EA045778B1 - NEW INSULIN ANALOGUES AND THEIR APPLICATION - Google Patents

Description

Field of invention

The present invention relates to a new insulin analog and, more specifically, to an insulin analog with improved in vitro effect compared to native insulin, and to the use thereof.

Prior Art

Insulin is a blood glucose-controlling hormone secreted by the pancreas that causes excess glucose to move from the blood into cells, whereby it provides them with a source of energy and maintains normal glucose levels. However, patients with diabetes are unable to maintain normal insulin functions due to insulin deficiency, insulin resistance, and loss of beta cell function. As a result, diabetic patients are unable to use blood glucose as an energy source but exhibit symptoms of hyperglycemia with high glucose levels and excrete glucose in the urine, leading to various complications. Accordingly, patients with diabetes and impaired insulin secretion (type I) or insulin resistance (type II) have a high need for insulin to help them maintain normal blood glucose levels.

Human insulin consists of two polypeptide chains, i.e. A-chain and B-chain, containing 21 and 30 amino acids, respectively, connected to each other by two disulfide bonds. Since insulin has a very short half-life in vivo, like other protein and peptide hormones, it is unable to exert a long-lasting therapeutic effect, and therefore there is the problem that it must be administered continuously and frequently in order for it to have its effect. Frequent insulin administration causes significant pain and discomfort to patients, and therefore there is a need to improve administration in terms of patient compliance, safety and convenience.

Accordingly, research has focused on developing various protein formulations, chemical conjugates, and so on to improve the therapeutic effects as well as the quality of life of patients by reducing the frequency of administration by increasing the half-life of these protein drugs, such as insulin, in vivo.

It is known that insulin removes glucose from the blood by binding to insulin receptors, and that the effect of insulin can be controlled by changing the sequence of native insulin. In vivo, the effect of insulin can be controlled by replacing the amino acid(s) of insulin with another amino acid(s) or by deleting certain amino acid(s) of insulin. Since highly active insulin derivatives can produce effects equivalent to or superior to those of native insulin, even in small amounts, they may thus be highly desirable from a therapeutic point of view. In particular, amino acid substitutions in the A-chain and/or B-chain of insulin have been widely studied from the point of view of the pharmacokinetic effect of insulin after subcutaneous injection.

Under these conditions, the inventors of the present invention have conducted extensive studies to improve the effect of insulin and, as a result, they have found that insulin analogues with modification(s) of a specific amino acid residue(s) of the A chain and/or B chain of insulin exhibit significantly improved in vitro effect, compared to native insulin, and that they can therefore be effectively used for the treatment of diabetes, thereby completing the present invention.

Description of the invention

Technical problem.

The object of the present invention is to provide a new insulin analog and, specifically, an insulin analog with improved in vitro effect compared to native insulin.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of diabetes containing an insulin analog as an active ingredient.

Another object of the present invention is to provide a method of treating diabetes comprising administering an insulin analog or a pharmaceutical composition containing an insulin analog as an active ingredient to a subject in need thereof.

Technical solution.

To solve the above objects, in one aspect, the present invention provides an insulin analog comprising an A chain of SEQ ID NO: 3 represented by the following general formula 1 and a B chain of SEQ ID NO: 4 represented by the following general formula 2.

General formula 1.

Xaa1-Ile-Val-Glu-Xaa2-Cys-Cys-Thr-Ser-Ile-Cys-Xaa3-Leu-Xaa4-Gln-Xaa5-Glu-Asn-Xaa6-Cys-Xaa7 (SEQ ID NO: 3)

In the above general formula 1:

Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid or asparagine;

Xaa2 is alanine, glutamic acid, glutamine, histidine or asparagine;

Xaa3 is alanine, serine, glutamine, glutamic acid, histidine or asparagine;

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Xaa4 is alanine, tyrosine, glutamic acid, histidine, lysine, aspartic acid or asparagine;

Xaa5 is alanine, leucine, tyrosine, histidine, glutamic acid or asparagine;

Xaa6 is alanine, tyrosine, serine, glutamic acid, histidine or asparagine;

And

Xaa7 is asparagine, glycine, histidine or alanine.

General formula 2.

Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Xaa8-Leu-Val-Cys-Gly-Glu-Arg-GlyPhe-Xaa9-Tyr- Xaa10-Xaa11-Lys-Thr (SEQ ID NO: 4)

In the above general formula 2:

Xaa8 is tyrosine, glutamic acid or aspartic acid or absent;

Xaa9 is phenylalanine or absent;

Xaa10 is threonine or absent; And

Xaa11 is proline, glutamic acid or aspartic acid or is absent (provided that the peptide containing the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2 is excluded).

In a more specific exemplary embodiment, the insulin analog is characterized in that it contains an A chain of general formula 1 and a B chain, wherein in the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is absent, and Xaa10 is threonine.

The insulin analogue is characterized in that it contains an A chain of general formula 1 and a B chain, wherein in Chain SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is phenylalanine and Xaa10 is absent.

In another exemplary embodiment, the insulin analog is characterized in that, in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is glutamic acid, Xaa5 is leucine, Xaa6 is tyrosine, and Xaa7 is asparagine, and in the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is threonine, and Xaa11 is proline.

In yet another exemplary embodiment, the insulin analog is characterized in that, in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is asparagine, Xaa5 is leucine, Xaa6 is tyrosine, and Xaa7 is asparagine, and in the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is threonine, and Xaa11 is proline.

In yet another exemplary embodiment, the insulin analog is characterized in that, in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is glutamic acid, Xaa5 is leucine, Xaa6 is tyrosine and Xaa7 is asparagine, and in the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is absent, Xaa10 is threonine, and Xaa11 is proline.

In yet another exemplary embodiment, the insulin analog is characterized in that, in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is alanine, Xaa5 is leucine, Xaa6 is tyrosine, and Xaa7 is asparagine, and in the B chain of SEQ ID NO: 4, Xaa8 is glutamic acid, Xaa9 is absent, Xaa10 is threonine, and Xaa11 is proline.

In yet another exemplary embodiment, the insulin analogue of the present invention is characterized in that:

in the A chain SEQ ID NO: 3 Xaa4 is glutamic acid, and in the B chain SEQ ID NO: 4 Xaa9 is phenylalanine; or in the A chain SEQ ID NO: 3 Xaa4 is asparagine, and in the B chain SEQ ID NO: 4 Xaa9 is phenylalanine; or in the A chain of SEQ ID NO: 3, Xaa4 is glutamic acid, and in the B chain of SEQ ID NO: 4, Xaa9 is absent; or in the A chain of SEQ ID NO: 3, Xaa4 is alanine, and in the B chain of SEQ ID NO: 4, Xaa8 is glutamic acid and Xaa9 is absent;

without limitation to the options indicated.

In another exemplary embodiment, the insulin analog of the present invention contains the amino acid sequence of SEQ ID NO: 16, 18, 20, or 22.

In another aspect, the present invention provides a nucleic acid encoding an insulin analog.

In a typical embodiment, the nucleic acid of the present invention contains nucleotide sequences selected from the group consisting of SEQ ID NOs: 15, 17, 19 and 21.

