EA041658B1 - NEW INSULIN ANALOGUES AND THEIR APPLICATIONS - Google Patents

Description

Field of invention

The present invention relates to a novel insulin analog, and more particularly to an insulin analog with improved in vitro performance over native insulin, and their uses.

Prior Art

Insulin is a blood glucose-controlling hormone secreted by the pancreas that causes excess glucose to move from the blood into the cells, whereby it provides them with an energy source and maintains normal glucose levels. However, diabetic patients cannot maintain normal insulin function due to insulin deficiency, insulin resistance, and loss of beta cell function. As a result, diabetic patients cannot use blood glucose as an energy source, but show symptoms of hyperglycemia with high glucose levels and excrete glucose in the urine, leading to various complications. Accordingly, patients with diabetes and disorders of insulin secretion (type I) or insulin resistance (type II) highly require insulin to allow them to maintain normal blood glucose levels.

Human insulin consists of two polypeptide chains, i.e. A-chains and B-chains, containing 21 and 30 amino acids, respectively, connected to each other by two disulfide bonds. Since insulin has a very short in vivo half-life, like other protein and peptide hormones, it is not able to exert a long-term therapeutic effect, and therefore there is a problem that it must be administered continuously and frequently in order to have its effect. Frequent administration of insulin causes severe pain and discomfort to patients, and therefore there is a need to improve administration in terms of patient adherence to treatment, their safety and convenience.

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

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. The in vivo effect of insulin can be controlled by replacing the amino acid(s) of insulin with another amino acid(s) or by deleting a specific amino acid(s) of insulin. Since highly potent insulin derivatives can have 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 extensively studied in terms of the pharmacokinetic effect of insulin action after subcutaneous injection.

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

Description of the invention

Technical problem.

It is an object of the present invention to provide a novel insulin analog, and in particular an insulin analog with an 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 for treating diabetes comprising administering an insulin analogue or a pharmaceutical composition containing an insulin analogue as an active ingredient to a subject in need thereof.

Technical solution.

To solve the above problems, in one aspect, according to the present invention, there is provided an insulin analog containing the A chain of SEQ ID NO: 3, represented by the following general formula 1, and the 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-AsnXaa6-Cys-Xaa7 (SEQ ID NO: 3)

In the above general formula 1:

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

Xa2 is alanine, glutamic acid, glutamine, histidine or asparagine;

- 1 041658

Xa3 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;

Xa6 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-CysGly-Glu-Arg-Gly-Phe-Xaa9-Tyr- Xaal0-Xaall-Lys-Thr (SEQ W NO: 4)

In the above general formula 2:

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

Xaa9 is phenylalanine or absent;

Xaa10 is threonine or absent; And

Xaa11 is proline, glutamic acid or aspartic acid, or 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 analogue is characterized in that it contains an A chain of general formula 1 and a B chain, where 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, where Xaa8 is tyrosine, Xaa9 is phenylalanine and Xa10 is absent in the Chain of SEQ ID NO: 4.

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

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 phenylalanine; or in the A chain of SEQ ID NO: 3, Xaa4 is asparagine and in the B chain of 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 being limited to the above options.

In yet another exemplary embodiment, the insulin analog of the present invention comprises 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 analogue.

- 2 041658

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

In 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 process for preparing 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 the insulin analogue peptide; And

d) isolating and purifying the expressed insulin analogue peptide.

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

beneficial effects.

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

Description of graphic materials

In FIG. 1 shows the result of purity analysis of the insulin analogs of the present invention by protein electrophoresis and, as an example, the result for insulin analog 1 (lane 1 is a size marker; and lane 2 is insulin analog 1).

In FIG. 2 shows the result of the analysis of the purity of the insulin analogs of the present invention by reverse phase and size exclusion chromatography and, as an example, the result for insulin analog 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. Those. the scope of the present invention includes all possible combinations of the various elements disclosed here. In addition, the scope of the present invention should not be limited by the specific descriptions below.

The present invention relates to a new insulin and, in particular, 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, it includes various native insulin analogs with improved in vitro effect compared to native insulin.

