CN118063589A

CN118063589A - Ultra-long acting insulin analogue, and preparation method and application thereof

Info

Publication number: CN118063589A
Application number: CN202410159119.7A
Authority: CN
Inventors: 陈勇; 罗华; 李祥; 李红亮; 蒋利敏; 刘舒生; 皮世玉; 罗吉鸿
Original assignee: Lepujian Sugar Pharmaceutical Chongqing Co ltd
Current assignee: Lepujian Sugar Pharmaceutical Chongqing Co ltd
Priority date: 2024-02-04
Filing date: 2024-02-04
Publication date: 2024-05-24

Abstract

The invention relates to the technical field of polypeptide medicaments, in particular to an ultra-long-acting insulin analogue, a preparation method and application thereof. The technical scheme is modified aiming at the amino acid sequence of natural human insulin, and the action time of insulin analogues is prolonged and the compliance of patients is improved through double modification of fatty acid at proper sites. The technical scheme can further solve the technical problem that the action time of the insulin analogue is not ideal in the prior art. Experimental data show that the sequence of the insulin analogue is modified and adopts a double-modification structure, so that the acting time of the medicine can be effectively prolonged. In addition, the insulin of the scheme can adopt a recombinant expression method, the process route is simple, the cost is low, certain economic advantages are achieved, and the technical scheme has ideal popularization and application prospects.

Description

Ultra-long acting insulin analogue, and preparation method and application thereof

Technical Field

The invention relates to the technical field of polypeptide medicaments, in particular to an ultra-long-acting insulin analogue, a preparation method and application thereof.

Background

Insulin is a protein hormone secreted by islet beta cells within the pancreas by stimulation with endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, and the like. Insulin is the only hormone in the body that reduces blood glucose while promoting glycogen, fat, protein synthesis. Human Insulin consists of A, B peptide chains, 11 Human Insulin (Insulin Human) A chains of 21 amino acids, 15 Human Insulin (Insulin Human) B chains of 30 amino acids, and 51 amino acids. Wherein the sulfhydryl groups in the four cysteines of A ₇(Cys)-B₇(Cys)、A₂₀(Cys)-B₁₉ (Cys) form two disulfide bonds that join the A, B two chains. In addition, a disulfide bond is present between A ₆ (Cys) and A ₁₁ (Cys) in the A chain. In order to improve the action effect and time of insulin, scientific researchers have conducted a great deal of research on the amino acid composition and modification mode of insulin.

Degu insulin was developed by NovoNordisk, inc., marketed in Japan at 10 in 2012, approved for the treatment of type 1,2 diabetes. Degu-insulin is an ultra-long-acting basic insulin analogue obtained by removing threonine at position B ₃₀ on the basis of human insulin and connecting 1 16-carbon fatty diacid with lysine at position B ₂₉ through 1L-gamma-glutamic acid linker. This unique molecular structure allows it to exist in the formulation in a stable, soluble, bis-hexamer form prior to injection. The insulin deltoid has limited in-vivo action time and needs to be frequently administered, and the mode can bring certain side effects and reduce the compliance of patients, so that the requirements of clinical application of insulin cannot be met. Further research on the structure and modification of insulin analogues is needed to develop insulin analogues with longer duration of action and lower frequency of administration.

Disclosure of Invention

The invention aims to provide an ultra-long acting insulin analogue to solve the technical problem that the action time of the insulin analogue is not ideal in the prior art.

In order to achieve the above purpose, the invention adopts the following technical scheme:

An ultra-long acting insulin analogue comprising a chain a: GIVEQCCTSICSLX ₁QLENYCX₂X₃X₄, and B chain ;FVX₅QHLCGSHLVEALX₆LVCGERGFX₇X₈X₉X₁₀X₁₁X₁₂;

Wherein X ₁ is Y or E or D; x ₂ is G or N or K; x ₃ is K or absent; x ₄ is A or G or is absent;

X ₅ is N or D; x ₆ is Y or H or E; x ₇ is F or H or I; x ₈ is K or Y; x ₉ is T or G or is absent; x ₁₀ is P or G or K or is absent; x ₁₁ is K or P or is absent; x ₁₂ is T or absent;

Fatty acids are modified at the amino acid residues of X ₈ or X ₁₀ or X ₁₁.