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In yet another aspect, the present invention provides a recombinant expression vector containing said nucleic acid.

In yet another aspect, the present invention provides a transformant transformed with a recombinant expression vector.

In yet another aspect, the present invention provides a method for producing an insulin analog, comprising:

a) obtaining a recombinant expression vector containing a nucleic acid encoding a peptide analogue of insulin;

b) transforming a host cell with a recombinant expression vector and obtaining a transformant from said host cell;

c) cultivation of the transformant and expression of an insulin analogue peptide; And

d) isolation and purification of the expressed insulin analogue peptide.

In yet another aspect, the present invention provides a pharmaceutical composition for the treatment of diabetes comprising an insulin analog as an active ingredient and a pharmaceutically acceptable carrier.

Beneficial effects.

The insulin analogues of the present invention show significantly improved in vitro effect compared to native insulin, and it is therefore expected that the administration of these insulin analogues even in small amounts will be sufficient for treatment and thus can be effectively used for the treatment of diabetes.

Description of graphic materials

In fig. 1 shows the result of purity analysis of insulin analogues of the present invention by protein electrophoresis and, as an example, the result for insulin analogue 1 (lane 1 - size marker; and lane 2 - insulin analogue 1).

In fig. 2 shows the result of analysis of the purity of the insulin analogues of the present invention by reverse phase chromatography and size exclusion chromatography and, as an example, the result for insulin analogue 1.

In fig. 3 shows the result of peptide mapping of analogs of the present invention and, as an example, the result for insulin analog 1, where USP insulin corresponds to native insulin used as a control.

Best Mode for Carrying Out the Invention

Hereinafter, the present invention will be described in more detail.

At the same time, each of the explanations and exemplary embodiments disclosed herein is applicable to other explanations and exemplary embodiments. That is, the scope of the present invention includes all possible combinations of the various elements disclosed herein. Moreover, the scope of the present invention should not be limited to the specific descriptions given below.

The present invention relates to a new insulin and, specifically, to an insulin analogue with improved in vitro effect compared to native insulin.

As used herein, the term insulin analog refers to a modified native insulin analog obtained by modifying a portion of the amino acid(s) of native insulin in the form of an insertion, deletion or substitution, and in particular includes various native insulin analogs with improved in vitro effect compared to native insulin.

Native insulin is a hormone secreted by the pancreas and typically functions by stimulating cellular glucose absorption and inhibiting fat breakdown, thereby controlling blood glucose levels in vivo. Insulin is formed by processing its precursor, proinsulin, which has no function in controlling blood glucose levels. Insulin consists of two polypeptide chains, i.e. A-chain and B-chain, containing 21 and 30 amino acids, respectively, connected to each other by disulfide bonds. Each of the A chain and B chain may contain the amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 shown below.

A-chain.

Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 1)

B-chain.

Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-GlyPhe-Phe-Tyr- Thr-Pro-Lys-Thr (SEQ ID NO: 2)

The insulin of the present invention refers to insulin analogues obtained using genetic recombination technology, but insulin is not limited to these analogues and includes any insulin with improved in vitro effect compared to native insulin. Preferably, the insulin of the present invention includes inverted insulin, insulin variants, insulin fragments, and so on. Insulin can be produced not only by recombinant method, but also by solid-phase synthesis, and the production method is not limited to these methods.

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These insulin analogues, which are peptides having in vivo blood glucose control properties equivalent or equal to native insulin, include any insulin agonists, insulin derivatives, insulin fragments, insulin variants, and so on.

As used herein, the term insulin agonist refers to a substance that can bind to the insulin receptor in vivo independent of the structure of insulin and, thereby, exhibits biological activity equivalent to the biological activity of insulin.

As used herein, the term insulin derivative may refer to a peptide homologous to each of the amino acid sequences of the A chain and B chain of native insulin, in a form having the ability to control blood glucose levels in vivo, wherein a portion of the groups on the amino acid residue are modified by chemical substitution (eg , alpha-methylation, alpha-hydroxylation), removal (eg, deamination) or modification (eg, Nmethylation).

Additionally, as used herein, the term insulin derivative can refer to a peptide mimic and low or high molecular weight compound that can control blood glucose levels in vivo by binding to the insulin receptor despite lacking amino acid sequence homology to native insulin.

As used herein, the term insulin fragment refers to a form of insulin where at least one amino acid is added or removed, and the added amino acid may be an amino acid not naturally present (eg, a D-type amino acid), and the insulin fragment has the ability to control glucose levels in blood in vivo.

As used herein, the term insulin variant refers to a peptide that differs from insulin in at least one amino acid sequence and also has the ability to control blood glucose levels in vivo.

Methods used in the preparation of insulin agonists, derivatives, fragments and variants can be used individually or in combination. For example, peptides having the ability to control blood glucose levels in vivo, differing in at least one amino acid sequence in which the amino acid residue at the N-terminus is deaminated, may be included within the scope of the present invention.

The insulin analogue of the present invention exclusively includes any peptide the in vitro effect of which is improved over native insulin by introducing substitution, insertion or deletion of amino acid(s) or post-translational modification (e.g., methylation, acylation, ubiquitination and intermolecular covalent linkage) of amino acid(s). sequences (SEQ ID NO: 1 and 2) of the A-chain and B-chain of native insulin. Not only the 20 amino acids commonly found in human proteins, but also atypical or artificial amino acids can be used to replace or insert amino acid(s). Atypical amino acids can be purchased from Sigma-Aldrich, ChemPep, Genzymepharmaceuticals and so on. Peptides containing these amino acids and conventional peptide sequences can be synthesized or purchased from commercial peptide synthesis companies such as American Peptide Company, Bachem (USA) and Anygen (Korea).

Specifically, the insulin analogs of the present invention may be insulin analogs comprising a modification or deletion of certain amino acid residue(s) of the A chain and B chain of native insulin, and preferably may be insulin analogs wherein certain amino acid residue(s) A- the native insulin chains are modified(s) and certain amino acid residue(s) of the native insulin B chain are modified(s) and/or deleted(s).

Preferably, the insulin analogs of the present invention may be an analogue wherein the 14th amino acid residue, tyrosine, of the A chain amino acid sequence represented by SEQ ID NO: 1 is replaced by glutamic acid, asparagine or alanine, or an analogue wherein the 16th the amino acid residue, tyrosine, is replaced by glutamic acid and/or the 25th amino acid residue, phenylalanine, of the B chain amino acid sequence represented by SEQ ID NO: 2 is deleted, or all of these analogues may be included.

More preferably, the insulin analogs of the present invention may be insulin analogs comprising an A chain of SEQ ID NO: 3 represented by the following general formula 1 and a B chain of SEQ ID NO: 4 represented by the following general formula 2.

General formula 1.