Native insulin is a hormone secreted by the pancreas and normally functions by stimulating cellular uptake of glucose and inhibiting the breakdown of fats, thereby controlling blood glucose levels in vivo. Insulin is formed from the processing of its precursor, proinsulin, which does not have the function of controlling blood glucose levels. Insulin consists of two polypeptide chains, i.e. A-chains and B-chains, containing 21 and 30 amino acids, respectively, connected to each other by disulfide bonds. Each of the A chain and the B chain may contain the amino acid sequences shown in 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-CysAsn (SEQ III NO: 1)

B-chain

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

The insulin of the present invention relates to insulin analogs obtained using genetic recombination technology, but the insulin is not limited to these analogs and includes any insulin with an 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.

These insulin analogs, which are peptides having an in vivo blood glucose control capability equivalent to or corresponding 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 regardless of the structure of insulin and thereby exhibit biological activity equivalent to that 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 in the amino acid residue are modified by chemical substitution (e.g. , alpha-methylation, alpha-hydroxylation), removal (eg, deamination) or modification (eg, N-methylation).

In addition, as used herein, the term insulin derivative can refer to a peptide mimic and a low or high molecular weight compound that can control blood glucose levels in vivo by binding to the insulin receptor, despite the lack of amino acid sequence homology of 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 occurring (e.g., 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 may be used alone or in combination. For example, peptides having the ability to control blood glucose levels in vivo that differ 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 whose in vitro effect is improved relative to native insulin by the introduction of substitution, insertion or deletion of amino acid(s) or post-translational modification (e.g., methylation, acylation, ubiquitination and intermolecular covalent binding) of amino acids. sequences (SEQ ID NO: 1 and 2) of the A-chain and B-chain of native insulin. Not only the 20 amino acids commonly seen 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 modification or deletion of the specified amino acid residue(s) of Acepi and the B chain of native insulin, and preferably may be insulin analogs wherein the specified amino acid residue(s) of A- the native insulin chains are modified (modified) and certain amino acid residue(s) of the native insulin B chain are modified (modified) and/or removed (deleted).

Preferably, the insulin analogs of the present invention may be an analog where the 14th amino acid residue, tyrosine, of the A chain amino acid sequence shown in SEQ ID NO: 1 is replaced with glutamic acid, asparagine or alanine, or an analog where the 16th the amino acid residue, tyrosine, has been replaced with glutamic acid and/or the 25th amino acid residue, phenylalanine, of the B chain amino acid sequence shown in SEQ ID NO: 2 has been deleted, or may include all of these analogs.

More preferably, the insulin analogs of the present invention may be insulin analogs comprising the A chain of SEQ ID NO: 3 represented by the following general formula 1 and the 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-AsnXaa6-Cys-Xaa7 (SEQ ID NO: 3)

- 4 041658

In the above general formula 1:

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

Xa2 is alanine, glutamic acid, glutamine, histidine or asparagine;

Xa3 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;

Xa6 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-CysGly-Glu-Arg-Gly-Phe-Xaa9-Tyr- Xaal0-Xaall-Lys-Thr (SEQ W NO: 4)

In the above general formula 2:

Xaa8 is tyrosine, glutamic acid, or aspartic acid, or is 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 analog may be an insulin analog, where:

in general formula 1:

Xaa1 is a glycine,

Xaa2 is a 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 is absent;

without being limited to the above options.

In another specific exemplary embodiment, the insulin analog may be an insulin analog, where:

in general formula 1:

Xaa1 is a glycine,

Xaa2 is a 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 tyrosine,

Xaa9 is phenylalanine or absent,

Xaa10 is threonine, and

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

without being limited to the above options.

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

The insulin analogue may contain an A chain of general formula 1 and a B chain, where in the B chain of SEQ ID NO: 4 Xaa8 is tyrosine, Xaa9 is phenylalanine, Xaa10 is absent and Xaa11

- 5 041658 is proline, glutamic acid or aspartic acid, or absent, without limitation to these options.

An insulin analogue can 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 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 is not limited to these options.

An insulin analogue can be 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, glutamic acid, or aspartic acid, or is not limited to these options.

In yet another specific exemplary embodiment, an 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 of SEQ ID NO: 4, Xaa8 is tyrosine, Xaa9 is absent, Xaa10 is threonine, and Xaa11 is proline, glutamic acid, or aspartic acid, or is not limited to these options.

In yet another specific exemplary embodiment, an 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, Xaa8 is glutamic acid, Xaa9 is absent, Xaa10 is threonine, and Xaa11 is proline, glutamic acid or aspartic acid, or is not limited to these options.

