7mL of water for injection are placed in a 10mL container and stirred to be dissolved, 1mol/L of citric acid or sodium hydroxide is added to adjust the pH value of the solution to 7.0, the solution is cooled to 0 ℃, the solid powder of the pharmaceutical composition prepared in example 4 is added into the solution, stirring is continued to fully dissolve the solid powder of the pharmaceutical composition, 1mol/L of citric acid or sodium hydroxide is added to adjust the pH value of the solution to 7.0, and the water for injection is supplemented to 10 mL. Adding 10mg of activated carbon, stirring at 0-4 deg.C for 20 min, filtering to remove activated carbon, filtering with microporous membrane to remove bacteria, packaging the filtrate into prefilled syringes (100 μ L each), and storing at 5 deg.C with specification of 50 μ g/tube.

Example 15: in vivo half-life assay of pharmaceutical compositions comprising SEQ ID No. 3 and GLP-1

SD rats were used in 5 groups of 10 animals each. SD rats were injected with GLP-1 alone or with a pharmaceutical composition comprising the polypeptide represented by SEQ ID No:3 and GLP-1 (wherein GLP-1 is administered in an amount of 1000. mu.g/kg per rat, the molar ratios of GLP-1 to polypeptide in the pharmaceutical composition are 1:5, 1:10, 1:20 and 1:50, respectively, at 0.5, 1, 3, 6, 9, 12, 24, 36, 48, 72 and 96 hours before and after injection, and about 0.2mL of blood was taken from the ocular plexus vein and serum was prepared for use.

The concentration of the pharmaceutical composition in the rat serum was determined by enzyme-linked immunosorbent assay (ELISA). The method comprises the following steps: the serum was obtained by centrifugation at 4 ℃ and at 13,000rpm/min for 20 minutes. The serum was incubated with 100mM ammonium acetate for 10 minutes at room temperature, and the concentration of GLP-1 was determined using the GLP-1EIA kit. The test method was performed with reference to the instructions of the kit, and GLP-1 stability was evaluated based on the results.

The results of the pharmacokinetics of GLP-1 and the pharmaceutical composition in vivo are shown in Table 1, and the results show that the pharmaceutical composition containing GLP-1 of the application has obviously prolonged half-life in vivo compared with GLP-1 alone and has long-acting property.

Table 1: GLP-1 and half-life period of pharmaceutical composition containing GLP-1 and SEQ ID No. 3 in rat body

Example 16: long-lasting hypoglycemic experiments of the pharmaceutical composition prepared in example 4 (GLP-1+ SEQ ID No:3)

SD rats were used in 5 groups of 10 animals each. Blood glucose levels were measured (0h) immediately before glucose stimulation and oral glucose stimulation of 2g/kg body weight was performed, and blood glucose levels were measured at 0.5 hours after glucose administration while separately intraperitoneally injecting GLP-1 alone and a pharmaceutical composition containing GLP-1 to SD rats and following the change in blood glucose levels, the results are shown in FIG. 1. The results show that the half-life period of GLP-1 in vivo is only 2-5 minutes, so that the GLP-1 alone can be rapidly degraded in vivo and cannot effectively reduce the blood sugar. After the pharmaceutical composition is formed by the polypeptide, the degradation speed of GLP-1 is greatly reduced, and the blood sugar can be controlled within a normal range within 8 hours.

Example 17: pharmaceutical compositions comprising GLP-1 and SEQ ID No. 5 improve the in vivo stability of GLP-1

Diabetic rats were used in 5 groups of 10 rats each.

Rats were injected with GLP-1 (control) and a pharmaceutical composition comprising GLP-1 and SEQ ID No:5 (GLP-1 administered to each rat is 100. mu.g/kg, the molar ratio of GLP-1 to SEQ ID No:5 is 1:5), and approximately 0.2mL of blood was taken from the ocular plexus vein at different time points before and after injection, respectively, and sera were prepared for use.

The blood concentration of GLP-1 in rat serum is detected by adopting an enzyme-linked immunosorbent assay (ELISA), and the operation is as follows: the serum was obtained by centrifugation at 4 ℃ and at 13,000rpm/min for 20 minutes. After incubating the serum with 100mM ammonium acetate for 10 minutes at room temperature, the GLP-1 concentration was determined using the GLP-1EIA kit. The test method refers to the instruction of the kit and evaluates the stability of GLP-1 according to the result.