Xaal-Ile-Val-Glu-Xaa2-Cys-Cys-Thr-Ser-Ile-Cys-Xaa3-Leu-Xaa4-Gln-Xaa5-Glu-Asn-Xaa6-Cys-Xaa7 (SEQ ID NO: 3)

In the above general formula 1:

Xaa1 is alanine, glycine, glutamine, histidine, glutamic acid or asparagine;

Xaa2 is alanine, glutamic acid, glutamine, histidine or asparagine;

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Xaa3 is alanine, serine, glutamine, glutamic acid, histidine or asparagine;

Xaa4 is alanine, tyrosine, glutamic acid, histidine, lysine, aspartic acid or asparagine;

Xaa5 is alanine, leucine, tyrosine, histidine, glutamic acid or asparagine;

Xaa6 is alanine, tyrosine, serine, glutamic acid, histidine or asparagine; And

Xaa7 is asparagine, glycine, histidine or alanine.

General formula 2.

Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Xaa8-Leu-Val-Cys-Gly-Glu-Arg-GlyPhe-Xaa9-Tyr- Xaa10-Xaall-Lys-Thr (SEQ ID NO: 4)

In the above general formula 2:

Xaa8 is tyrosine, glutamic acid or aspartic acid or absent;

Xaa9 is phenylalanine or absent;

Xaa10 is threonine or absent; And

Xaa11 is proline, glutamic acid or aspartic acid or is absent (where peptides containing the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2 may be excluded).

In a more specific exemplary embodiment, the insulin analogue may be an insulin analogue, wherein:

in general formula 1

Xaa1 is glycine,

Xaa2 is glutamine

Xaa3 is serine,

Xaa4 is alanine, glutamic acid or asparagine,

Xaa5 is leucine,

Xaa6 is a tyrosine and

Xaa7 is asparagine; and in general formula 2

Xaa8 is tyrosine or glutamic acid,

Xaa9 is phenylalanine or absent,

Xaa10 is threonine and

Xaa11 is proline, glutamic acid or aspartic acid or absent;

without limitation to the options indicated.

In another specific exemplary embodiment, the insulin analogue may be an insulin analogue, wherein:

in general formula 1

Xaa1 is glycine,

Xaa2 is glutamine

Xaa3 is serine,

Xaa4 is glutamic acid or asparagine,

Xaa5 is leucine,

Xaa6 is a tyrosine and

Xaa7 is asparagine; and in general formula 2

Xaa8 is a tyrosine,

Xaa9 is phenylalanine or absent,

Xaa10 is threonine and

Xaa11 is proline, glutamic acid or aspartic acid or absent;

without limitation to the options indicated.

In yet another specific exemplary embodiment, the insulin analog may comprise an A chain of general formula 1 and a B chain, wherein in the B chain SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is absent, Xaa10 is threonine, and Xaa11 is proline, glutamine acid or aspartic acid or absent, without limitation to these options.

The insulin analogue may comprise an A chain of general formula 1 and a B chain, wherein in the B chain SEQ ID NO: 4 Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is absent and Xaa11 is proline, glutamic acid or aspartic acid or absent , without limitation to the indicated options.

The insulin analogue may be characterized in that in the A chain SEQ ID NO: 3 Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is glutamic acid, Xaa5 is leucine, Xaa6 is tyrosine and Xaa7

- 5 045778 is asparagine, and in the B chain SEQ ID NO: 4 Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is threonine and Xaa11 is proline, glutamic acid or aspartic acid or none, without limitation thereof.

The insulin analogue may be characterized in that in the A chain SEQ ID NO: 3 Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is asparagine, Xaa5 is leucine, Xaa6 is tyrosine and Xaa7 is asparagine and in the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is threonine, and Xaa11 is proline, glutamic acid, or aspartic acid or none, without limitation.

In yet another specific exemplary embodiment, the insulin analog may be characterized in that, in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is glutamic acid, Xaa5 is leucine, Xaa6 is is tyrosine and Xaa7 is asparagine, and in the B chain SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is absent, Xaa10 is threonine, and Xaa11 is proline, glutamic acid, or aspartic acid or absent, without limitation.

In yet another specific exemplary embodiment, the insulin analog may be characterized in that, in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is alanine, Xaa5 is leucine, Xaa6 is tyrosine and Xaa7 is asparagine, and in the B chain of SEQ ID NO: 4, Xa8 is glutamic acid, Xaa9 is absent, Xaa10 is threonine, and Xaa11 is proline, glutamic acid, or aspartic acid or absent, without limitation.

In a typical embodiment, the insulin analogs of the present invention may include the following analogs:

1) insulin analog 1: a peptide, where the 14th amino acid residue of the A-chain amino acid sequence represented by SEQ ID NO: 3 is glutamic acid and the 25th amino acid residue of the B-chain amino acid sequence represented by SEQ ID NO: 4, is a phenylalanine having the amino acid sequence represented by SEQ ID NO: 16, encoded by a nucleic acid containing the nucleotide sequence represented by SEQ ID NO: 15;

2) insulin analog 2: a peptide, wherein the 14th amino acid residue of the A-chain amino acid sequence represented by SEQ ID NO: 3 is asparagine and the 25th amino acid residue of the B-chain amino acid sequence represented by SEQ ID NO: 4 is is a phenylalanine having the amino acid sequence represented by SEQ ID NO: 18, encoded by a nucleic acid containing the nucleotide sequence represented by SEQ ID NO: 17;

3) insulin analog 3: a peptide, where the 14th amino acid residue of the A-chain amino acid sequence represented by SEQ ID NO: 3 is glutamic acid and the 25th amino acid residue of the B-chain amino acid sequence represented by SEQ ID NO: 4, deleted, having the amino acid sequence represented by SEQ ID NO: 20, encoded by a nucleic acid containing the nucleotide sequence represented by SEQ ID NO: 19;

4) insulin analog 4: a peptide, where the 14th amino acid residue of the A-chain amino acid sequence represented by SEQ ID NO: 3 is alanine, the 16th amino acid residue of the B-chain amino acid sequence represented by SEQ ID NO: 4 is is glutamic acid and the 25th amino acid residue is missing, having the amino acid sequence represented by SEQ ID NO: 22, encoded by a nucleic acid containing the nucleotide sequence represented by SEQ ID NO: 21.

As used herein, the term in vitro effect refers to the uptake of glucose by the action of an insulin analogue and is measured by the EC ₅₀ measured against glucose uptake in murine 3T3-L1 adipocyte-derived cells.

In a typical embodiment, when measuring the in vitro effect of insulin analogs 1-3, insulin analog 1 showed a 238.4% increase in glucose uptake, insulin analog 2 showed a 241.7% increase, and insulin analog 3 showed a 705% increase compared to native insulin, respectively, thereby confirming that the insulin analogues of the present invention demonstrate an outstanding in vitro effect, 2-7 times greater than the effect of native insulin (Table 1).

In another aspect, the present invention provides nucleic acids encoding the above insulin analogs.

As used herein, the term nucleic acid refers to a deoxyribonucleotide (DNA) or ribonucleotide (RNA), including genomic DNA, cDNA and RNA produced by their transcription, and the nucleotide as the basic structural unit includes not only naturally occurring nucleotides but also analogues with sugar or base modifications (Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Uhlman and Peyman, Chemical Reviews, 90: 543-584, 1990). The nucleic acid of the present invention can be isolated and produced using standard molecular biology techniques. For example, the nucleic acid of the present invention can be obtained by PCR amplification using suitable primer sequences based on the sequence of the native insulin gene (NM_000207.2, NCBI), and can be obtained by standard synthesis technology using an automatic DNA synthesizer.