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

1) insulin analog 1: a peptide, wherein 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 analogue 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 wherein 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 analogue 4: a peptide, wherein the 14th amino acid residue of the A chain amino acid sequence represented by SEQ ID NO: 3 is alanine, the 16 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 absent, 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 an insulin analogue and is measured by the measurement of EC ₅₀ relative to glucose uptake by adipocyte differentiated murine 3T3-L1 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 analogs of the present invention show an outstanding in vitro effect, 2-7 times greater than that of native insulin (Table 1).

- 6 041658

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

As used herein, the term nucleic acid refers to deoxyribonucleotide (DNA) or ribonucleotide (RNA), including genomic DNA, cDNA, and RNA resulting from their transcription, and nucleotide as a basic structural unit includes not only naturally occurring nucleotides, but also analogues with sugar modifications or bases (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 shown in SEQ ID NOs: 15, 17, 19, and 21. The nucleic acid of the present invention includes not only the nucleotide sequences shown in SEQ ID NOs: 15, 17, 19, and 21, but also all sequences 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 demonstrating substantially the same biological activity as insulin.

As used herein, the term homology refers to the degree of similarity to a given native wild-type protein amino acid sequence or polynucleotide sequence encoding it, and includes sequences that are as described above or greater than the percentage of 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 analysis of homology by aligning the sequences in question for comparison. The homology of two given amino acid sequences can be indicated 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 sequence alignment algorithms,

Pearson-Lipman (Pearson & Lipman) and Smith-Waterman (Smith & Waterman). 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 to allow expression of the introduced nucleic acid. The present invention makes it possible to obtain 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 said 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 a functional association 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 translation of another nucleotide sequence.

As used herein, the term promoter refers to an untranslated nucleic acid sequence located upstream of the coding region, containing a polymerase binding site, and having the activity of initiating transcription of a gene located downstream of the promoter to form mRNA, i.e. the DNA domain to which the polymerase binds to initiate gene transcription, and is located in the 5' transcription initiation domain to form mRNA.

For example, when the vector of the present invention is a recombinant vector and

- 7 041658 a prokaryotic cell is used as the host cell, a strong promoter (e.g. tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, tip promoter, T7 promoter, etc.) capable of conducting transcription, a ribosome binding site for translation initiation, and transcription/translation termination sequences.

In addition, a vector for use in the present invention can be obtained by manipulation with plasmids (e.g., pSC101, pGV1106, pACYC177, ColEl, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFRl, pHV14, pGEX, pET series, pPICZa series, pUC19, etc.), phages (eg, λgt4 λΒ, λ-Charon, λΔζ1, 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. (e.g., adenovirus late promoter, papillomavirus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk promoter), and typically the vector contains a polyadenylated sequence (e.g., a bovine growth hormone terminator and a polyadenylated sequence derived from SV40) as a 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, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline resistance genes.

The recombinant vector of the present invention may additionally contain another sequence to facilitate the purification of the resulting target proteins, i.e. single chain insulin analog, proinsulin or analogues thereof. An optional sequence may be a marker sequence for protein purification, e.g., glutathione-S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), 6-histidine, etc., but sequence types required to purify the 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 glutathione-S-transferase is attached, glutathione, which is a substrate of this enzyme, can be used, and when 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 replicate in the host cell in the form of a chromosomal factor or as a result of integration into a chromosome, which is a phenomenon of artificial genetic change by introducing exogenous DNA into the 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 CaCl ₂ precipitation method, Hanahan method with improved efficiency using dimethyl sulfoxide (DMSO) as a reducing agent in CaCl ₂ precipitation method, electroporation, CaPO ₄ precipitation method, protoplast fusion method, agitation method using silicon carbide fibers, Agrobacteria-mediated transformation, polyethylene glycol (PEG) transformation, dextran sulfate, lipofectamine and drying/suppression mediated transformation, and so on.