The technical scheme also provides a preparation method of the ultra-long acting insulin analogue, which comprises the following steps in sequence:

s1: obtaining an insulin analog a precursor;

s2: insulin analogues were subjected to fatty acid modification.

The technical scheme also provides application of the ultra-long acting insulin analogue in preparing a medicament for treating diabetes or a medicament for reducing blood sugar.

Further, a fatty acid is also modified at the amino acid residue of X ₃.

Further, the fatty acid comprises a C ₁₈ modifier with a structural formula shown in a formula (2) and a C ₂₀ modifier with a structural formula shown in a formula (3); c ₂₂ modifier with structural formula shown in formula (4);

further, X ₁ is E; x ₂ is N or K; x ₃ is K or absent; x ₄ is absent; x ₅ is N; x ₆ is H; x ₇ is H; x ₈ is Y; x ₉ is T or G; x ₁₀ is P or G; x ₁₁ is K; x ₁₂ is absent; fatty acids are modified at amino acid residues X ₁₁ and fatty acids are also modified at amino acid residues X ₂ or X ₃.

Further, the sequence of the A chain is shown as SEQ ID NO.5 or SEQ ID NO.7, and the sequence of the B chain is shown as SEQ ID NO.6 or SEQ ID NO. 8.

Further, the A chain and the B chain are linked by disulfide bonds.

Further, in S1, recombinant expression is performed using escherichia coli or yeast to obtain an insulin analog a precursor.

The technical scheme can be obtained by means of engineering bacteria expression (escherichia coli and saccharomycetes), and can be obtained by means of chemical synthesis. The above-mentioned insulin analogues can be obtained by the conventional means of the prior art, either by self synthesis/expression or by the biotechnological company.

Further, the molar ratio of modifier to insulin analogue is 3:1-6:1.

The fatty acid modification process is as follows: dissolving a modifier in N-methyl pyrrolidone to obtain a modifier solution; dissolving insulin analogue with sodium carbonate to obtain insulin analogue solution; the insulin analogue solution and the modifier solution are reacted and purified to obtain the fatty acid modified insulin analogue.

To sum up, the technical principle of the technical scheme has the following beneficial effects:

The technical scheme adopts a double-modification structure to prolong the action time of insulin analogues and improve the compliance of patients. The double-modified structure is effective and has prolonged action, and has higher stability, so that the compound can be prepared into oral preparations with high potential. The insulin of the scheme can adopt a recombinant expression method, has simple process route and low cost, and has certain economic advantages. According to the technical scheme, through proper insulin amino acid design and corresponding fatty acid modification, the blood glucose reduction maintenance time in the medicine body is greatly improved.

More specifically, in the technical scheme, lysine (K) is newly introduced into the A ₂₂ position of insulin or the amino acid residue is replaced by lysine (K) at the A ₂₁ position, and the sites are used for connecting a fatty acid modifier to realize the fatty acid modification of the A chain. Compared with the modification of the G residue on the conventional A ₁ site, the operation mode (A ₂₁ site and A ₂₂ site) of the technical scheme can greatly improve the blood glucose reduction maintenance time in the medicine. According to the technical scheme, fatty acid modification can be carried out on the B ₂₆ position (the amino acid residue (Y) is required to be replaced by lysine (K)), the B ₂₈ position (the amino acid residue (P) is required to be replaced by lysine (K)) and the B ₂₉ position (lysine (K)) of insulin, so that the fatty acid modification of a B chain is realized, and the blood glucose reducing maintenance time in a medicine body is further improved. According to the technical scheme, the A chain and the B chain can be subjected to fatty acid modification simultaneously (one of A ₂₁ -site fatty acid modification and A ₂₂ -site fatty acid modification is selected, and one of B ₂₆ -site fatty acid modification, B ₂₈ -site fatty acid modification and B ₂₉ -site fatty acid modification is selected), so that the in-vivo acting time of the medicine is prolonged through double modification. In addition, threonine (T) at position B ₂₇ is replaced by glycine (G) and proline (P) at position B ₂₈ is replaced by glycine (G), and the change of the amino acid residue of the peptide chain can further improve the in-vivo acting time of the medicament on the basis of fatty acid modification.