Preferably, the nucleic acid of the present invention includes the nucleotide sequences represented by SEQ ID NOs: 15, 17, 19 and 21. The nucleic acid of the present invention includes not only the nucleotide sequences represented by SEQ ID NOs: 15, 17, 19 and 21, but also all sequences that are at least 70% homologous to the above sequences, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 98%, and the peptide encoded by the above nucleic acid can bind to insulin receptors in vivo, thereby exhibiting substantially the same biological activity as insulin.

As used herein, the term homology refers to the degree of similarity to a given amino acid sequence of a native wild-type protein or the polynucleotide sequence encoding it, and includes sequences that are the above-described or greater percentage identical to the amino acid sequences or polynucleotide sequences of the present invention. Homology can be determined by comparing two given sequences with the naked eye or can be determined using a bioinformatics algorithm that allows homology analysis by aligning the sequences in question for comparison. The homology of two given amino acid sequences can be reported as a percentage. A useful automated algorithm is available for use in the GAP, BESTFIT, FASTA, and TFASTA modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI, USA). The alignment algorithm automated in the above modules includes the Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Other useful sequence alignment and homology determination algorithms are automated in software including FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W.

In another aspect, the present invention provides a recombinant vector containing a nucleic acid encoding an insulin analog. The recombinant vector of the present invention can be designed as a vector for conventional cloning or expression, and can be designed as a vector for using a prokaryotic cell or a eukaryotic cell as a host cell.

As used herein, the term vector refers to a recombinant vector capable of expressing a target protein in a suitable host cell, which is a gene construct containing the necessary operably linked regulatory factors for expression of the introduced nucleic acid. The present invention provides a recombinant vector containing a nucleic acid encoding an insulin analog, and the insulin analog of the present invention can be obtained by transforming or transfecting a host cell with the recombinant vector.

In the present invention, a nucleic acid encoding an insulin analog is operably linked to a promoter. As used herein, the term operably linked refers to the functional linkage of a regulatory sequence that regulates the expression of a nucleic acid (e.g., a promoter, a signal sequence, a ribosome binding site, a transcription termination sequence, etc.) with another nucleotide sequence, whereby the regulatory sequence can regulate transcription and/or or translation of another nucleotide sequence.

As used herein, the term promoter refers to an untranslated nucleic acid sequence located upstream of a coding region that contains a polymerase binding site and has the activity of initiating transcription of a gene located downstream of the promoter to produce mRNA, that is, a domain of DNA to which the polymerase binds, initiating transcription of the gene , and it is located in the 5' transcription initiation domain to produce mRNA.

For example, when the vector of the present invention is a recombinant vector and a prokaryotic cell is used as the host cell, generally a strong promoter should be included (for example, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter and so on) capable of performing transcription, ribosome binding site for translation initiation and

- 7 045778 transcription/translation termination sequences.

In addition, the vector for use in the present invention can be obtained by manipulation of plasmids (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, series pGEX, pET series, pPICZa series, pUC19, etc.), phages (eg, λgt4λB, λ-Charon, λΔz1, M13, etc.) or viruses (eg, SV40, etc.) commonly used in the art.

At the same time, when the vector of the present invention is a recombinant vector and a eukaryotic cell is used as the host cell, promoters derived from mammalian cell genomes (for example, metallothionein promoter) or promoters derived from mammalian viruses can be used. (eg, adenovirus late promoter, papillomavirus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and HSV tk promoter), and typically the vector contains a polyadenylated sequence (eg, bovine growth hormone terminator and SV40-derived polyadenylated sequence) as the sequence transcription termination.

In addition, the recombinant vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selectable marker, and may contain, for example, resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

The recombinant vector of the present invention may further contain another sequence to facilitate the purification of the resulting target proteins, ie single chain insulin analog, proinsulin or analogs thereof. The additionally included sequence may be a marker sequence for protein purification, for example, glutathione-S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6-histidine and so on, but the types of sequences required for the purification of target proteins are not limited to these options.

Fusion proteins expressed using a recombinant vector containing the above marker sequence can be purified by affinity chromatography. For example, when attaching glutathione S-transferase, glutathione, which is a substrate of this enzyme, can be used, and by using a 6-histidine tag, the desired target protein can be easily obtained using a Ni-NTA column.

In yet another aspect, the present invention provides a transformant transformed with a recombinant vector containing a nucleic acid encoding an insulin analog.

As used herein, the term transformation refers to the process of introducing DNA into a host cell and allowing said DNA to be replicated in the host cell in the form of a chromosomal factor or as a result of integration into a chromosome, which is the phenomenon of artificial genetic change through the introduction of exogenous DNA into a cell.

The transformation method used in the present invention may be any transformation method, and it can be easily carried out by a conventional method used in the art. Examples of commonly used transformation methods may include the CaCl ₂ precipitation method, the Hanahan method with improved efficiency and the use of dimethyl sulfoxide (DMSO) as a reducing agent in the CaCl ₂ precipitation method, electroporation, the CaPO ₄ precipitation method, protoplast fusion method, stirring method using silicon carbide fibers, Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated transformation, dextran sulfate-mediated transformation, lipofectamine and drying/suppression, and so on.

The method of transformation with a recombinant vector containing a nucleic acid encoding an insulin analog of the present invention may not be limited to these methods, and any, without limitation, transformation or transfection method commonly used in the art may be used.

The transformant of the present invention can be obtained by introducing a recombinant vector containing a target nucleic acid encoding an insulin analog into a host cell. There may be no significant restrictions on a suitable host used in the present invention, as long as it can express the nucleic acid of the present invention. Examples of suitable hosts may include bacteria belonging to the genus Escherichia such as E. coli, bacteria belonging to the genus Bacillus such as Bacillus subtilis, bacteria belonging to the genus Pseudomonas such as Pseudomonas putida, yeasts such as Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces pombe, insect cells such as Spodoptera frugiperda (SF9), and animal cells such as CHO, COS and BSC. Preferably, E. coli is used as the host cell.

In a typical embodiment, the corresponding nucleotide sequence encoding insulin analogs 1-3 of the present invention is amplified by PCR and amplified

- 8 045778 bathed gene fragments were cloned into the pET22b vector (Novagen). To express insulin analogues in the form of intracellular inclusions, the pET22b vector was treated with restriction enzymes NdeI and BamHI, removing the signal sequence from it, PCR-amplified products of insulin analogues were treated with the same restrictases NdeI and BamHI, and the corresponding isolated DNA was introduced into the pET22b cloning vector using DNA ligase T4. The expression vectors thus obtained were named pET22b insulin analog 1-4, respectively.

The expression vectors pET22b-insulin analog 1-4 encode the amino acid sequences represented by SEQ ID NO: 16, 18, 20 and 22, respectively, under the control of the T7 promoter, and each of the insulin analogs was expressed as inclusions in the host cell, respectively.

E. coli was transformed with recombinant pET22b-insulin analog 1-4 vectors containing nucleic acids encoding each of the insulin analogs of SEQ ID NO: 16, 18, 20, and 22, respectively, and thereby producing transformants expressing them in inclusion form.