The method of transformation with a recombinant vector containing a nucleic acid encoding an insulin analogue of the present invention may not be limited to these methods, and any, without limitation, transformation or transfection method commonly used in this field can 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, provided that 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, the host cell is

- 8 041658

E. coli.

In a typical embodiment, the corresponding nucleotide sequence encoding the insulin analogs 1-3 of the present invention was amplified by PCR and the amplified gene fragments were cloned into the pET22b vector (Novagen). For the expression of insulin analogs in the form of intracellular inclusions, the pET22b vector was treated with NdeI and BamHI restrictases, removing the signal sequence from it, the PCR-amplified products of insulin analogs were treated with the same restriction enzymes 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 pET22b insulin analog expression vectors 1-4 encode the amino acid sequences shown in SEQ ID NOs: 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 were transformed with pET22b insulin analog 1-4 recombinant vectors containing the nucleic acids encoding each of the insulin analogs of SEQ ID NOs: 16, 18, 20, and 22, respectively, and thereby obtained transformants expressing them as inclusions.

In yet another aspect, the present invention provides a process for preparing an insulin analogue using said transformants.

Preferably, according to the present invention, there is provided a process for preparing an insulin analog, 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) isolating and purifying 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. Carbon sources to be included in the host cell growth medium can be appropriately selected according to the judgment of one skilled in the art according to the transformants obtained from said host cell, and suitable culture conditions can be selected to control the culture duration and the number of cultured cells.

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 glycerol and ethanol; and organic acids such as acetic acid. These substances may be used singly or in combination.

Examples of nitrogen sources used may include peptone, yeast extract, meat broth, malt extract, liquid corn extract, soy flour, urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may be used singly 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 iron 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 at the time of culture by batch culture or continuous culture. 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, a defoamer such as a polyglycol fatty acid ester may be added to prevent foaming. In addition, oxygen or an oxygen-containing gas (eg, air) may be added to the culture to maintain an aerobic state. The transformant of the present invention can be cultivated at 20-45°C and preferably at 25-40°C. In addition, the culture is continued until the maximum amount of the desired insulin analogs is obtained, and, therefore, the culture time can usually be from 10 to 160 hours.

As described above, the transformant of the present invention can produce insulin analogs under culture conditions suitable for host cells, and the peptide-N-glycosidase produced by them according to the vector composition and characteristics of the host cells can enter the cytoplasm or periplasmic space of the host cells. hosts or into the extracellular environment.

Expressed proteins located inside or outside the host cell can be purified

- 9 041658 by the usual method.

Examples of purification methods may include salting out (for example, ammonium sulfate precipitation, ammonium phosphate precipitation, and so on), solvent precipitation (for example, precipitation of the protein fraction 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 singly or in combination.

The transformant of the present invention is characterized by the expression of insulin analogues 1-3 from the recombinant vector pET22b-analogue of insulin 1-3 in the form of inclusions under the control of the T7 promoter. Accordingly, it is preferable to convert the insulin analogs 1-3 expressed as inclusions into a soluble form, followed by their isolation and purification.

In a typical embodiment, the present invention may further include the following steps for isolating and purifying insulin analogs expressed as inclusions from a transformant:

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

d-2) isolating the expressed insulin analogue peptide from the minced cell lysate with its subsequent refolding;

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

d-4) treating the purified insulin analog peptide with trypsin and carboxypeptidase B; and d-5) sequentially purifying the treated insulin analog 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 comprising the above insulin analogues.

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

Meanwhile, examples of suitable carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, 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 a filler, an anticoagulant, a lubricating agent, a humectant, a flavoring agent, an emulsifier, a preservative, and so on.

In yet another aspect, the present invention provides a method for 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 a significantly improved in vitro effect compared to native insulin, and therefore it is expected that the administration of a pharmaceutical composition containing the above insulin analogs can be effective for the treatment of diabetes.

As used herein, the term administration refers to the administration of a particular substance to a patient by a suitable route, and the conjugate of the present invention may be administered by any known route of administration, provided that the drug can enter the target tissue. For example, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, and rectal administration may be administered, but the route of administration is not limited to these. However, due to the degradation of peptides upon oral administration, the active ingredients of the 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 may be administered using a specific device to transport the active ingredients into the target cell.

In addition, the pharmaceutical composition of the present invention can be determined by the type of drug used as the active ingredient, as well as a number of relevant factors, including the types of diseases to be treated, the route of administration, the age, sex and body weight of the patient, and the severity of the disease. . Since the pharmaceutical composition of the present invention has an excellent in vivo shelf life and titer, the administration frequency and dosage of the pharmaceutical drugs of the present invention can be significantly reduced.