Drawings

FIG. 1 shows the mass spectrum of analog 2 of example 4.

FIG. 2 shows the mass spectrum of analog 5 of example 4.

FIG. 3 shows the mass spectrum of analog 6 of example 4.

FIG. 4 is a mass spectrometry detection result of analog 10 of example 4.

FIG. 5 is the mass spectrometry results for analog 11 of example 4.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.

The structure of the ultra-long acting insulin analogue in the scheme is shown in a formula (1):

In formula (1), the amino acids represented by code numbers X ₁-X₁₂ respectively are shown in Table 1; a represents insulin A chain and B represents insulin B chain. The A chain amino acid sequence is shown in SEQ ID NO.1: GIVEQCCTSICSLNQLENYCNNN; the amino acid sequence of the B chain is shown in SEQ ID NO.2: FVNQHLCGSHLVEALNLVCGERGFNNNNNN. The A chain and the B chain are connected through disulfide bonds, and are a conventional connection mode between two subunits of insulin, and are not described in detail herein.

The human insulin sequence is: the A chain amino acid sequence is shown in SEQ ID NO.3: GIVEQCCTSICSLYQLENYCN; the amino acid sequence of the B chain is shown in SEQ ID NO.4: FVNQHLCGSHLVEALYLVCGERGFFYTPKT.

Table 1: composition of ultra-long acting insulin analogues

The double modification site is the K residue of the A chain end X ₂、X₃ and the K residue of the B chain end X ₈、X₁₀、X₁₁, and the modifier can be selected from fatty acid modification such as C ₁₈、C₂₀、C₂₂, namely the fatty acid modification of two sites is carried out on the insulin A chain and the insulin B chain. The structural formula of the C ₁₈、C₂₀、C₂₂ fatty acid modifier is shown in the formula (2), the formula (3) and the formula (4) respectively.

Example 1: blood glucose reducing effect in normal mice

Single administration of insulin analogues shown in tables 2 and 3 was performed in normal mice for maintenance of hypoglycemic effect. The detection process comprises the following steps: SPF grade KM mice, 10 weeks old, weighing about 35g, were given sufficient food and water for free feeding. After 1 week of environmental adaptation, the experiment was started. The mice are randomly divided into a physiological saline group and a drug test group, 6 mice in each group are injected according to the dose of 25U/kg, and meanwhile, 0.1 ml/serving as a control is given to the physiological saline group, and blood is taken from the tail of the mice at different times after the administration, and the blood glucose is detected by a high-strength glucometer. The maintenance time of the blood glucose reducing effect of the insulin analogues in mice is obtained by comparing the blood glucose values (comparing the blood glucose values of the experimental group and the control group), and the detection results are shown in table 3.

Table 2: the base composition of the insulin analogue to be tested (sequence of analogue is referred to in formula (1), specific amino acid residue selections are referred to in this table, "/" indicates the absence of amino acid residues)

Site(s)