In yet another aspect, the present invention provides a method for producing an insulin analog using these transformants.

Preferably, the present invention provides a method for producing an insulin analogue, comprising:

a) obtaining a recombinant expression vector containing a nucleic acid encoding an insulin analogue;

b) transforming a host cell with a recombinant expression vector and obtaining a transformant from said host cell;

c) cultivation of the transformant and expression of the insulin analogue; And

d) isolation and purification of the expressed insulin analogue peptide.

The medium used for culturing the transformants of the present invention must meet the requirements for proper culturing of the host cell. The carbon sources to be included in the host cell growth medium may be suitably selected by those skilled in the art according to the transformants produced from said host cell, and suitable culture conditions may be selected to control the culture duration and the number of cells cultured.

Examples of sugar sources used may include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil and coconut oil; fatty acids such as palmitic acid, stearic acid and linoleic acid; alcohols such as glycerin and ethanol; and organic acids such as acetic acid. These substances can be used individually or in combination.

Examples of nitrogen sources used may include peptone, yeast extract, meat broth, malt extract, liquid corn extract, soybean meal, urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can be used individually or in combination.

Examples of phosphorus sources used may include potassium dihydrogen phosphate, potassium hydrogen phosphate, or the corresponding sodium salt. In addition, the culture medium may contain a metal salt, such as magnesium sulfate or ferrous sulfate, necessary for the growth of the transformant. In addition, substances necessary for growth, such as amino acids and vitamins, can be used. Moreover, suitable precursors can be used in culture media. The above sources can be suitably added to the culture during cultivation by batch culture method or continuous culture method. The pH of the culture can be suitably adjusted using a basic compound such as sodium hydroxide, potassium hydroxide and ammonia, or an acidic compound such as phosphoric acid and/or sulfuric acid. In addition, an antifoam agent such as a polyglycol fatty acid ester may be added to prevent foaming. In addition, to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas (for example, air) can be added to it. The transformant of the present invention can be cultured at 20-45°C and preferably at 25-40°C. In addition, the culture is continued until the maximum amount of desired insulin analogues is obtained, and therefore the culture duration can typically be from 10 to 160 hours.

As described above, the transformant of the present invention can produce insulin analogues by providing culture conditions suitable for the host cells, and the peptide-Nglycosidase produced by them in accordance with the composition of the vector and the characteristics of the host cells can enter the cytoplasm or periplasmic space of the cells. hosts or into the extracellular environment.

Expressed proteins located inside or outside the host cell can be purified by conventional methods.

Examples of purification methods may include salting out (for example, ammonium sulfate precipitation, ammonium phosphate precipitation, etc.), solvent precipitation (for example, protein precipitation

- 9 045778 fractions using acetone or ethanol and so on), dialysis, gel filtration, ion exchange or chromatography such as reverse phase column chromatography, ultrafiltration and so on, and these methods can be applied individually or in combination.

The transformant of the present invention is characterized by the expression of insulin analogues 1-3 from the recombinant vector pET22b-insulin analogue 1-3 in the form of inclusions under the control of the T7 promoter. Accordingly, it is preferable to convert insulin analogues 1-3 expressed in inclusion form into a soluble form and then isolate and purify them.

In a typical embodiment, the present invention may further include the following steps of isolating and purifying inclusion-expressed insulin analogs from the transformant:

d-1) obtaining transformant cells from the culture and grinding them;

d-2) isolation of the expressed peptide analogue of insulin from the lysate of crushed cells with its subsequent refolding;

d-3) purifying the refolded insulin analogue peptide by cation exchange chromatography;

d-4) treating the purified insulin analogue peptide with trypsin and carboxypeptidase B; and d-5) sequential purification of the treated insulin analogue peptide by cation exchange chromatography and anion exchange chromatography.

In yet another aspect, the present invention provides a pharmaceutical composition for the treatment of diabetes containing the above insulin analogues.

The pharmaceutical composition containing the insulin analogs of the present invention may contain a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier for oral administration may include a binding agent, a glidant, a disintegrating agent, an excipient, a solubilizing agent, a dispersing agent, a stabilizing agent, a suspending agent, a coloring agent, a flavoring agent, and the like; for injection compositions, a buffering agent, a preservative, an analgesic, an isotonic agent, a stabilizing agent and so on can be mixed for use; and in topical compositions, a base, an excipient, a lubricant, a preservative, and the like may be used. The pharmaceutical composition of the present invention can be prepared in combination with various pharmaceutically acceptable carriers described above. For example, for oral administration, the pharmaceutical composition may be formulated as tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and so on. For injection, the pharmaceutical composition can be formulated in the form of single-dose ampoules or multi-dose containers. In addition, the pharmaceutical composition can also be formulated in the form of solutions, suspensions, tablets, pills, capsules and sustained release formulations.

However, examples of suitable carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and so on. In addition, the pharmaceutical composition of the present invention may further contain an excipient, an anticoagulant, a lubricant, a humectant, a flavoring agent, an emulsifier, a preservative, and so on.

In yet another aspect, the present invention provides a method of treating diabetes, comprising administering a pharmaceutical composition containing the insulin analogs of the present invention to a subject in need thereof.

The insulin analogs of the present invention show significantly improved in vitro effect compared to native insulin, and therefore it is expected that administration of a pharmaceutical composition containing the above insulin analogs may be effective for the treatment of diabetes.

As used herein, the term administration refers to the administration of a specific substance to a patient by a suitable route, and the conjugate of the present invention can be administered by any known route of administration, provided that the drug can penetrate the target tissue. For example, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary and rectal administration may be performed, but the route of administration is not limited to these options. However, due to the degradation of peptides upon oral administration, the active ingredients of an oral composition must be coated or included in the composition to protect against degradation in the stomach. Preferably, the present composition may be administered in an injection form. In addition, the pharmaceutical composition can be administered using a specific device that transports the active ingredients into the target cell.

In addition, the pharmaceutical composition of the present invention may be determined by the type of drug used as the active component, as well as a number of relevant factors, including the types of diseases being treated, route of administration, age, gender and body weight

- 10 045778 patient and severity of disease. Since the pharmaceutical composition of the present invention has excellent shelf life and titer in vivo, it can significantly reduce the frequency of administration and dose of the pharmaceutical drugs of the present invention.

Embodiment of the invention.

The present invention will now be described in more detail with reference to the following examples. However, these examples are provided for illustrative purposes only, and the invention should not be limited to these examples.

Example 1: Construction and expression of a vector for insulin analogues.

To construct insulin analogues with modification of the amino acid(s) of the A-chain and/or B-chain of native insulin, primer pairs consisting of a forward primer and a reverse primer were synthesized to amplify insulin analogues with the introduction of the corresponding modification and then PCR was performed using proinsulin cDNA in as a matrix. In particular, a template was used where the proinsulin cDNA (SC128255, OriGene) (see sequences BC005255.1 and AAH05255) was cloned into the pET22b vector (Novagen) and for optimal recombinant expression of insulin the nucleotide sequence SEQ ID NO: 23 (ATG GCA ACA ACA TCA ACA GCA ACT ACG CGT), encoding the amino acid sequence Met Ala Thr Thr Ser Thr Ala Thr Thr Arg (SEQ ID NO: 24), was introduced into the cloned proinsulin cDNA as an N-terminal fusion partner.