The in vivo pharmacokinetic results for GLP-1 and the pharmaceutical composition comprising GLP-1 and SEQ ID No:5 are shown in FIG. 2. As shown in FIG. 2, GLP-1 is measured in the animal after several minutes of administration of GLP-1 and the pharmaceutical composition comprising GLP-1 and SEQ ID No: 5. As shown in FIG. 2, GLP-1 is not substantially detectable in animals administered with GLP-1 alone (as indicated by the solid black circle ● in FIG. 2), whereas a significant amount of GLP-1 can be detected in animals administered with the pharmaceutical composition comprising GLP-1 and SEQ ID No:5 (as indicated by the white circle O in FIG. 2), indicating that the pharmaceutical composition comprising GLP-1 and SEQ ID No:5 increases the half-life of GLP-1 in animals, which greatly decreases the proteolysis rate of GLP-1, resulting in a substantial increase in the stability of GLP-1 molecules which are otherwise stable for about 2-5 minutes.

Example 18: the pharmaceutical composition prepared in example 6 (GLP-1+ SEQ ID No:6) has long-lasting hypoglycemic effect

Diabetic rats were used in 5 groups of 10 rats each. Blood glucose values were measured (0h) immediately before glucose challenge and diabetic rats were injected intraperitoneally with GLP-1 alone and the pharmaceutical composition prepared in example 6, respectively. Then, oral glucose stimulation of 2g/kg body weight was immediately performed, and blood glucose value was measured 0.5 hours after the sugar administration and blood glucose value change was followed (GLP-1 dose was 0.3mg/kg bw), and the results are shown in FIG. 3. The results show that GLP-1 alone is degraded rapidly in animals and cannot effectively reduce blood sugar because GLP-1 has an in vivo half-life of only 2-5 minutes. However, the pharmaceutical composition of the application enables the degradation rate of GLP-1 molecules to be greatly reduced, and can control the blood sugar to be in a normal range within 8 hours.

Example 19: in vivo half-life assay for combinations of insulin and SEQ ID No:4 at different molar ratios

The pharmaceutical compositions of insulin and SEQ ID No:4 were prepared in different molar ratios according to the preparation method shown in example 7, example 8 and example 9, and the dosage of insulin administered was 2mg/kg rat body weight. Rats were injected with different molar ratios of insulin to the pharmaceutical composition of SEQ ID No:4, 0.5, 1, 3, 6, 9, 12 and 24 hours before and after injection, approximately 0.2mL of blood was taken from the ocular plexus vein and incubated with 100mM ammonium acetate for 10 minutes for future use.

The concentration of the pharmaceutical composition in the rat serum is detected by adopting an enzyme-linked immunosorbent assay, and the operation is as follows: serum was obtained by centrifugation at 4 ℃ and at 13,000rpm/min for 20 minutes. The concentration of insulin was measured using an insulin EIA kit. The test method was performed with reference to the instructions of the kit, and insulin stability was evaluated based on the results.

The in vivo pharmacokinetic results of insulin and the pharmaceutical composition containing insulin are shown in table 2, and the results show that the pharmaceutical composition containing insulin of the present application has a significantly longer half-life in vivo than insulin alone, and has a long-acting property.

Table 2: insulin and pharmaceutical composition containing insulin half-life period in rat body

Example 20: the pharmaceutical composition prepared in example 9 (insulin + SEQ ID No:4) improves the serum stability of insulin

The solid powder of the pharmaceutical composition prepared in example 9 was dissolved in 10mL of human plasma, left at 37 ℃ for various periods of time (0, 0.5, 1, 4 hours), and the plasma insulin content was measured by the insulin enzyme-linked immunosorbent assay to calculate the stability. And compared with the stability of the same concentration of insulin in plasma, the results are shown in fig. 4. As shown in FIG. 4, the pharmaceutical composition comprising insulin and SEQ ID No:4 increased the serum stability of insulin and decreased the degradation of insulin.