A₁₄

A₂₁

A₂₂

A₂₃

B₃

B₁₆

B₂₅

B₂₆

B₂₇

B₂₈

B₂₉

B₃₀

Code number

X₁

X₂

X₃

X₄

X₅

X₆

X₇

X₈

X₉

X₁₀

X₁₁

X₁₂

Human insulin

Y

N

/

N

Y

F

Y

T

P

K

T

Analogue 1

E

N

/

N

H

Y

T

P

K

/

Analogue 2

E

N

/

N

H

Y

T

P

K

/

Analogue 3

E

N

K

/

N

H

Y

T

P

K

/

Analogue 4

E

N

K

/

N

H

Y

T

P

K

/

Analogue 5

E

N

K

/

N

H

Y

T

P

K

/

Analogue 6

E

N

K

/

N

H

Y

T

P

K

/

Analogue 7

E

N

K

/

N

H

Y

T

P

K

/

Analogue 8

E

N

K

/

N

H

Y

T

P

K

/

Analogue 9

E

N

K

/

N

H

Y

G

K

/

Analog 10

E

N

K

/

N

H

Y

G

K

/

Analogue 11

E

N

K

/

N

H

Y

G

K

/

Analog 12

E

N

K

/

N

H

Y

G

K

/

Analogue 13

E

N

/

N

H

Y

T

P

K

/

Analog 14

E

N

K

/

N

H

Y

T

P

K

/

Table 3: the insulin analogues to be tested were modified with fatty acids, administered amounts and in vivo activity in mice (amino acid composition of the analogues, see Table 2; "/" non-selected modification sites and fatty acid modifications)

From tables 2 and 3, it can be seen that the amino acid sequences of analogue 1 (B ₂₉ single modification), analogue 2 (a ₁、B₂₉ double modification), analogue 13 (precursor without fatty acid modification) are identical, the a chain sequence of which is shown in SEQ ID No.5: GIVEQCCTSICSLEQLENYCN; the B chain sequence is shown in SEQ ID NO.6: FVNQHLCGSHLVEALHLVCGERGFHYTPK. The A1 position of the A chain is G, and the analogue 2 is subjected to fatty acid modification on the residual amino group.

The amino acid sequences of analogues 3-8 and analogue 14 are identical, and the A chain sequence is shown in SEQ ID NO.7: GIVEQCCTSICSLEQLENYCNK; the B chain sequence is shown in SEQ ID NO.6: FVNQHLCGSHLVEALHLVCGERGFHYTPK. In the amino acid sequence, analogues 3-8, analogue 14, as compared to analogue 1 (analogue 2, analogue 13), have a new lysine (K) introduced at position A22. In the analogue 14, glu-free means that L-alpha-glutamic acid (Glu) is not contained in the fatty acid chain, and the rest of the fatty acid chains all contain L-alpha-glutamic acid (Glu), so that no obvious difference in synthesis process exists, and the specific structure of the fatty acid for modification is shown as a formula (5).

The amino acid sequences of analogues 9-12 are identical, the A chain sequence of which is shown in SEQ ID NO.7: GIVEQCCTSICSLEQLENYCNK; the B chain sequence is shown in SEQ ID NO.8: FVNQHLCGSHLVEALHLVCGERGFHYGGK. In amino acid sequence, analogs 9-12 have 3 amino acid substitutions compared to analog 1 (analog 2, analog 13): lysine (K) is newly introduced at the A ₂₂ position, threonine (T) at the B ₂₇ position is replaced by glycine (G), and proline (P) at the B ₂₈ position is replaced by glycine (G).

The results of the in vivo activity assay for insulin analogues were analyzed as follows:

(1) Insulin analogues (i.e. precursors) not modified by fatty acids have short in vivo blood glucose lowering maintenance times

Wherein, analogue 8 (A chain SEQ ID NO.7, B chain SEQ ID NO. 6), analogue 9 (A chain SEQ ID NO.7, B chain SEQ ID NO. 8) and analogue 13 (A chain SEQ ID NO.5, B chain SEQ ID NO. 6) are insulin analogues not modified by fatty acid. The three insulin analogues were substantially identical (2 h) in the mice with the same dose (25U/kg), indicating that the precursor of the insulin analogue had a short maintenance time for lowering blood glucose, which could be increased by modification with fatty acids.