Embodiment of the invention

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are 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 analogs with modification of the amino acid (amino acids) 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 analogs with the introduction of the appropriate modification, and then PCR was performed using cDNA of proinsulin in as a matrix. In particular, a template was used where the cDNA of proinsulin (SC128255, OriGene) (see sequences BC005255.1 and AAH05255) was cloned into the vector pET22b (Novagen) and for optimal recombinant expression of insulin, the nucleotide sequence of 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, the following insulin analogs containing the amino acid modifications shown in Table 1 were synthesized in the present invention. 1.

Table 1

Amino acid modifications Insulin analogue 1 A ¹⁴ Tug Glu Insulin analogue 2 A ¹⁴ Tug Asn Insulin analogue 3 A ¹⁴ Tyr -> Glu + deletion B ^2E Insulin analogue 4 A ¹⁴ Tyr -> Ala + B ¹⁶ Tyr -> Glu, deletion B ²⁵

In table. 1 above, insulin analog 1 is the 14th amino acid substitution analog of the native insulin A chain amino acid sequence shown in SEQ ID NO: 1, i.e. tyrosine, glutamic acid;

insulin analog 2 is a 14th amino acid substitution analog of the native insulin A chain amino acid sequence shown in SEQ ID NO: 1, ie. tyrosine, to asparagine;

insulin analog 3 is a 14th amino acid substitution analog of the native insulin A chain amino acid sequence shown in SEQ ID NO: 1, ie. tyrosine, to glutamic acid and deletion of the 25th amino acid of the native insulin B chain amino acid sequence shown in SEQ ID NO: 2, i.e. phenylalanine; and insulin analog 4 is a 14th amino acid substitution analog of the native insulin A chain amino acid sequence shown in SEQ ID NO: 1, i.e. tyrosine to alanine by replacing the 16th amino acid of the native insulin B chain amino acid sequence shown in SEQ ID NO: 2, i.e. tyrosine, to glutamic acid and deletion of the 25th amino acid of the native insulin B chain amino acid sequence shown in SEQ ID NO: 2, i.e. phenylalanine.

The respective pairs of forward primers and reverse primers designed to amplify insulin analogs 1-3 are shown in Table 1. 2 below.

- 11 041658

table 2

Subsequence SEQID NO: Insulin analogue 1 5'-ccagcatctgctccctcgaacagctggagaactactg-3' 5 5'-cagtagttctctccagctgttcgagggagcagatgctgg-3' 6 Insulin analogue 2 5'-cagcatctgctccctcaaccagctggagaactac-3' 7 5'-gtagttctccagctggttgagggagcagatgctg-3' 8 Insulin analogue 3 5'-ccagcatctgctccctcgaacagctggagaactactg-3' 5 5'-cagtagttctctccagctgttcgagggagcagatgctgg-3' 6 5'-gcggggaacgaggcttctacacacccaagacccg-3' 9 5'-cgggtcttgggtgtgtagaagcctcgttccccgc-3' 10 Insulin analogue 4 5'-cagcatctgctccctcgcccagctggagaactac-3' eleven 5'-gtagttctctccagctgggcgagggagcagatgctg-3' 12 5'-ctggtggaagctctcgagctagtgtgcggggaac-3' 13 5'-gttccccgcacactagctcgagagcttccaccag-3' 14 5'-gcggggaacgaggcttctacacacccaagacccg-3' 9 5'-cgggtcttgggtgtgtagaagcctcgttccccgc-3' 10

In table. 2 above, the 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. tyrosine, 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, to 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, i.e. 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, to 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, i.e. tyrosine to glutamic acid.

For PCR to amplify insulin analogues with appropriate modifications, a reaction solution was prepared by mixing 150 ng of DNA template, 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 an initial denaturation at 95°C for 30 s followed by 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. at 5 m. The PCR amplified products thus obtained were isolated using a gel isolation kit (Qiagen, Germany) and digested with NdeI and BamHI to obtain fragments for administration. The pET22b vector (Novagen, USA) was then digested with the same restriction enzymes, and the resulting fragments were isolated using the same gel isolation kit. The above fragments for introduction were ligated with the thus prepared vector using T4 ligase to obtain pET22b-insulin analogue expression vectors 1-4. These expression vectors contain nucleic acids encoding the amino acid sequences of insulin analogues 1-4 under the control of the T7 promoter, and these vectors allow the expression of insulin analogue proteins in the form of inclusions in the host cell.