Specifically, in the present invention, the following insulin analogs were synthesized containing the amino acid modifications shown in table. 1.

Table 1

Amino acid modifications Insulin analogue 1 A ¹⁴ Tyr Glu Insulin analogue 2 A ¹⁴ Tug —► Asn Insulin analogue 3 A ¹⁴ Tug —► Glu + deletion B ²⁵ Insulin analogue 4 A ¹⁴ Tug —► Ala + B ¹⁶ Tug —► Glu, deletion B ²⁵

In table 1 above, insulin analog 1 is an analog replacing the 14th amino acid of the amino acid sequence of the native insulin A chain represented by SEQ ID NO: 1, that is, tyrosine, with glutamic acid;

insulin analog 2 is an analog replacing the 14th amino acid of the amino acid sequence of the native insulin A chain represented by SEQ ID NO: 1, that is, tyrosine, with asparagine;

Insulin analog 3 is an analog with a replacement of the 14th amino acid of the amino acid sequence of the native insulin A chain represented by SEQ ID NO: 1, i.e., tyrosine, with glutamic acid and a deletion of the 25th amino acid of the amino acid sequence of the native insulin B chain represented by SEQ ID NO: 2, that is, phenylalanine; and insulin analog 4 is an analog replacing the 14th amino acid of the native insulin A chain amino acid sequence represented by SEQ ID NO: 1, that is, tyrosine, with alanine, replacing the 16th amino acid of the native insulin B chain amino acid sequence represented by SEQ ID NO: 2, that is, tyrosine, to glutamic acid and deletion of the 25th amino acid of the amino acid sequence of the native insulin B chain represented by SEQ ID NO: 2, that is, phenylalanine.

The corresponding pairs of forward primers and reverse primers designed for the amplification of insulin analogues 1-3 are shown in Table. 2 below.

table 2

Subsequence SEQ Ш NO: Insulin analogue 1 5'-ccagcatctgctccctcgaacagctggagaactactg-3' 5

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5'-cagtagttctccagctgttcgagggagcagatgctgg-3' 6 Insulin analogue 2 5'-cagcatctgctccctcaaccagctggagaactac-3' 7 5 '-gtagttctccagctggttgagggagcagatgctg-3' 8 Insulin analogue 3 5'-ccagcatctgctccctcgaacagctggagaactactg-3' 5 5'-cagtagttctccagctgttcgagggagcagatgctgg-3' 6 5'-gcggggaacgaggcttctacacacccaagacccg-3' 9 5 '-cgggtcttgggtgtgtagaagcctcgttccccgc-3' 10 Insulin analogue 4 5'-cagcatctgctccctcgcccagctggagaactac-3' eleven 5'-gtagttctccagctgggcgagggagcagatgctg-3' 12 5'-ctggtggaagctctcgagctagtgtgcggggaac-3' 13 5'-gttccccgcacactagctcgagagcttccaccag-3' 14 5'-gcggggaacgaggcttctacacacccaagacccg-3' 9 5 '-cgggtcttgggtgtgtagaagcctcgttccccgc-3' 10

In table 2 above, a primer pair consisting of SEQ ID NOs: 5 and 6 was designed to replace the 14th amino acid of the native insulin A chain amino acid sequence, i.e., tyrosine, with glutamic acid; a primer pair consisting of SEQ ID NOs: 7 and 8 was designed to replace the 14th amino acid of the native insulin A chain amino acid sequence, i.e., tyrosine, with asparagine; a primer pair consisting of SEQ ID NOs: 9 and 10 was designed to delete the 25th amino acid of the native insulin B chain amino acid sequence, that is, phenylalanine; a primer pair consisting of SEQ ID NOs: 11 and 12 was designed to replace the 14th amino acid of the native insulin A chain amino acid sequence, i.e. tyrosine, with alanine; and a primer pair consisting of SEQ ID NOs: 13 and 14 was designed to replace the 16th amino acid of the native insulin B chain amino acid sequence, that is, tyrosine, with glutamic acid.

To carry out PCR for amplification of insulin analogues with appropriate modifications, a reaction solution was prepared by mixing 150 ng of template DNA, 1 ml of each 100 pM primer, 5 ml of 2.5 mM dNTP, 10 units of pfx polymerase (Invitrogen, USA) and 10X buffer solution . The reaction solution was subjected to initial denaturation at 95°C for 30 s, followed by repeating 18 cycles of annealing at 95°C for 30 s, 55°C for 30 s and 68°C for 6 min and finally left at 68°C for 5 min. The PCR-amplified products thus obtained were isolated using a gel extraction kit (Qiagen, Germany) and treated with restriction enzymes NdeI and BatHI to obtain fragments for introduction. Vector pET22b (Novagen, USA) was then digested with the same restriction enzymes and the resulting fragments were isolated using the same gel extraction kit. The above injection fragments were ligated to the thus prepared vector using T4 ligase to obtain pET22b-insulin analogue 1-4 expression vectors. These expression vectors contain nucleic acids encoding the amino acid sequences of insulin analogs 1-4, under the control of the T7 promoter, and these vectors allow the expression of insulin analog proteins in the form of inclusions in the host cell.

The thus obtained expression vector pET22b-insulin analog 1 of the present invention contains a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 15, encoding an insulin analog having the amino acid sequence represented by SEQ ID NO: 16; the thus obtained expression vector pET22b-insulin analog 2 of the present invention contains a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 17, encoding an insulin analog having the amino acid sequence represented by SEQ ID NO: 18; the thus obtained expression vector pET22b-insulin analog 3 of the present invention contains a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 19, encoding an insulin analog having the amino acid sequence represented by SEQ ID NO: 20; and the thus obtained expression vector pEt22b-insulin analog 4 of the present invention contains a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 21, encoding an insulin analog having the amino acid sequence represented by SEQ ID NO: 22.

The DNA sequences and protein sequences of each of the insulin analogs 1-4 are shown in table. 3 below.