(2) The fatty acid modification on the K residues of the A chain and the B chain can improve the blood glucose reduction and maintenance time in the medicine body to be short

To increase the maintenance time of insulin analogue activity, insulin represented by analogue 1 was tried to have a C ₂₀ modification added to the K residue on the B chain of analogue 13. The modification plays a very key role in improving the blood glucose reduction maintenance time. The inventor further researches other fatty acid modification sites, and firstly tries to modify fatty acid in A ₁ of A chain SEQ ID NO.5, so that the in-vivo blood glucose reduction maintenance time of the medicine can be further improved, and compared with the analogue 1, the in-vivo blood glucose reduction maintenance time is improved from 40h to 48h. If an amino acid residue K is added at the end of the A chain SEQ ID NO.5, a new A chain sequence SEQ ID NO.7 is formed, on which residue K a fatty acid modification is performed. A study was performed on a class of insulin analogues of the A chain SEQ ID NO.7+B chain SEQ ID NO.6 (i.e. analogues 3-8, analogue 14). Analogue 2 was compared to analogue 6, which differed in the C ₂₀ modification of the K residue at position a ₂₂ and the C ₂₀ modification of the G residue at position a ₁, the latter improving the in vivo hypoglycemic maintenance time by 8h compared to the former. C ₂₀ modification of K residue on A ₂₂ site can effectively promote the activity of drug mice in vivo and prolong the activity maintenance time. The replacement of the A ₂₂ site of the A chain of the insulin analogue with a K residue and the modification of the residue with a fatty acid were the first attempts by the inventors to obtain a significant effect in improving the duration of action of the drug.

(3) The fatty acid modification type has obvious influence on the in-vivo blood glucose reduction maintenance time of the medicine

The modification of analogue 14 with C ₂₀ only means that L-alpha-glutamic acid (Glu) is not contained in the fatty acid chain, and compared with the modification of analogue 6 with C ₂₀ containing Glu, the maintenance time of glucose reduction in mice of analogue 14 is only 25h, which is reduced by more than twice compared with 56h of analogue 6.

The carbon chain length of fatty acid can also have a significant effect on the in vivo hypoglycemic maintenance time of the drug. Generally, the larger the molecular weight of the modified molecule, the longer the circulation time of the drug in vivo, and thus the longer the drug action time. However, in the present technical solution, for the analogues of a-chain SEQ ID No.7 and B-chain SEQ ID No.6, C ₂₀ (analogue 6) can greatly increase the in vivo blood glucose reduction maintenance time (56 h), whereas both C ₂₂ (analogue 7) and C ₁₈ (analogue 5) modifications can only obtain the in vivo blood glucose reduction maintenance time of 35h, and only two carbon changes can bring about an effect increase of 60%, which was not predicted by the inventors before the experiment. For the analogues of A-chain SEQ ID NO.7, B-chain SEQ ID NO.8, the C ₂₀ (analogue 10) modification may result in an analogue with in vivo blood glucose lowering maintenance time of up to 80h, whereas C ₂₂ (analogue 11) has in vivo blood glucose lowering maintenance time of only 40h. Only two carbon changes can bring about a 50% improvement in effect, which the inventors have failed to predict before the experiment. Experimental data shows that the modification effect of C ₂₀ modification (containing Glu) is optimal when a plurality of fatty acid modifiers are screened.

(4) The B chain amino acid sequence has obvious influence on the in-vivo blood glucose reduction maintenance time of the medicine modified by fatty acid

The inventors adjusted for the B chain, obtained an analogue of the A chain SEQ ID NO.7+B chain SEQ ID NO.8, and tried fatty acid modification thereof. That is, the B chain has two amino acid residues TP replaced with GG based on SEQ ID NO. 6. Analogue 6 (amino acid residue TP) maintains blood glucose in vivo for 56h with the same C ₂₀ modification; analog 10 (amino acid residue GG) has a blood glucose lowering maintenance time of 80h. Analogue 7 (amino acid residue TP) maintains blood glucose in vivo for 35h with the same C ₂₂ modification; analog 11 (amino acid residue GG) has a blood glucose lowering maintenance time of 40h. Comparing analogue 10 with analogue 6, it can be seen that the in vivo blood glucose lowering maintenance time is greatly improved by 24 hours due to the replacement of two amino acid residues, and the improvement rate is 43%, which is unexpected by the inventor before the experiment. Compared with analogue 11 and analogue 7, the amino acid sequence of the A chain SEQ ID NO.7+B chain SEQ ID NO.8 is more advantageous for C ₂₂ modification, and although the substitution of two amino acid residues fails to produce more remarkable change than the C ₂₀ modification, the in vivo blood glucose reduction maintenance time is improved by 5h, and the improvement rate is 14%. For single fatty acid modification of the K residue of B ₂₉, analogue 12 (amino acid residue GG) and analogue 4 (amino acid residue TP) are compared, analogue 12 shows longer drug action time, 8h improvement and 20% improvement rate. It follows that the analogues of the A chain SEQ ID NO.7+B chain SEQ ID NO.6, the analogues of the A chain SEQ ID NO.7+B chain SEQ ID NO.8 only differ in two amino acids at the end of the B chain, but that the two types of analogues have very significant differences in the effect of the fatty acid modification, in particular in respect of the C ₂₀ fatty acid double modification, the B chain SEQ ID NO.8 has achieved unexpected technical effects.