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

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

- 12 041658

Table 3

Subsequence SEQ ID NO: Insulin analogue 1 DNA TTC GTT AAS CAA CAC TTG TGT GGC TCA CAC CTG GIG GAA GCT CTC TAC STA GTG TGC GGG GAA CGA GGC 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 15 Protein Phe Va1 Asn G1n His Leu Cys Gly Ser His Leu Vai Glu A1a 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 He Vai Glu Gin Cys Cys Thr Ser lie 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 CTC TAC STA GTG TGC GGG GAA CGA GGC 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 AAC CAG CTG GAG AAC TAC TGC AAC TGA 17 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 18

- 13 041658

Lys Arg Gly lie Val Glu Gin Cys Cys Thr Ser Ile 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 CTC TAC STA GTG TGC GGG GAA CGA GGC TTC TAC ACA 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 CAA TGC TGT ACC AGC ATC TGC TCC CTC GAA CAG CTG GAG AAC TAC TGC AAC TGA 19 Protein Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gin Val Gly Gin Val 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 Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Glu Gin Leu Glu Asn Tyr Cys Asn 20 Insulin analog 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 TAC ACA 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 CAA TGC TGT ACC AGC ATC TGC TCC CTC GCC CAG CTG GAG AAC TAC TGC AAC TGA 21 Protein Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Glu Leu Val Cys Gly Glu Arg Gly Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gin Val Gly Gin Val Glu Leu Gly Gly Gly Pro Gly Ala Gly 22 Ser Leu Gin Pro Leu Ala Leu Glu Gly Ser Leu Gin Lys Arg Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Ala Gin Leu Glu Asn Tyr Cys Asn

Example 2 Expression of Recombinant Insulin Analogs.

Recombinant expression of the insulin analogs of the present invention under the control of the T7 promoter was performed 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 performed using the method recommended Novagen, manufacturer of E. coli BL21-DE3. Each individual colony transformed with insulin analog expression vectors was harvested, seeded in 2X liquid Luria medium (Luria Broth, LB) containing 50 μg/ml ampicillin, and cultured at 37°C for 15 h. Culture of recombinant E. coli and 2X LB medium containing 30% glycerol was mixed in a ratio of 1:1 (v/v), 1 ml aliquots of the mixture were placed in each cryovial, respectively, and stored at -140°C. The resulting cells were used as starting cells for the production of recombinant insulin analogues.

For the expression of recombinant insulin analogs, 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. used as a seed crop. The inoculum was inoculated into 17 L of fermentation medium using a 50 L fermenter (MSJ-U2, VE 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 v/v/min) and maintaining a pH of 6.70 with 30% ammonia water. As fermentation decreases with a decrease in the amount of nutrients in the culture medium, fermentation

- 14 041658 was carried out in fed-batch culture by adding a feeding solution. Bacterial growth was monitored by OD values, and when an OD value of 100 or more was reached, IPTG was added at a final concentration of 500 μM. After the addition, the cultivation was continued for approximately 23-25 hours. Upon completion of the cultivation, the recombinant bacteria were isolated by centrifugation and stored at

-80°C before use.

Example 3 Isolation and Purification of Recombinant Insulin Analogs.

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

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

Specifically, each cell pellet was resuspended in 1 L of solubilizing 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 the cells were lysed using an M-110EH 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 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 pellet was resuspended in distilled water, followed by centrifugation in the same manner. Each of the precipitates obtained 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 analogs, 400 ml of 8 M urea and stirred at 40°C for 1 hour. To refold the solubilized recombinant insulin analogues, the resulting mixture was centrifuged at 7000 rpm and 4°C for 20 min 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.

3-2. Purification by cation exchange chromatography.

Samples refolded in Example 3-1 were loaded appropriately on a cation exchange column (Source S, GE Healthcare) equilibrated with 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol 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 a 20 mM sodium citrate buffer (pH 2.0) containing 0.5 M potassium chloride and 45% ethanol. .

3-3. Trypsin and carboxypeptidase B.

Salts were removed from the samples eluted in Example 3-2 using a desalting column, followed by their replacement with a buffer solution (10 mM Tris-HCl, pH 8.0). The samples were treated with trypsin according to a molar ratio of 1000 by the amount of protein in the sample and carboxypeptidase B according to the molar ratio of 2000 by the amount of protein in the sample and stirred at 16°C for 16 hours. The reaction was stopped by lowering the pH to 3.5 using 1 M sodium citrate (pH 2.0).