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Table 3

Subsequence SEQ ID NO: Insulin analogue 1 DNA TTC GTT AAC CAA CAC TTG TGT GGC TCA CAC CTG GTG GAA GCT CTC TAS STA GTG TGC GGG GAA CGA GGC TTC TTC TAS ASA CCC AAG ACC CGC CGG GAG GCA GAG GAC CTG CAG GTG GGG CAG GTG GAG CTG GGC GGG GGC CCT GGT GCA GGC AGC CTG CAG CCC TTG GCC CTG GAG GGG TCC CTG CAG AAG CGT GGC ATT GTG GAA C AA TGC TGT ACC AGC ATC TGC TCC CTC GAA CAG CTG GAG AAC TAC TGC AAC TGA 15 Protein Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gin Vai Gly Gin Vai Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg Gly lie Vai Glu Gin Cys Cys Thr Ser He Cys Ser Leu Glu Gin Leu Glu Asn Tyr Cys Asn 16 Insulin analogue 2 DNA TTC GTT AAC CAA CAC TTG TGT GGC TCA CAC CTG GTG GAA GCT STS TAS STA GTG TGC GGG GAA CGA GGC TTC TTC TAS ASA CCC AAG ACC CGC CGG GAG GCA GAG GAC CTG CAG GTG GGG CAG GTG GAG CTG GGC GGG GGC CCT GGT GCA GGC AGC CTG CAG CCC TTG GCC CTG GAG GGG TCC CTG CAG AAG CGT GGC ATT GTG GAA C AA TGC TGT ACC AGC ATC TGC TCC CTC AAC CAG CTG GAG AAC TAC TGC AAC TGA 17 Protein Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai 18

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Glu Ala Leu Thug Leu Vai Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gin Vai Gly Gin Vai Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg Gly lie Vai Glu Gin Cys Cys Thr Ser He Cys Ser Leu Asn Gin Leu Glu Asn Tyr Cys Asn Insulin analogue 3 DNA TTC GTT AAC CAA CAC TTG TGT GGC TCA CAC CTG GTG GAA GCT STS TAS STA GTG TGC GGG GAA CGA GGC TTC TAS ASA CCC AAG ACC CGC CGG GAG GCA GAG GAC CTG CAG GTG GGG C AG GTG GAG CTG GGC GGG GGC CCT GGT GCA GGC AGC CTG CAG CCC TTG GCC CTG GAG GGG TCC CTG CAG AAG CGT GGC ATT GTG GAA CAA TGC TGT ACC AGC ATC TGC TCC CTC GAA CAG CTG GAG AAC TAC TGC AAC TGA 19 Protein Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Cys Gly Glu Arg Gly Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gin Vai Gly Gin Vai Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg Gly He Vai Glu Gin Cys Cys Thr Ser He Cys Ser Leu Glu Gin Leu Glu Asn Tyr Cys Asn 20 Insulin analogue 4 DNA TTC GTT AAC CAA CAC TTG TGT GGC TCA CAC CTG GTG GAA GCT CTC GAG STA GTG TGC GGG GAA CGA GGC TTC TAS ASA CCC AAG ACC CGC CGG GAG GCA GAG GAC CTG CAG GTG GGG CAG GTG GAG CTG GGC GGG GGC CCT GGT GCA GGC AGC CTG CAG CCC TTG GCC CTG GAG GGG TCC CTG CAG 21

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AAG CGT GGC ATT GTG GAA CAA TGC TGT ACC AGC ATS TGC TCC STS GCC CAG CTG GAG AAC TAC TGC AAC TGA Protein Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Glu Leu Vai Cys Gly Glu Arg Gly Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gin Vai Gly Gin Vai Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg Gly He Vai Glu Gin Cys Cys Thr Ser He Cys Ser Leu Ala Gin Leu Glu Asn Tyr Cys Asn 22

Example 2. Expression of recombinant insulin analogues.

Recombinant expression of the insulin analogues of the present invention under the control of the T7 promoter was carried out as follows. E. coli BL21-DE3 (E. coli B F-dcm ompT hsdS(rB-mB-) gal λDE3) (Novagen, USA) was transformed with each of the insulin analog expression vectors obtained in example 1. Transformation was carried out using the method recommended Novagen, manufacturer of E. coli BL21-DE3. Each individual colony transformed with insulin analogue expression vectors was collected, inoculated in 2X liquid Luria Broth (LB) medium containing 50 μg/ml ampicillin, and cultured at 37°C for 15 hours. Culture of recombinant E. coli and 2X LB medium containing 30% glycerol was mixed in a 1:1 (v/v) ratio, and 1 ml aliquots of the mixture were placed in each cryovial, respectively, and stored at -140°C. The resulting cells were used as starting materials for the production of recombinant insulin analogues.

For the expression of recombinant insulin analogues, one tube of each original cell was diluted in 500 ml of 2X LB and incubated in a water bath with a shaker at 37°C for 14-16 hours. The incubation was stopped when the OD value reached 5.0 or higher, and the resulting culture used as a seed crop. The seed culture was sown in 17 L of fermentation medium using a 50 L fermenter (MSJ-U2, B.E. MARUBISHI, Japan) and the first batch fermentation was started. Cultivation was carried out at 37°C and a stirring speed of 500 rpm with an air supply of 20 l/min (1 rpm) and maintaining a pH of 6.70 with 30% ammonia water. As fermentation progressed and the amount of nutrients in the culture medium decreased, fermentation was carried out in a fed culture by adding a feeding solution. Bacterial growth was monitored by OD values, and IPTG was added at a final concentration of 500 μM when an OD value of 100 or more was reached. After application, cultivation was continued for approximately 23-25 hours. Upon completion of cultivation, recombinant bacteria were isolated by centrifugation and stored at 80°C until use.

Example 3. Isolation and purification of recombinant insulin analogues.

To isolate and purify the recombinant insulin analogs expressed in Example 2 from the transformants, cells were lysed as follows, followed by refolding to convert the insulin analogs expressed as water-insoluble inclusions to a water-soluble form.

<3-1> Isolation and refolding of recombinant insulin analogues.

Specifically, each cell pellet was resuspended in 1 L of solubilization buffer solution (50 mM Tris-HCl (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M NaCl and 0.5% Triton X-100) and cells were lysed using an M-110EN microfluidizer (AC Technology Corp. Model M1475C) at 15,000 psi (103.4 MPa). Cell lysates were centrifuged at 7000 rpm and 4°C for 20 min and the supernatant was removed. The resulting pellet was resuspended in 3 L of wash buffer (0.5% Triton X100, 50 mM Tris (pH 8.0), 0.2 M NaCl and 1 mM EDTA). Centrifugation was carried out at 7000 rpm and 4°C for 20 minutes and the resulting sediment was resuspended in distilled water, followed by centrifugation in the same way. Each of the resulting sediments was resuspended in 400 ml of a buffer solution (1 M glycine, 3.78 g of cysteine-HCl, pH 10.6) and stirred at room temperature for 1 hour. To isolate the resuspended recombinant insulin analogues, 400 ml of 8 M urea and stirred at 40°C for 1 hour. To refold solubilized recombinant insulin analogs, the resulting mixture was centrifuged at 7000 rpm and 4°C for 20 minutes and the supernatant was collected. The supernatant was stirred at 4°C for 16 h with the addition of 7.2 L of distilled water using a peristaltic pump at a flow rate of 1000 ml/h.

- 15 045778 <3-2> Purification by cation exchange chromatography.

Samples that were refolded in Example <3-1> were suitably applied to a cation exchange column (Source S, GE Healthcare) equilibrated with 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol for conjugation. Insulin analog proteins were then eluted from the column with a linear concentration gradient from 0% to 100% in 10 column volumes using a 20 mM sodium citrate buffer (pH 2.0) containing 0.5 M potassium chloride and 45% ethanol

<3-3> Treatment with trypsin and carboxypeptidase B.

From the samples eluted in Example <3-2>, salts were removed using a desalting column and then replaced with a buffer solution (10 mM Tris-HCl, pH 8.0). Samples were treated with trypsin at a molar ratio of 1000 based on the amount of protein in the sample and with carboxypeptidase B at a molar ratio of 2000 based on the amount of protein in the sample and stirred at 16°C for 16 hours. The reaction was stopped by reducing the pH to 3.5 using 1 M sodium citrate (pH 2.0).