In summary, analogs 9-12 have 3 amino acid substitutions compared to analog 1 (analog 2, analog 13): lysine (K) is newly introduced at the A ₂₂ position, threonine (T) at the B ₂₇ position is replaced by glycine (G), and proline (P) at the B ₂₈ position is replaced by glycine (G). Wherein, after amino acid exchange at position B ₂₇、B₂₈, the single modified analogue at position B ₂₉ has obviously prolonged activity time, and the double modified analogues at positions A ₂₂ and B ₂₉ have obviously prolonged activity time. Lysine (K) at position a ₂₂ can provide a new modification site, and the double modification activity time of positions a ₂₂ and B ₂₉ is higher than that of positions a ₁ and B ₂₉.

In addition to the above sequences (analogues 9-12), it is also possible to replace the N residue at the A chain end with a K residue, the B chain end X ₈(B₂₆) Y residue with a K residue, the B chain end X ₁₀(B₂₈) P residue with a K residue on the basis of human insulin, and introduce double modification sites (K residue is the linking site of the fatty acid modifier) to form a new insulin sequence. On the basis of these novel insulin sequences, a double fatty acid modification can also be carried out to obtain an effect similar to that of analogue 10.

Example 2: recombinant expression of insulin analogues in E.coli

Based on the amino acid sequences of insulin analog A (see Table 1 and Table 2 for insulin analogs containing A and B chains), cDNA sequences were designed based on E.coli preferred codons, and were synthesized by the Probiotechnological engineering (Shanghai) Co., ltd. The synthesized cDNA fragment (inserted into pET-26b (+) plasmid) was digested with Nde I and Hind III, and the recombinant fusion-expressed pET-26b (+) plasmid was digested with the same enzymes to recover a large fragment. The TOP10 strain was transformed by ligation of the insulin analogue A gene fragment with pET-26b (+) under the action of T4 ligase. The recombinant plasmid containing the inserted insulin analogue A gene was selected by plate screening and designated pET-26b-A. Then the recombinant expression strain is obtained by transforming the expression host bacterium BL21 (DE 3) by CaCl ₂ method, and the fusion protein of the insulin analogue A is produced by 1mmol/L IPTG induction expression. The fusion protein is subjected to inclusion body recovery, renaturation and ion exchange chromatography, then is subjected to enzyme digestion, and finally is purified by reverse phase chromatography and is freeze-dried to obtain an insulin analogue A precursor. The preparation process of the insulin analogue a precursor (i.e. insulin analogue not modified by fatty acid) is specifically as follows: after recombinant expression by escherichia coli, the thalli are collected by high-speed centrifugation, the thalli are crushed by water suspension thalli under the pressure of 60MPa, and the insulin analogue A inclusion body is obtained by centrifugation. Insulin analogue inclusion bodies are mixed according to the mass-volume ratio of 1:10 adding a dissolving solution (8 mol/L urea, 20mmol/L beta-mercaptoethanol, 20mol/L Tris, pH 10.0) for dissolving, stirring until the precipitate is completely dissolved, and centrifuging to obtain an insulin analogue A fusion protein dissolving solution; dissolving solution in a volume ratio of 1:10 adding pre-cooling water at 2-8 ℃ for dilution, standing and renaturation for 40 hours at 2-8 ℃, regulating pH to 5.0-6.0 by dilute hydrochloric acid, standing and precipitation for 2 hours, and centrifuging to obtain insulin analogue A fusion protein; dissolving with 50mmol/L Tris, pH9.0, and digestion for 8 hours at 25℃using a C8 reverse phase chromatography column, mobile phase A:20% acetonitrile, 0.1% tfa, mobile phase B: gradient elution of 60% acetonitrile gave insulin analogue a precursor.