Example 21: the pharmaceutical composition prepared in example 9 (insulin + SEQ ID No:4) has an oral hypoglycemic effect

Insulin alone or the pharmaceutical composition prepared in example 9, wherein insulin was used in an amount of 2mg/kg bw and the molar ratio of insulin to carrier protein was 1:5, was orally administered daily to rats with diabetes. Rats were orally administered glucose (2g/kg) daily and blood glucose levels were measured daily, and the results are shown in FIG. 5. The results show that orally administered insulin does not have any blood glucose regulating function at all, but that the pharmaceutical composition comprising insulin has oral activity.

Example 22: oral hypoglycemic assay of the pharmaceutical composition prepared in example 7 (insulin + SEQ ID No:3)

Beagle 3 groups of 3 dogs each were used. The experiment was stimulated with oral glucose: the results of measuring the blood glucose level (0h) immediately before glucose challenge and separately intraperitoneally injecting insulin alone (as shown in box ■ in fig. 6) into beagle dogs, orally administering the pharmaceutical composition of example 7 (as shown in circle ● in fig. 6. animals immediately underwent oral glucose challenge of 2g/kg body weight, and measuring the blood glucose level 0.5 hours after sugar administration and tracking the change in blood glucose level are shown in fig. 6. the results show that, by forming a pharmaceutical composition with the polypeptide of the present application, insulin can be protected from the acidic environment or degradation by proteases in the intestinal tract, and the pore size of the formed pharmaceutical composition meets the requirement of intestinal absorption (i.e., 60-120 nm). furthermore, intraperitoneally injected insulin normally exerts its hypoglycemic function as a positive control.

Example 23: long-lasting effect of the pharmaceutical composition prepared in example 11 (EPO + SEQ ID No:4) on erythropoiesis in mice

Kunming mice weighing 25-30g were used, 10 mice per group.

Mice were subcutaneously injected with erythropoietin (EPO, once a day) and the pharmaceutical composition prepared in example 11 (once every three days), respectively, and after 3 days, the mice were sacrificed, and whole blood was taken for peripheral blood cell and reticulocyte counting, in which blood cell counting was performed by a fully automatic hemocytometer. See table 3 for results.

The results indicate that both EPO and the pharmaceutical composition containing EPO stimulate the increase of the mouse peripheral blood reticulocyte count, indicating that they stimulate erythropoiesis. However, the pharmaceutical composition containing EPO administered once every three days is significantly superior in drug efficacy to EPO administered once a day.

Table 3: pharmaceutical composition containing EPO with long-acting stimulation to generate mouse reticulocyte

Experimental group	Dosage form	Number of reticulocytes
			Blank group	PBS buffer	109.75±3.45
Pharmaceutical composition group 1	4.5 mg/kg/every 3 days	786.94±3.86
			Pharmaceutical composition group 2	4.5 mg/kg/every 3 days	768.64±4.86
Pharmaceutical composition group 3	4.5 mg/kg/every 3 days	818.65±4.01
			Pharmaceutical composition group 4	4.5 mg/kg/every 3 days	687.95±5.76
Pharmaceutical composition group 5	4.5 mg/kg/every 3 days	789.75±4.01
			Pharmaceutical composition group 6	4.5 mg/kg/every 3 days	756.35±2.46
Pharmaceutical composition group 7	4.5 mg/kg/every 3 days	698.64±3.91
			Pharmaceutical composition group 8	4.5 mg/kg/every 3 days	704.81±4.01
Pharmaceutical composition group 9	4.5 mg/kg/every 3 days	803.95±3.37
			Pharmaceutical composition group 10	4.5 mg/kg/every 3 days	734.65±2.86
EPO alone group	4.5 mg/kg/day	683.19±4.82

Example 24: solubility comparison of the polypeptides shown in SEQ ID Nos 1-4

The four proteins (i.e., the polypeptide shown in SEQ ID No:1, the polypeptide shown in SEQ ID No:2, the polypeptide shown in SEQ ID No:3 and the polypeptide shown in SEQ ID No:4, each 10mg) were dissolved in 1mL of physiological saline, and it was observed by visual observation that the other three proteins (i.e., the polypeptide shown in SEQ ID No:2, the polypeptide shown in SEQ ID No:3 and the polypeptide shown in SEQ ID No:4) had better solubility than that of SEQ ID No:1, and the observation results are shown in FIG. 7.