<3-4> Purification by cation exchange chromatography.

Samples that completed the reaction in Example <3-3> were suitably reapplied to a cation exchange column (Source S, GE Healthcare) equilibrated with 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol for conjugation. Insulin analog proteins were then eluted from the column with a linear concentration gradient from 0% to 100% in 10 column volumes using a 20 mM sodium citrate buffer (pH 2.0) containing 0.5 M potassium chloride and 45% ethanol <3-5> Purification by anion exchange chromatography.

From the samples eluted in example <3-4>, salts were removed using a desalting column followed by their replacement with a buffer solution (10 mM Tris-HCl, pH 7.5). To isolate pure insulin analogues from the samples thus obtained, the obtained samples were suitably applied to an anion exchange column (Source Q, GE Healthcare) equilibrated with 10 mM Tris buffer solution (pH 7.5) for conjugation. Insulin analogue proteins were then eluted from the column with a linear concentration gradient from 0% to 100% in 10 column volumes using 10 mM Tris buffer (pH 7.5) containing 0.5 M sodium chloride.

The purity of the purified insulin analogues was analyzed by protein electrophoresis (SDS-PAGE) and reverse phase size exclusion chromatography, and the results are shown in FIG. 1 and 2, respectively. In addition, amino acid modifications were confirmed by peptide mapping and molecular weight analysis of each peak, and the results are shown in FIG. 3.

As a result, the desired target modifications to the amino acid sequence of each of the insulin analogues were confirmed.

Example 4. Comparison of the in vitro effect of native insulin and insulin analogues.

To evaluate the in vitro effect of the insulin analogues isolated and purified in Example 3, a glucose absorption capacity experiment (the ability to absorb glucose or lipid synthesis) was performed using the mouse-derived 3T3-L1 cell line with adipocyte differentiation. The 3T3-L1 cell line (ATCC, CL-173) was subcultured using Dulbecco's Modified Eagle's Medium (DMEM, Gibco, catalog number 12430) containing 10% bovine newborn calf serum (NBCS). ) two to three times a week. The 3T3-L1 cell line was suspended in differentiation medium (DMEM containing 10% FBS), seeded in a 48-well plate at a concentration of 5x10 ⁴ cells per well and cultured at 37°C for 48 hours. To differentiate the 3T3-L1 cell line with adipocyte formation, 1 μg/ml human insulin (Sigma, catalog no. I9278), 0.5 mM IBMX (3-isobutyl-1-methylxanthine, Sigma, catalog no. I5879), and 1 μM dexamethasone (Sigma, catalog no. I5879) were added to the differentiation medium according to catalog D4902), the old medium was removed and an aliquot of the resulting mixture was added to each well in an amount of 250 μl per well. After forty-eight hours, the medium was replaced with differentiation medium supplemented with only 1 μg/ml human insulin. Induction of differentiation of the 3T3-L1 cell line to form adipocytes was then confirmed over a period of 7 to 9 days by replacing the medium with differentiation medium containing 1 μg/ml human insulin at 48-hour intervals.

For the glucose uptake capacity experiment, cells that had completed their differentiation to form adipocytes were washed once with serum-free DMEM and then treated with 250 μL of serum-free DMEM for 4 h to remove serum.

Human insulin and insulin analogs, respectively, were serially diluted 10-fold from 5 μM to 0.005 nM with serum-free DMEM for further use as samples. The insulin samples thus obtained were added to the corresponding wells in an amount of 250 μl and cultured at 37°C for 24 hours in an incubator with 5% CO ₂ . To estimate the amount of glucose remaining in the medium upon completion of cultivation, from each culture

Claims (8)

Then, a 200 µl sample was obtained, diluted 5 times using D-PBS, and subjected to GOPOD analysis (GOPOD Assay Kit, Megazyme, catalog number K-GLUC). The concentration of remaining glucose was calculated from the optical density of a standard glucose solution, the corresponding EC50 values were calculated relative to the glucose uptake capacity of insulin analogues, and the results are shown in Table 1. 4 below. Table 4 Glucose uptake capacity (compared to native insulin) (%) Native human insulin 100 Insulin analogue 1 238.4 Insulin analogue 2 241.7 Insulin analogue 3 705 As shown in table. 4, insulin analog 1 showed a 238.4% increase in glucose uptake capacity, insulin analog 2 showed a 241.7% increase, and insulin analog 3 showed a 705% increase compared to native insulin, respectively. Based on the results presented above, it has been confirmed that the insulin analogs of the present invention exhibit outstanding in vitro effects, 2-7 times superior to those of native insulin, and these results demonstrate that these insulin analogs can have a significantly increased serum half-life in vivo and can thus be presented in the form of stable insulin compositions and effectively used as therapeutic agents for the treatment of diabetes. Those skilled in the art will appreciate that the present invention may be embodied in other specific forms without departing from its spirit or essential features. The described embodiments are to be considered in all respects only as illustrative and non-limiting. Accordingly, the scope of the present invention is defined by the appended claims and not by the preceding description. All changes within the meaning and range of equivalence of the claims are to be considered as included within the scope of the present invention. CLAIM 1. A proinsulin peptide comprising an A chain of SEQ ID NO: 3 as set forth in the following general formula 1 and a B chain of SEQ ID NO: 4 as set forth in the following general formula 2, wherein an insulin analogue containing an A chain and The B-chain can be formed during the processing of the indicated proinsulin peptide: (general formula 1) Xaa1-Ile-Val-Glu-Xaa2-Cys-Cys-Thr-Ser-Ile-Cys-Xaa3-Leu-Xaa4-Gln-Xaa5-Glu-Asn-Xaa6-Cys-Xaa7 (SEQ ID NO: 3), ( general formula 2) Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Xaa8-Leu-Val-Cys-Gly-Glu-Arg-GlyPhe-Xaa9-Tyr- Xaa10-Xaa11-Lys-Thr (SEQ ID NO: 4), where: (a) in the A chain of SEQ ID NO: 3, Xaa1 is glycine, Xaa2 is glutamine, Xaa3 is serine, Xaa4 is aspartic acid, Xaa5 is leucine, Xaa6 is tyrosine and Xaa7 is asparagine; and in the B chain of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is threonine, and Xaa11 is proline. 2. Nucleic acid encoding the proinsulin peptide according to claim 1. 3. Recombinant expression vector containing the nucleic acid according to claim 2. 4. A host cell containing the recombinant expression vector according to claim 3. 5. The host cell according to claim 4, which is E. coli. 6. A method for producing an insulin analogue, including: a) obtaining a recombinant expression vector containing a nucleic acid encoding the proinsulin peptide according to claim 1; b) transforming the host cell with a recombinant expression vector and obtaining its transformant; c) cultivation of the transformant and expression of the nucleic acid encoding the proinsulin peptide; And d) isolating and purifying the expressed proinsulin peptide, and then processing it to produce an insulin analogue. 7. The method according to claim 6, where the transformant is E. coli. 8. The method according to claim 6, where step (d) includes: -