Example 3: recombinant expression of insulin analogues in Pichia pastoris

According to the amino acid sequence of insulin analogue A, its cDNA sequence was designed according to Pichia pastoris preferred codon, and total gene artificial synthesis was carried out by the division of biological engineering (Shanghai). The synthesized cDNA fragment (inserted into pPICZ alpha A plasmid) was digested with Not I and Xho I, and the recombinant fusion-expressed pPICZ alpha A plasmid was digested with the same enzymes to recover the large fragment. The TOP10 strain was transformed by ligation of the insulin analogue A gene fragment with pPICZαA under the action of T4 ligase. The recombinant plasmid containing the inserted insulin analogue A gene was selected by plate screening and designated pPICZ alpha A-A. And then transforming the expression host bacterium X-33 by an electrotransformation method to obtain a recombinant expression strain, and carrying out methanol-glycerol mixed induction expression to generate the fusion protein of the insulin analogue A. The fusion protein is subjected to ion exchange chromatography, enzyme digestion, and purification by reverse phase chromatography to obtain recombinant insulin analogue A, and freeze-drying to obtain dry powder. After recombinant expression of pichia pastoris, high-speed centrifugation is carried out to obtain a thallus supernatant, cation exchange chromatography is used, and a mobile phase A:20mmol/L sodium acetate, pH3.0, mobile phase B: ion-purifying 20mmol/L sodium acetate, 0.5MNaCl and pH3.0 to obtain insulin analogue A fusion protein, and performing digestion at 25 ℃ and pH8.0 for 8 hours, using a C8 reverse phase chromatographic column, and obtaining a mobile phase A:20% acetonitrile, 0.1% tfa, mobile phase B: gradient elution is carried out by 60% acetonitrile to obtain the insulin analogue A precursor. This example is a general procedure for obtaining insulin analog A precursors. The obtained precursor is then subjected to subsequent modification.

Example 4: modification of insulin analogue precursors with modifier

The single-modified sample and the double-modified sample are mainly obtained by controlling the modification proportion, are conventional means in the prior art, and the A ₂₂ site and the B ₂₉ site can be preferentially modified by the modifier under the condition of pH9.5-11.5, and can be separated by reversed phase chromatography. In modification ratio 1:4 (insulin analogue precursor: modifier), a double modified sample of A ₂₂ and B ₂₉ can be obtained and recovered by reverse phase chromatography. To obtain the a22 single modification analogue (analogue 3), insulin analogue precursor: the molar ratio of the modifier is controlled to be 1:1.5-1:2.5; to obtain the B29 single modified analogues (analogues 4, 12), insulin analogue precursors: the modification proportion of the modifier is controlled to be 1:1.5-1:2.5. then, the target sample was isolated by reverse phase chromatography.

Dissolving modifier (C ₁₈、C₂₀ or C ₂₂) in N-methylpyrrolidone at 100mg/ml, weighing a certain amount of insulin analogue precursor, dissolving the insulin analogue precursor into 10mg/ml by using 0.1mol/L sodium carbonate, controlling pH to 10.5-11.5, and stirring at room temperature according to the modifier: insulin analogue precursor molar ratio is 4:1-6:1, after 0.5 hour of reaction, pH7.5 was adjusted with acetic acid. Purifying by reverse phase chromatography after the reaction is completed to obtain insulin analogues, freeze-drying and preserving, and analyzing by utilizing LC-MS to obtain molecular weight information. In addition, the synthesis method of the modifier (C ₁₈、C₂₀ or C ₂₂) can be seen in my prior patent CN116655770A (fatty acid modified thrombopoietin mimetic peptide homotetramer and preparation method and application thereof). The molecular weight information of the synthesized analogues 1-14 of this scheme is shown in Table 4, and the mass spectrum detection results of analogues 2, 5, 6, 10 and 11 are shown in FIGS. 1-5.