Finally, it should be understood that while the various aspects of the present specification describe specific embodiments, those skilled in the art will readily appreciate that the disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Accordingly, it is to be understood that, unless explicitly stated otherwise, the disclosed subject matter is not limited to the particular compositions, methods, and/or formulations, etc., described herein. Moreover, those of ordinary skill in the art will recognize that certain changes, modifications, permutations, variations, additions, subtractions and sub-combinations may be made in accordance with the teachings herein without departing from the spirit of the present specification. It is therefore intended that the following appended claims be interpreted as including all such alterations, modifications, permutations, variations, additions, subtractions and sub-combinations as fall within the true spirit and scope thereof.

Sequence listing

<110> Borui Biotechnology Ltd

<120> a polypeptide, a pharmaceutical composition comprising the same and applications thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 83

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> SITE

<222> (24)..(24)

<223> Xaa is Glu or Lys

<400> 1

Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr

1 5 10 15

Ala Asn Asp Pro Ala Thr Ser Xaa Leu Leu Gly Leu Ile Gly Val Val

20 25 30

Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser

35 40 45

Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys

50 55 60

Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro

65 70 75 80

Val Asn Val

<210> 2

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> SITE

<222> (51)..(51)

<223> Xaa is Glu or Lys

<400> 2

Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp

1 5 10 15

Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg Gln Gln Cys Thr Thr

20 25 30

Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala Asn Asp Pro Ala

35 40 45

Thr Ser Xaa Leu Leu Gly Leu Ile Gly Val Val Ile Ser Asp Val Asp

50 55 60

Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val Ile Gly Val Gly

65 70 75 80

Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys Asp Ser Ser Pro

85 90 95

Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val Asn Val

100 105 110

<210> 3

<211> 149

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ser Ser Met Ala Thr Lys Ala Val Ser Val Leu Lys Gly Asp Gly Pro

1 5 10 15

Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val

20 25 30

Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe

35 40 45

His Val His Glu Phe Gly Asp Asn Thr Ala Gly Ser Thr Ser Ala Gly

50 55 60

Pro Arg Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr

65 70 75 80

Ser Thr Ala Asn Asp Pro Ala Thr Ser Glu Leu Leu Gly Leu Ile Gly

85 90 95

Val Val Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro

100 105 110

Ile Ser Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro

115 120 125

Val Cys Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys

130 135 140

Val Pro Val Asn Val

145

<210> 4

<211> 149

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ser Ser Met Ala Thr Lys Ala Val Ser Val Leu Lys Gly Asp Gly Pro

1 5 10 15

Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val

20 25 30

Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe

35 40 45

His Val His Glu Phe Gly Asp Asn Thr Ala Gly Ser Thr Ser Ala Gly

50 55 60

Pro Arg Gln Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr

65 70 75 80

Ser Thr Ala Asn Asp Pro Ala Thr Ser Lys Leu Leu Gly Leu Ile Gly

85 90 95

Val Val Ile Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro

100 105 110

Ile Ser Val Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro

115 120 125

Val Cys Cys Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys

130 135 140

Val Pro Val Asn Val

145

<210> 5

<211> 194

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp

1 5 10 15

Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg Gln Gln Cys Thr Thr

20 25 30

Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala Asn Asp Pro Ala

35 40 45

Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val Ile Ser Asp Val Asp

50 55 60

Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val Ile Gly Val Gly

65 70 75 80

Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys Asp Ser Ser Pro

85 90 95

Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val Asn Val Arg Gln

100 105 110

Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala

115 120 125

Asn Asp Pro Ala Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val Ile

130 135 140

Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val

145 150 155 160

Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys

165 170 175

Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val

180 185 190

Asn Val

<210> 6

<211> 194

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp

1 5 10 15

Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg Gln Gln Cys Thr Thr

20 25 30

Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala Asn Asp Pro Ala

35 40 45

Thr Ser Glu Leu Leu Gly Leu Ile Gly Val Val Ile Ser Asp Val Asp

50 55 60

Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val Ile Gly Val Gly

65 70 75 80

Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys Asp Ser Ser Pro

85 90 95

Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val Asn Val Arg Gln

100 105 110

Gln Cys Thr Thr Gly Gln Leu Gln Cys Cys Glu Ser Thr Ser Thr Ala

115 120 125

Asn Asp Pro Ala Thr Ser Lys Leu Leu Gly Leu Ile Gly Val Val Ile

130 135 140

Ser Asp Val Asp Ala Leu Val Gly Leu Thr Cys Ser Pro Ile Ser Val

145 150 155 160

Ile Gly Val Gly Ser Gly Ser Ala Cys Thr Ala Asn Pro Val Cys Cys

165 170 175

Asp Ser Ser Pro Ile Gly Gly Leu Val Ser Ile Gly Cys Val Pro Val

180 185 190

Asn Val

<210> 7

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Leu Thr Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp

1 5 10 15

Asn Thr Ala Gly Ser Thr Ser Ala Gly Pro Arg

20 25

<210> 8

<211> 39

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ser Ser Met Ala Thr Lys Ala Val Ser Val Leu Lys Gly Asp Gly Pro

1 5 10 15

Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val

20 25 30

Lys Val Trp Gly Ser Ile Lys

35

Claims

1. A polypeptide, wherein the polypeptide comprises S ¹ and S ² , wherein

S ¹ and S ² are connected in series by peptide bonds along the N-terminal to C-terminal direction;

The S ¹ is the amino acid sequence shown in SEQ ID No: 1 or a functional variant thereof;

The S ² is the amino acid sequence shown in SEQ ID No: 7 or a functional variant thereof;

Preferably, the polypeptide is the amino acid sequence shown in SEQ ID No:2.

2. The polypeptide of claim 1, wherein the polypeptide comprises S ¹ , S ² and S ³ , wherein

S ¹ , S ² and S ³ are connected in series with each other by peptide bonds along the N-terminal to C-terminal direction;

The S ³ is the amino acid sequence shown in SEQ ID No: 8 or a functional variant thereof;

Preferably, the polypeptide is the amino acid sequence shown in SEQ ID No:3 or SEQ ID No:4.

3. The polypeptide of claim 1, wherein the polypeptide comprises S ¹ , X ¹ and S ² , wherein

Along the N-terminal to C-terminal direction, S ¹ , S ² , X ¹ are connected in series with each other by peptide bonds;

The X ¹ is any amino acid, preferably lysine or arginine, more preferably arginine;

Optionally, the polypeptide comprises two S ¹ in addition to X ¹ and S ² , preferably, the polypeptide is the amino acid sequence shown in SEQ ID No:5 or SEQ ID No:6.

4. The polypeptide of any one of claims 1 to 3, wherein the polypeptide has increased solubility relative to the amino acid sequence shown in SEQ ID No: 1;

Preferably, the solubility of the polypeptide is about 2 times, about 5 times, about 10 times, about 20 times, about 50 times, about 100 times, or more times the solubility of the amino acid sequence shown in SEQ ID No: 1.

5. A pharmaceutical composition comprising

(1) the polypeptide of any one of claims 1-4, and

(2) Active drugs.

6. The pharmaceutical composition of claim 5, wherein

The active drug is a small molecule drug, nucleic acid drug or protein drug; preferably, the active drug is selected from one or more of the following: penicillins, cephalosporins, GLP-1, EPO, Erylysin A , Exendin-4, PKA competitive peptide inhibitors, EGFR siRNA, insulin, monoclonal antibody drugs or fragments thereof; and/or

The molar ratio of the polypeptide to the active drug is 10:1 to 1:10; and/or

The dosage form of the pharmaceutical composition is freeze-dried powder or solution injection.

7. The pharmaceutical composition of claim 5, further comprising one or more pharmaceutically acceptable adjuvants;

Preferably, the pharmaceutically acceptable adjuvant is one or more selected from the group consisting of soluble fillers, pH adjusters, stabilizers, nonionic surfactants and osmotic pressure adjusters.

8. A method for preparing a pharmaceutical composition, comprising the steps of:

mixing the peptide with the active drug;

Preferably,

The mixing is sonication for 1-15 minutes; and/or

The mixing is stirring for 60-180 minutes; and/or

The mixing is about 6-12 hours at room temperature; and/or

The mixing ratio of the polypeptide and the active drug is 10:1 to 1:10 on a molar basis; and/or

The polypeptide and active drug are mixed in physiological saline, purified water or PBS buffer.

9. Use of the polypeptide according to any one of claims 1-3 in the preparation of a pharmaceutical composition for the treatment of a disease;

Preferably, the disease is bacterial infection, diabetes, obesity, anemia or non-alcoholic fatty liver disease.

10. A nucleic acid encoding the polypeptide of any one of claims 1-3.