3-4. Purification by cation exchange chromatography.

Samples completed in Example 3-3 were reapplied as appropriate to a cation exchange column (Source S, GE Healthcare) equilibrated with a 20 mM sodium citrate buffer (pH 2.0) containing 45% ethanol to conjugation. Insulin analogue 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 thus obtained samples, the obtained samples were suitably applied to an anion exchange column (Source Q, GE Healthcare) equilibrated with 10 mM Tris buffer (pH 7.5) for conjugation. The insulin analog proteins were then eluted from the column with a linear concentration gradient from 0 to 100% in 10 column volumes using a 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 and 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 analysis of the molecular weight of each peak, and the results are shown in FIG. 3.

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

-

Claims (13)

Example 4 Comparison of the in vitro effect of native insulin and insulin analogues. To evaluate the in vitro effect of the insulin analogs isolated and purified in Example 3, a glucose absorption capacity (glucose uptake or lipid synthesis capacity) experiment was performed using a murine adipocyte differentiation cell line 3T3-L1. 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 33-Ll cell line was suspended in differentiation medium (DMEM containing 10% FBS), seeded in a 48-well plate at a concentration of 5x10 cells per well, and cultured at 37°C for 48 hours. adipocytes, 1 µg/mL human insulin (Sigma, cat. no. 19278), 0.5 mM IBMX (3-isobutyl-1-methylxanthine, Sigma, cat. no. 15879), and 1 µM dexamethasone (Sigma, cat. no. 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. Forty-eight hours later, the medium was replaced with differentiation medium supplemented with only 1 μg/ml human insulin. The induction of differentiation of the 3TZ-Ll cell line into 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 hours to remove serum from them. Human insulin and insulin analogues, respectively, were subjected to a 10-fold serial dilution from 5 μM to 0.005 nM serum-free DMEM medium for further use as samples. The thus obtained insulin samples were added to the respective wells in the 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 at the end of the culture, a 200 μl sample was obtained from each culture, diluted 5-fold with D-PBS, and subjected to GOPOD assay (GOPOD Assay Kit, Megazyme, catalog number K-GLUC) . The remaining glucose concentration was calculated from the absorbance of the standard glucose solution, the corresponding EC ₅₀ values were calculated relative to the ability to absorb glucose under the influence of insulin analogues, and the results are shown in table. 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 l 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 above results, it was confirmed that the insulin analogs of the present invention exhibit an outstanding in vitro effect, 2 to 7 times that 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 not restrictive. Thus, 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 being included within the scope of the present invention. CLAIM 1. A proinsulin peptide comprising an A chain of SEQ ID NO: 3 as indicated in the following general formula 1 and a B chain of SEQ ID NO: 4 as indicated in the following general formula 2, wherein an insulin analogue comprising an A chain and B-chain, can be formed during the processing of the specified proinsulin peptide: - 16 041658 (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 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. 2. The proinsulin peptide according to claim 1, wherein the peptide (a) contains the amino acid sequence shown in SEQ ID NO: 20. 3. Nucleic acid encoding the proinsulin peptide according to claim 1 or 2. 4. Nucleic acid according to claim 3, containing the nucleotide sequence of SEQ ID NO: 19. 5. Recombinant expression vector containing the nucleic acid according to claim 3. 6. A host cell containing a recombinant expression vector according to claim 5. 7. The host cell according to claim 6, which is E. coli. 8. A method for producing an insulin analog, comprising: 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. 9. The method of claim 8, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 19. 10. The method of claim 8 wherein the transformant is E. coli. 11. The method according to claim 8, where stage (d) includes: d-1) obtaining transformant cells from the culture and grinding them; d-2) isolation of the expressed proinsulin peptide from the crushed cell lysate with its subsequent refolding; d-3) purification of the refolded proinsulin peptide by cation exchange chromatography; d-4) treating the purified proinsulin peptide with trypsin and carboxypeptidase B; and d-5) sequentially purifying the insulin analog by cation exchange chromatography and anion exchange chromatography. 12. An insulin analogue comprising the A chain of SEQ ID NO: 3 as set forth in the following general formula 1 and the B chain of SEQ ID NO: 4 as set forth in 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), ( 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 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. 13. A pharmaceutical composition for the treatment of diabetes, comprising the insulin analog of claim 12 and a pharmaceutically acceptable carrier. -