Table 4: molecular weight information for analogues 1-4

Example 5: blood glucose reduction experiment for mouse type I diabetes model

(1) Mouse modeling

The Streptozotocin (STZ) is injected into the abdominal cavity to construct a model of the type I diabetes of the mice, the blood sugar measurement reaches more than 16.8mmol/L, and the model is formed.

(2) Administration to mice

The molded mice were divided into three groups of model group, analogue 1, analogue 6. Normal mice served as blank. PBS was administered 6 times to the normal and model groups, with 0.2ml of PBS per dose. Analogue 1 was administered at a frequency of 1/36 hours; analogue 6 was administered at a frequency of 1/36 hours at a dose of 25U/kg. After 6 consecutive administrations, blood glucose was measured for each group of mice at 0h, 12h, 24h, 36h, 48h, 72h, 78h, 84h, 96h post-administration until the blood glucose of the administered group was restored to the pre-administration blood glucose level. See table 5 for experimental data. Using analogue 1, blood glucose levels were restored to pre-dosing levels at 72 h. While blood glucose levels were maintained below pre-dose levels for 96 hours after dosing using analogue 6, indicating that analogue 6 was longer acting and better.

Table 5: blood glucose level measurement result (mmol/L)

The foregoing is merely exemplary of the present application, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims

1. An ultra-long acting insulin analogue comprising a chain a: GIVEQCCTSICSLX ₁QLENYCX₂X₃X₄, and B chain ;FVX₅QHLCGSHLVEALX₆LVCGERGFX₇X₈X₉X₁₀X₁₁X₁₂;

2. The ultra-long acting insulin analogue of claim 1 wherein the fatty acid is also modified at the amino acid residue of X ₂ or X ₃.

3. The ultra-long acting insulin analogue of claim 2, wherein the fatty acid comprises a C ₁₈ modifier of formula (2) and a C ₂₀ modifier of formula (3); c ₂₂ modifier with structural formula shown in formula (4);

4. A very long acting insulin analogue according to claim 3, wherein X ₁ is E; x ₂ is N or K; x ₃ is K or absent; x ₄ is absent; x ₅ is N; x ₆ is H; x ₇ is H; x ₈ is Y; x ₉ is T or G; x ₁₀ is P or G; x ₁₁ is K; x ₁₂ is absent; fatty acids are modified at amino acid residues X ₁₁ and fatty acids are also modified at amino acid residues X ₂ or X ₃.

5. The ultra-long acting insulin analogue of claim 4, wherein the sequence of the a chain is shown in SEQ ID No.5 or SEQ ID No.7 and the sequence of the B chain is shown in SEQ ID No.6 or SEQ ID No. 8.

6. The ultra-long acting insulin analogue of claim 5 wherein the a-chain and the B-chain are linked by disulfide bonds.

7. The method for preparing a very long acting insulin analogue according to any one of claims 1-6, comprising the steps of, in order:

s1: obtaining an insulin analog a precursor;

s2: insulin analogues were subjected to fatty acid modification.

8. The method for preparing ultra-long acting insulin analogue according to claim 7, wherein in S1, the precursor of insulin analogue a is obtained by recombinant expression using escherichia coli or yeast.

9. The method for preparing ultra-long acting insulin analogue according to claim 8, wherein in S2, the molar ratio of modifier to insulin analogue is 3:1-6:1, a step of; the fatty acid modification process is as follows: dissolving a modifier in N-methyl pyrrolidone to obtain a modifier solution; dissolving insulin analogue with sodium carbonate to obtain insulin analogue solution; the insulin analogue solution and the modifier solution are reacted and purified to obtain the fatty acid modified insulin analogue.

10. Use of an ultralong acting insulin analogue according to any one of claims 1-6 for the preparation of a medicament for the treatment of diabetes or a hypoglycemic agent.